Fluorine-18 Radiochemistry, Labeling Strategies and Synthetic Routes

PubMed Central

2015-01-01

Fluorine-18 is the most frequently used radioisotope in positron emission tomography (PET) radiopharmaceuticals in both clinical and preclinical research. Its physical and nuclear characteristics (97% β+ decay, 109.7 min half-life, 635 keV positron energy), along with high specific activity and ease of large scale production, make it an attractive nuclide for radiochemical labeling and molecular imaging. Versatile chemistry including nucleophilic and electrophilic substitutions allows direct or indirect introduction of 18F into molecules of interest. The significant increase in 18F radiotracers for PET imaging accentuates the need for simple and efficient 18F-labeling procedures. In this review, we will describe the current radiosynthesis routes and strategies for 18F labeling of small molecules and biomolecules. PMID:25473848

Quantitative Whole Body Biodistribution of Fluorescent-Labeled Agents by Non-Invasive Tomographic Imaging

PubMed Central

Vasquez, Kristine O.; Casavant, Chelsea; Peterson, Jeffrey D.

2011-01-01

When small molecules or proteins are injected into live animals, their physical and chemical properties will significantly affect pharmacokinetics, tissue penetration, and the ultimate routes of metabolism and clearance. Fluorescence molecular tomography (FMT) offers the ability to non-invasively image and quantify temporal changes in fluorescence throughout the major organ systems of living animals, in a manner analogous to traditional approaches with radiolabeled agents. This approach is best used with biotherapeutics (therapeutic antibodies, or other large proteins) or large-scaffold drug-delivery vectors, that are minimally affected by low-level fluorophore conjugation. Application to small molecule drugs should take into account the significant impact of fluorophore labeling on size and physicochemical properties, however, the presents studies show that this technique is readily applied to small molecule agents developed for far-red (FR) or near infrared (NIR) imaging. Quantification by non-invasive FMT correlated well with both fluorescence from tissue homogenates as well as with planar (2D) fluorescence reflectance imaging of excised intact organs (r2 = 0.996 and 0.969, respectively). Dynamic FMT imaging (multiple times from 0 to 24 h) performed in live mice after the injection of four different FR/NIR-labeled agents, including immunoglobulin, 20–50 nm nanoparticles, a large vascular imaging agent, and a small molecule integrin antagonist, showed clear differences in the percentage of injected dose per gram of tissue (%ID/g) in liver, kidney, and bladder signal. Nanoparticles and IgG1 favored liver over kidney signal, the small molecule integrin-binding agent favored rapid kidney and bladder clearance, and the vascular agent, showed both liver and kidney clearance. Further assessment of the volume of distribution of these agents by fluorescent volume added information regarding their biodistribution and highlighted the relatively poor extravasation into tissue by IgG1. These studies demonstrate the ability of quantitative FMT imaging of FR/NIR agents to non-invasively visualize and quantify the biodistribution of different agents over time. PMID:21731618

Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.

PubMed

Altai, Mohamed; Honarvar, Hadis; Wållberg, Helena; Strand, Joanna; Varasteh, Zohreh; Rosestedt, Maria; Orlova, Anna; Dunås, Finn; Sandström, Mattias; Löfblom, John; Tolmachev, Vladimir; Ståhl, Stefan

2014-11-24

Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of (188)Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental (188)Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of (188)Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs. Copyright © 2014 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

Dextran hydrogel coated surface plasmon resonance imaging (SPRi) sensor for sensitive and label-free detection of small molecule drugs

NASA Astrophysics Data System (ADS)

Li, Shaopeng; Yang, Mo; Zhou, Wenfei; Johnston, Trevor G.; Wang, Rui; Zhu, Jinsong

2015-11-01

The label-free and sensitive detection of small molecule drugs on SPRi is still a challenging task, mainly due to the limited surface immobilization capacity of the sensor. In this research, a dextran hydrogel-coated gold sensor chip for SPRi was successfully fabricated via photo-cross-linking for enhanced surface immobilization capacity. The density of the dextran hydrogel was optimized for protein immobilization and sensitive small molecule detection. The protein immobilization capacity of the hydrogel was 10 times greater than a bare gold surface, and 20 times greater than an 11-mercaptoundecanoic acid (MUA) surface. Such a drastic improvement in immobilization capacity allowed the SPRi sensor to detect adequate response signals when probing small molecule binding events. The binding signal of 4 nM liquid-phase biotin to streptavidin immobilized on the dextran surface reached 435 RU, while no response was observed on bare gold or MUA surfaces. The dextran hydrogel-coated SPRi sensor was also applied in a kinetic study of the binding between an immunosuppressive drug (FK506) and its target protein (FKBP12) in a high-throughput microarray format. The measured binding affinity was shown to be consistent with reported literature values, and a detection limit of 0.5 nM was achieved.

Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs.

PubMed

Gao, Jinxu; Mfuh, Adelphe; Amako, Yuka; Woo, Christina M

2018-03-28

Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by photoaffinity labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope.

PubMed

Fei, Yiyan; Landry, James P; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope

PubMed Central

Fei, Yiyan; Landry, James P.; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S.

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm×4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide. PMID:20210464

Phosphatidylglycerol Synthesis in Spinach Chloroplasts: Characterization of the Newly Synthesized Molecule

PubMed Central

Sparace, Salvatore A.; Mudd, J. Brian

1982-01-01

Intact chloroplasts from spinach (Spinacia oleracea L., hybrid 424) readily incorporate [14C]glycerol-3-phosphate and [14C]acetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. Glycerol-3-phosphate-labeled phosphatidylglycerol is equally labeled in the two glycerol moieties of the molecule. Acetate-labeled phosphatidylglycerol is equally labeled in both acyl groups. Position one contains primarily oleate, linoleate and small amounts of palmitate. Position two contains primarily palmitate. No radioactive trans-Δ3-hexadecenoate was detected. The labeling patterns indicate that the radioactive phosphatidylglycerol is the product of de novo chloroplast lipid biosynthesis and furthermore, phosphatidylglycerol may be a substrate for fatty acid desaturation. Images Fig. 1 PMID:16662664

Formylbenzene diazonium hexafluorophosphate reagent for tyrosine-selective modification of proteins and the introduction of a bioorthogonal aldehyde

PubMed Central

Gavrilyuk, Julia; Ban, Hitoshi; Nagano, Masanobu; Hakamata, Wataru; Barbas, Carlos F.

2012-01-01

4-Formylbenzene diazonium hexafluorophosphate (FBDP) is a novel bench-stable crystalline diazonium salt that reacts selectively with tyrosine to install a bioorthogonal aldehyde functionality. Model studies with N-acyl-tyrosine methylamide allowed us to identify conditions optimal for tyrosine ligation reactions with small peptides and proteins. FBDP-based conjugation was used for the facile introduction of small molecule tags, poly(ethylene) glycol chains (PEGylation), and functional small molecules onto model proteins and to label the surface of living cells. PMID:23181702

Formylbenzene diazonium hexafluorophosphate reagent for tyrosine-selective modification of proteins and the introduction of a bioorthogonal aldehyde.

PubMed

Gavrilyuk, Julia; Ban, Hitoshi; Nagano, Masanobu; Hakamata, Wataru; Barbas, Carlos F

2012-12-19

4-Formylbenzene diazonium hexafluorophosphate (FBDP) is a novel bench-stable crystalline diazonium salt that reacts selectively with tyrosine to install a bioorthogonal aldehyde functionality. Model studies with N-acyl-tyrosine methylamide allowed us to identify conditions optimal for tyrosine ligation reactions with small peptides and proteins. FBDP-based conjugation was used for the facile introduction of small molecule tags, poly(ethylene glycol) chains (PEGylation), and functional small molecules onto model proteins and to label the surface of living cells.

A new fluorogenic small molecule labeling tool for surface diffusion analysis and advanced fluorescence imaging of β-site amyloid precursor protein (APP)-cleaving enzyme 1 based on silicone rhodamine: SiR-BACE1.

PubMed

Karch, Sandra; Broichhagen, Johannes; Schneider, Julia; Böning, Daniel; Hartmann, Stephanie; Schmid, Benjamin; Tripal, Philipp; Palmisano, Ralf; Alzheimer, Christian; Johnsson, Kai; Huth, Tobias

2018-06-25

β-site APP-cleaving enzyme 1 (BACE1) is a major player in the pathogenesis of Alzheimer's disease. Structural and functional fluorescence microscopy offers a powerful approach to learn about the physiology and pathophysiology of this protease. Up to now, however, common labeling techniques either require genetic manipulation, use large antibodies, or are not compatible with live cell imaging. Fluorescent small molecules that specifically bind to the protein of interest can overcome these limitations. Herein, we introduce SiR-BACE1, a conjugate of the BACE1 inhibitor S-39 and SiR647, as a novel fluorogenic, tag-free, and antibody-free label for BACE1. We present its chemical development, characterize its photo-physical and pharmacologic properties, and evaluate its behavior in solution, in over-expression systems, and in native brain tissue. We demonstrate its applicability in confocal, stimulated emission depletion (STED), and dynamic single molecule microscopy. First functional studies with SiR-BACE1 on the surface mobility of BACE1 revealed a markedly confined diffusion pattern.

A Simple Picaxe Microcontroller Pulse Source for Juxtacellular Neuronal Labelling.

PubMed

Verberne, Anthony J M

2016-10-19

Juxtacellular neuronal labelling is a method which allows neurophysiologists to fill physiologically-identified neurons with small positively-charged marker molecules. Labelled neurons are identified by histochemical processing of brain sections along with immunohistochemical identification of neuropeptides, neurotransmitters, neurotransmitter transporters or biosynthetic enzymes. A microcontroller-based pulser circuit and associated BASIC software script is described for incorporation into the design of a commercially-available intracellular electrometer for use in juxtacellular neuronal labelling. Printed circuit board construction has been used for reliability and reproducibility. The current design obviates the need for a separate digital pulse source and simplifies the juxtacellular neuronal labelling procedure.

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based β-lactam probes.

PubMed

Sadhu, Kalyan K; Mizukami, Shin; Watanabe, Shuji; Kikuchi, Kazuya

2011-05-01

Development of protein labeling techniques with small molecules is enthralling because this method brings promises for triumph over the limitations of fluorescent proteins in live cell imaging. This technology deals with the functionalization of proteins with small molecules and is anticipated to facilitate the expansion of various protein assay methods. A new straightforward aggregation and elimination-based technique for a protein labeling system has been developed with a versatile emissive range of fluorophores. These fluorophores have been applied to show their efficiency for protein labeling by exploiting the same basic principle. A genetically modified version of class A type β-lactamase has been used as the tag protein (BL-tag). The strength of the aggregation interaction between a fluorophore and a quencher plays a governing role in the elimination step of the quencher from the probes, which ultimately controls the swiftness of the protein labeling strategy. Modulation in the elimination process can be accomplished by the variation in the nature of the fluorophore. This diversity facilitates the study of the competitive binding order among the synthesized probes toward the BL-tag labeling method. An aggregation and elimination-based BL-tag technique has been explored to develop an order of color labeling from the equimolar mixture of the labeling probe in solutions. The qualitative and quantitative determination of ordering within the probes toward labeling studies has been executed through SDS-PAGE and time-dependent fluorescence intensity enhancement measurements, respectively. The desirable multiple-wavelength fluorescence labeling probes for the BL-tag technology have been developed and demonstrate broad applicability of this labeling technology to live cell imaging with coumarin and fluorescein derivatives by using confocal microscopy.

LABELLING OF NUCLEIC ACID WITH RADIOACTIVE ISOTOPES AND THEIR APPLICATION (in Yugoslavian)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Becarevic, A.; Hudnik-Plevnik, T.; Glisin, V.

The DNA of the liver and spleen of rats was labeled with P/sup 32/. The specific activity obtained was high enough to be able to follow the fate of these acids after injection in irradiated and non-irradiated rats. The results show that, after injection in the rats, the labeled kids were degraded into small fragmnts, which were utilized as building blocks for the synthesis of the big molecules of the cells. (auth)

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA.

PubMed

Razvi, F; Gargiulo, G; Worcel, A

1983-08-01

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

Pharmacogenomic Biomarkers: an FDA Perspective on Utilization in Biological Product Labeling.

PubMed

Schuck, Robert N; Grillo, Joseph A

2016-05-01

Precision medicine promises to improve both the efficacy and safety of therapeutic products by better informing why some patients respond well to a drug, and some experience adverse reactions, while others do not. Pharmacogenomics is a key component of precision medicine and can be utilized to select optimal doses for patients, more precisely identify individuals who will respond to a treatment and avoid serious drug-related toxicities. Since pharmacogenomic biomarker information can help inform drug dosing, efficacy, and safety, pharmacogenomic data are critically reviewed by FDA staff to ensure effective use of pharmacogenomic strategies in drug development and appropriate incorporation into product labels. Pharmacogenomic information may be provided in drug or biological product labeling to inform health care providers about the impact of genotype on response to a drug through description of relevant genomic markers, functional effects of genomic variants, dosing recommendations based on genotype, and other applicable genomic information. The format and content of labeling for biologic drugs will generally follow that of small molecule drugs; however, there are notable differences in pharmacogenomic information that might be considered useful for biologic drugs in comparison to small molecule drugs. Furthermore, the rapid entry of biologic drugs for treatment of rare genetic diseases and molecularly defined subsets of common diseases will likely lead to increased use of pharmacogenomic information in biologic drug labels in the near future. In this review, we outline the general principles of therapeutic product labeling and discuss the utilization of pharmacogenomic information in biologic drug labels.

Calcium phosphate nanoparticles as versatile carrier for small and large molecules across cell membranes

NASA Astrophysics Data System (ADS)

Sokolova, Viktoriya; Rotan, Olga; Klesing, Jan; Nalbant, Perihan; Buer, Jan; Knuschke, Torben; Westendorf, Astrid M.; Epple, Matthias

2012-06-01

The successful transport of molecules across the cell membrane is a key point in biology and medicine. In most cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100-250 nm, depending on the functionalization) were loaded with fluorescent oligonucleotides, peptide, proteins, antibodies, polymers or porphyrins and characterized by dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. Any excess of molecules was removed by ultracentrifugation, and the dissolved molecules at the same concentration were used as control. The uptake of such fluorescence-labeled nanoparticles into HeLa cells was monitored by fluorescence microscopy and confocal laser scanning microscopy. Calcium phosphate nanoparticles were able to transport all molecules across the cell membrane, whereas the dissolved molecules alone were taken up only to a very small extent or even not at all.

Fluorescence quenching by TEMPO: a sub-30 A single-molecule ruler.

PubMed

Zhu, Peizhi; Clamme, Jean-Pierre; Deniz, Ashok A

2005-11-01

A series of DNA molecules labeled with 5-carboxytetramethylrhodamine (5-TAMRA) and the small nitroxide radical TEMPO were synthesized and tested to investigate whether the intramolecular quenching efficiency can be used to measure short intramolecular distances in small ensemble and single-molecule experiments. In combination with distance calculations using molecular mechanics modeling, the experimental results from steady-state ensemble fluorescence and fluorescence correlation spectroscopy measurements both show an exponential decrease in the quenching rate constant with the dye-quencher distance in the 10-30 A range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules.

Evolution and Protein Packaging of Small Molecule RNA Aptamers

PubMed Central

Lau, Jolene L.; Baksh, Michael M.; Fiedler, Jason D.; Brown, Steven D.; Kussrow, Amanda; Bornhop, Darryl J.; Ordoukhanian, Phillip

2011-01-01

A high-affinity RNA aptamer (Kd = 50 nM) was efficiently identified by SELEX against a heteroaryl dihydropyrimidine structure, chosen as a representative drug-like molecule with no cross reactivity with mammalian or bacterial cells. This aptamer, its weaker-binding variants, and a known aptamer against theophylline were each embedded in a longer RNA sequence that was encapsidated inside a virus-like particle by a convenient expression technique. These nucleoprotein particles were shown by backscattering interferometry to bind to the small-molecule ligands with affinities similar to those of the free (non-encapsidated) aptamers. The system therefore comprises a general approach to the production and sequestration of functional RNA molecules, characterized by a convenient label-free analytical technique. PMID:21899290

A Simple Picaxe Microcontroller Pulse Source for Juxtacellular Neuronal Labelling †

PubMed Central

Verberne, Anthony J. M.

2016-01-01

Juxtacellular neuronal labelling is a method which allows neurophysiologists to fill physiologically-identified neurons with small positively-charged marker molecules. Labelled neurons are identified by histochemical processing of brain sections along with immunohistochemical identification of neuropeptides, neurotransmitters, neurotransmitter transporters or biosynthetic enzymes. A microcontroller-based pulser circuit and associated BASIC software script is described for incorporation into the design of a commercially-available intracellular electrometer for use in juxtacellular neuronal labelling. Printed circuit board construction has been used for reliability and reproducibility. The current design obviates the need for a separate digital pulse source and simplifies the juxtacellular neuronal labelling procedure. PMID:28952589

Improved Tumor Targeting and Longer Retention Time of NIR Fluorescent Probes Using Bioorthogonal Chemistry.

PubMed

Zhang, Xianghan; Wang, Bo; Zhao, Na; Tian, Zuhong; Dai, Yunpeng; Nie, Yongzhan; Tian, Jie; Wang, Zhongliang; Chen, Xiaoyuan

2017-01-01

The traditional labeling method for targeted NIR fluorescence probes requires directly covalent-bonded conjugation of targeting domains and fluorophores in vitro . Although this strategy works well, it is not sufficient for detecting or treating cancers in vivo , due to steric hindrance effects that relatively large fluorophore molecules exert on the configurations and physiological functions of specific targeting domains. The copper-free, "click-chemistry"-assisted assembly of small molecules in living systems may enhance tumor accumulation of fluorescence probes by improving the binding affinities of the targeting factors. Here, we employed a vascular homing peptide, GEBP11, as a targeting factor for gastric tumors, and we demonstrate its effectiveness for in vivo imaging via click-chemistry-mediated conjugation with fluorescence molecules in tumor xenograft mouse models. This strategy showed higher binding affinities than those of the traditional conjugation method, and our results showed that the tumor accumulation of click-chemistry-mediated probes are 11-fold higher than that of directly labeled probes. The tracking life was prolonged by 12-fold, and uptake of the probes into the kidney was reduced by 6.5-fold. For lesion tumors of different sizes, click-chemistry-mediated probes can achieve sufficient signal-to-background ratios (3.5-5) for in vivo detection, and with diagnostic sensitivity approximately 3.5 times that of traditional labeling probes. The click-chemistry-assisted detection strategy utilizes the advantages of "small molecule" probes while not perturbing their physiological functions; this enables tumor detection with high sensitivity and specific selectivity.

Shaping Small Bioactive Molecules to Untangle Their Biological Function: A Focus on Fluorescent Plant Hormones.

PubMed

Lace, Beatrice; Prandi, Cristina

2016-08-01

Modern biology overlaps with chemistry in explaining the structure and function of all cellular processes at the molecular level. Plant hormone research is perfectly located at the interface between these two disciplines, taking advantage of synthetic and computational chemistry as a tool to decipher the complex biological mechanisms regulating the action of plant hormones. These small signaling molecules regulate a wide range of developmental processes, adapting plant growth to ever changing environmental conditions. The synthesis of small bioactive molecules mimicking the activity of endogenous hormones allows us to unveil many molecular features of their functioning, giving rise to a new field, plant chemical biology. In this framework, fluorescence labeling of plant hormones is emerging as a successful strategy to track the fate of these challenging molecules inside living organisms. Thanks to the increasing availability of new fluorescent probes as well as advanced and innovative imaging technologies, we are now in a position to investigate many of the dynamic mechanisms through which plant hormones exert their action. Such a deep and detailed comprehension is mandatory for the development of new green technologies for practical applications. In this review, we summarize the results obtained so far concerning the fluorescent labeling of plant hormones, highlighting the basic steps leading to the design and synthesis of these compelling molecular tools and their applications. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Detection of protein-small molecule binding using a self-referencing external cavity laser biosensor.

PubMed

Meng Zhang; Peh, Jessie; Hergenrother, Paul J; Cunningham, Brian T

2014-01-01

High throughput screening of protein-small molecule binding interactions using label-free optical biosensors is challenging, as the detected signals are often similar in magnitude to experimental noise. Here, we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with sub-picometer wavelength accuracy. Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets with binding affinities or inhibition constants in the sub-nanomolar to low micromolar range. The demonstrated ability to perform detection in the presence of several interfering compounds opens the potential for increasing the throughput of the approach. As an example application, we performed a "needle-in-the-haystack" screen for inhibitors against carbonic anhydrase isozyme II (CA II), in which known inhibitors are clearly differentiated from inactive molecules within a compound library.

Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Kohl, Thomas O; Ascoli, Carl A

2017-07-05

The competitive enzyme-linked immunosorbent assay (ELISA) (cELISA; also called an inhibition ELISA) is designed so that purified antigen competes with antigen in the test sample for binding to an antibody that has been immobilized in microtiter plate wells. The same concept works if the immobilized molecule is antigen and the competing molecules are purified labeled antibody versus antibody in a test sample. Direct cELISAs incorporate labeled antigen or antibody, whereas indirect assay configurations use reporter-labeled secondary antibodies. The cELISA is very useful for determining the concentration of small-molecule antigens in complex sample mixtures. In the direct cELISA, antigen-specific capture antibody is adsorbed onto the microtiter plate before incubation with either known standards or unknown test samples. Enzyme-linked antigen (i.e., labeled antigen) is also added, which can bind to the capture antibody only when the antibody's binding site is not occupied by either the antigen standard or antigen in the test samples. Unbound labeled and unlabeled antigens are washed away and substrate is added. The amount of antigen in the standard or the test sample determines the amount of reporter-labeled antigen bound to antibody, yielding a signal that is inversely proportional to antigen concentration within the sample. Thus, the higher the antigen concentration in the test sample, the less labeled antigen is bound to the capture antibody, and hence the weaker is the resultant signal. © 2017 Cold Spring Harbor Laboratory Press.

Evaluation of a maleimido derivative of CHX-A” DTPA for site-specific labeling of Affibody molecules

PubMed Central

Tolmachev, Vladimir; Xu, Heng; Wållberg, Helena; Ahlgren, Sara; Hjertman, Magnus; Sjöberg, Anna; Sandström, Mattias; Abrahmsén, Lars; Brechbiel, Martin W.; Orlova, Anna

2008-01-01

Affibody molecules are a new class of small targeting proteins based on a common threehelix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule ZHER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A” DTPA was site-specifically conjugated at the C-terminal cysteine of ZHER2:2395-C, a variant of ZHER2:342, providing a homogenous conjugate with a dissociation constant of 56 pM. The yield of labeling with 111In was > 99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of 111In-CHX-A” DTPAZ2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all non-specific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of 111In-CHX-A” DTPAZ2395-C was 17.3 ± 4.8 % IA/g and the tumor-to-blood ratio 86 ± 46 (4 h post-injection). HER2-exprssing xenografts were clearly visualized 1 h post-injection. In conclusion, coupling of maleimido-CHX-A” DTPA to cysteine-containing Affibody molecules provides welldefined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo. PMID:18620447

Low Copy Numbers of DC-SIGN in Cell Membrane Microdomains: Implications for Structure and Function

PubMed Central

Liu, Ping; Wang, Xiang; Itano, Michelle S.; Neumann, Aaron K.; de Silva, Aravinda M.; Jacobson, Ken; Thompson, Nancy L.

2014-01-01

Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy (TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC-SIGN, a C-type lectin, in membrane microdomains. DC-SIGN or its mutants were labeled with primary monoclonal antibodies (mAbs) in either dendritic cells (DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC-SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1μm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3T3 cells contains only 4-8 molecules of DC-SIGN, consistent with our preliminary super-resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (~50nm) pathogen, dengue virus, leading to infection of host cells. PMID:24313910

Single-molecule fluorescence study of the inhibition of the oncogenic functionality of STAT3

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Badali, Daniel; Fletcher, Steven; Avadisian, Miriam; Gunning, Patrick; Gradinaru, Claudiu

2009-06-01

Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein plays an important role in the onset of cancers such as leukemia and lymphoma. In this study, we aim to test the effectiveness of a novel peptide drug designed to tether STAT3 to the phospholipid bilayer of the cell membrane and thus inhibit unwanted transcription. As a first step, STAT3 proteins were successfully labelled with tetramethylrhodamine (TMR), a fluorescent dye with suitable photostability for single molecule studies. The effectiveness of labelling was determined using fluorescence correlation spectroscopy in a custom built confocal microscope, from which diffusion times and hydrodynamic radii of individual proteins were determined. A newly developed fluorescein derivative label (F-NAc) has been designed to be incorporated into the structure of the peptide drug so that peptide-STAT3 interactions can be examined. This dye is spectrally characterized and is found to be well suited for its application to this project, as well as other single-molecule studies. The membrane localization via high-affinity cholesterol-bound small-molecule binding agents can be demonstrated by encapsulating TMR-labeled STAT3 and inhibitors within a vesicle model cell system. To this end, unilaminar lipid vesicles were examined for size and encapsulation ability. Preliminary results of the efficiency and stability of the STAT3 anchoring in lipid membranes obtained via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope are reported here.

Fluorescence Quenching by TEMPO: A Sub-30 Å Single-Molecule Ruler

PubMed Central

Zhu, Peizhi; Clamme, Jean-Pierre; Deniz, Ashok A.

2005-01-01

A series of DNA molecules labeled with 5-carboxytetramethylrhodamine (5-TAMRA) and the small nitroxide radical TEMPO were synthesized and tested to investigate whether the intramolecular quenching efficiency can be used to measure short intramolecular distances in small ensemble and single-molecule experiments. In combination with distance calculations using molecular mechanics modeling, the experimental results from steady-state ensemble fluorescence and fluorescence correlation spectroscopy measurements both show an exponential decrease in the quenching rate constant with the dye-quencher distance in the 10–30 Å range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules. PMID:16199509

HaloTag technology for specific and covalent labeling of fusion proteins.

PubMed

Benink, Hélène A; Urh, Marjeta

2015-01-01

Appending proteins of interest to fluorescent protein tags such as GFP has revolutionized how proteins are studied in the cellular environment. Over the last few decades many varieties of fluorescent proteins have been generated, each bringing new capability to research. However, taking full advantage of standard fluorescent proteins with advanced and differential features requires significant effort on the part of the researcher. This approach necessitates that many genetic fusions be generated and confirmed to function properly in cells with the same protein of interest. To lessen this burden, a newer category of protein fusion tags termed "self-labeling protein tags" has been developed. This approach utilizes a single protein tag, the function of which can be altered by attaching various chemical moieties (fluorescent labels, affinity handles, etc.). In this way a single genetically encoded protein fusion can easily be given functional diversity and adaptability as supplied by synthetic chemistry. Here we present protein labeling methods using HaloTag technology; comprised of HaloTag protein and the collection of small molecules designed to bind it specifically and provide it with varied functionalities. For imaging purposes these small molecules, termed HaloTag ligands, contain distinct fluorophores. Due to covalent and rapid binding between HaloTag protein and its ligands, labeling is permanent and efficient. Many of these ligands have been optimized for permeability across cellular membranes allowing for live cell labeling and imaging analysis. Nonpermeable ligands have also been developed for specific labeling of surface proteins. Overall, HaloTag is a versatile technology that empowers the end user to label a protein of interest with the choice of different fluorophores while alleviating the need for generation of multiple genetic fusions.

ChemNet: A Transferable and Generalizable Deep Neural Network for Small-Molecule Property Prediction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goh, Garrett B.; Siegel, Charles M.; Vishnu, Abhinav

With access to large datasets, deep neural networks through representation learning have been able to identify patterns from raw data, achieving human-level accuracy in image and speech recognition tasks. However, in chemistry, availability of large standardized and labelled datasets is scarce, and with a multitude of chemical properties of interest, chemical data is inherently small and fragmented. In this work, we explore transfer learning techniques in conjunction with the existing Chemception CNN model, to create a transferable and generalizable deep neural network for small-molecule property prediction. Our latest model, ChemNet learns in a semi-supervised manner from inexpensive labels computed frommore » the ChEMBL database. When fine-tuned to the Tox21, HIV and FreeSolv dataset, which are 3 separate chemical tasks that ChemNet was not originally trained on, we demonstrate that ChemNet exceeds the performance of existing Chemception models, contemporary MLP models that trains on molecular fingerprints, and it matches the performance of the ConvGraph algorithm, the current state-of-the-art. Furthermore, as ChemNet has been pre-trained on a large diverse chemical database, it can be used as a universal “plug-and-play” deep neural network, which accelerates the deployment of deep neural networks for the prediction of novel small-molecule chemical properties.« less

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

Investigating rhodamine B-labeled peptoids: scopes and limitations of its applications.

PubMed

Birtalan, Esther; Rudat, Birgit; Kölmel, Dominik K; Fritz, Daniel; Vollrath, Sidonie B L; Schepers, Ute; Bräse, Stefan

2011-01-01

The fluorophore rhodamine B is often used in biological assays. It is inexpensive, robust under a variety of reaction conditions, can be covalently linked to bioactive molecules, and has suitable spectral properties in terms of absorption and fluorescence wavelength. Nonetheless, there are some drawbacks: it can readily form a spirolactam compound, which is nonfluorescent, and therefore may not be the dye of choice for all fluorescence microscopy applications. Herein this spirolactam formation was observed by purifying such a labeled peptoid with high performance liquid chromatography (HPLC) and monitored in detail by making a series of analytical HPLC runs over time. Additionally, a small library of eight peptoids with rhodamine B as label was synthesized. Analysis of the absorption properties of these molecules demonstrated that the problem of fluorescence loss can be overcome by coupling secondary amines with rhodamine B.

On-bead antibody-small molecule conjugation using high-capacity magnetic beads.

PubMed

Nath, Nidhi; Godat, Becky; Benink, Hélène; Urh, Marjeta

2015-11-01

Antibodies labeled with small molecules such as fluorophore, biotin or drugs play an important role in various areas of biological research, drug discovery and diagnostics. However, the majority of current methods for labeling antibodies is solution-based and has several limitations including the need for purified antibodies at high concentrations and multiple buffer exchange steps. In this study, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads. High-capacity magnetic antibody capture beads are key to this method and were developed by combining porous and hydrophilic cellulose beads with oriented immobilization of Protein A and Protein G using HaloTag technology. With a variety of fluorophores it is shown that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry. This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications. Copyright © 2015. Published by Elsevier B.V.

Enhanced Vibrational Spectroscopies as Tools for Small Molecule Biosensing

PubMed Central

Boujday, Souhir; Lamy de la Chapelle, Marc; Srajer, Johannes; Knoll, Wolfgang

2015-01-01

In this short summary we summarize some of the latest developments in vibrational spectroscopic tools applied for the sensing of (small) molecules and biomolecules in a label-free mode of operation. We first introduce various concepts for the enhancement of InfraRed spectroscopic techniques, including the principles of Attenuated Total Reflection InfraRed (ATR-IR), (phase-modulated) InfraRed Reflection Absorption Spectroscopy (IRRAS/PM-IRRAS), and Surface Enhanced Infrared Reflection Absorption Spectroscopy (SEIRAS). Particular attention is put on the use of novel nanostructured substrates that allow for the excitation of propagating and localized surface plasmon modes aimed at operating additional enhancement mechanisms. This is then be complemented by the description of the latest development in Surface- and Tip-Enhanced Raman Spectroscopies, again with an emphasis on the detection of small molecules or bioanalytes. PMID:26343666

Ultrasensitive molecular detection using thermal conductance of a hydrophobic gold-water interface.

PubMed

Green, Andrew J; Alaulamie, Arwa A; Baral, Susil; Richardson, Hugh H

2013-09-11

The thermal conductance from a hydrophobic gold aqueous interface is measured with increasing solute concentration. A small amount of aqueous solute molecules (1 solute molecule in 550 water molecules) dramatically increases the heat dissipation into the surrounding liquid. This result is consistent with a thermal conductance that is limited by an interface interaction where minority aqueous components significantly alter the surface properties and heat transport through the interface. The increase in heat dissipation can be used to make an extremely sensitive molecular detector that can be scaled to give single molecule detection without amplification or utilizing fluorescence labels.

Photoaffinity Labeling Studies on a Promoter of Dendritic Spine Formation

NASA Astrophysics Data System (ADS)

Sibucao, Kevin Carlo Abril

The small molecule BTA-EG4 has been shown to be a promoter of dendritic spine formation. The mechanism behind this phenomenon, however, is not well understood. The work in this dissertation is motivated by this gap in knowledge. The first part of this dissertation focuses on photoaffinity labeling studies to identify the cellular targets of BTA-EG4. Chapter 1 provides a summary of Alzheimer's disease, the rational design of BTA-EG 4, and methods to determine targets of small molecules. In Chapter 2, the synthesis of a BTA-EG4-based photoaffinity labeling probe and photodegradation studies are presented. Kinetic studies demonstrate that the probe photolyzes rapidly under UV light. In Chapter 3, photoaffinity labeling studies and subsequent protein identification experiments are reported. Competition experiments with the photoaffinity labeling probe and BTA-EG4 demonstrate that the probe labels a 55-kDa protein specifically. Tandem mass spectrometry revealed that the 55-kDa protein is the actin binding protein fascin 1. The second part of this dissertation focuses on the major protein identified from photoaffinity labeling studies, fascin 1. Chapter 4 provides a brief survey of the structure and function of fascin 1. In Chapter 5, characterizations of the interaction between BTA-EG4 and fascin 1 are reported. Isothermal titration calorimetry confirms the physical binding between fascin 1 and BTA-EG6, a BTA-EG4 analog. Slow speed sedimentation assays reveal that BTA-EG4 does not affect the actin-bundling activity of fascin 1. However, GST pull-down experiments show that BTA-EG4 inhibits the binding of fascin 1 with the GTPase Rab35. In addition, this work demonstrates that BTA-EG4 may be mechanistically distinct from the known fascin inhibitor G2.

Raman imaging of molecular dynamics during cellular events

NASA Astrophysics Data System (ADS)

Fujita, Katsumasa

2017-07-01

To overcome the speed limitation in Raman imaging, we have developed a microscope system that detects Raman spectra from hundreds of points in a sample simultaneously. The sample was illuminated by a line-shaped focus, and Raman scattering from the illuminated positions was measured simultaneously by an imaging spectrophotometer. We applied the line-illumination technique to observe the dynamics of intracellular molecules during cellular events. We found that intracellular cytochrome c can be clearly imaged by resonant Raman scattering. We demonstrated label-free imaging of redistribution of cytochrome c during apoptosis and osteoblastic mineralization. We also proposed alkyne-tagged Raman imaging to observe small molecules in living cells. Due to its small size and the unique Raman band, alkyne can tag molecules without strong perturbation to molecular functions and with the capability to be detected separately from endogenous molecules.

Highly sensitive detection of target molecules using a new fluorescence-based bead assay

NASA Astrophysics Data System (ADS)

Scheffler, Silvia; Strauß, Denis; Sauer, Markus

2007-07-01

Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

PubMed

Nakatsuka, Nako; Cao, Huan H; Deshayes, Stephanie; Melkonian, Arin Lucy; Kasko, Andrea M; Weiss, Paul S; Andrews, Anne M

2018-05-31

Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or L-tryptophan were selectively recognized by previously identified dopamine or L-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though slightly greater than the previously determined solution dissociation constant. Using pre-functionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with L-DOPA, L-DOPS, and L-5-HTP enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons, and future identification and characterization of novel aptamers targeting neurotransmitters or other important small-molecules.

Quantitative analysis of small molecule-nucleic acid interactions with a biosensor surface and surface plasmon resonance detection.

PubMed

Liu, Yang; Wilson, W David

2010-01-01

Surface plasmon resonance (SPR) technology with biosensor surfaces has become a widely-used tool for the study of nucleic acid interactions without any labeling requirements. The method provides simultaneous kinetic and equilibrium characterization of the interactions of biomolecules as well as small molecule-biopolymer binding. SPR monitors molecular interactions in real time and provides significant advantages over optical or calorimetic methods for systems with strong binding coupled to small spectroscopic signals and/or reaction heats. A detailed and practical guide for nucleic acid interaction analysis using SPR-biosensor methods is presented. Details of the SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips, and samples, as well as extensive information on experimental design, quantitative and qualitative data analysis and presentation. A specific example of the interaction of a minor-groove-binding agent with DNA is evaluated by both kinetic and steady-state SPR methods to illustrate the technique. Since the molecules that bind cooperatively to specific DNA sequences are attractive for many applications, a cooperative small molecule-DNA interaction is also presented.

Examining small molecule: HIV RNA interactions using arrayed imaging reflectometry

NASA Astrophysics Data System (ADS)

Chaimayo, Wanaruk; Miller, Benjamin L.

2014-03-01

Human Immunodeficiency Virus (HIV) has been the subject of intense research for more than three decades as it causes an uncurable disease: Acquired Immunodeficiency Syndrome, AIDS. In the pursuit of a medical treatment, RNAtargeted small molecules are emerging as promising targets. In order to understand the binding kinetics of small molecules and HIV RNA, association (ka) and dissociation (kd) kinetic constants must be obtained, ideally for a large number of sequences to assess selectivity. We have developed Aqueous Array Imaged Reflectometry (Aq-AIR) to address this challenge. Using a simple light interference phenomenon, Aq-AIR provides real-time high-throughput multiplex capabilities to detect binding of targets to surface-immobilized probes in a label-free microarray format. The second generation of Aq-AIR consisting of high-sensitivity CCD camera and 12-μL flow cell was fabricated. The system performance was assessed by real-time detection of MBNL1-(CUG)10 and neomycin B - HIV RNA bindings. The results establish this second-generation Aq-AIR to be able to examine small molecules binding to RNA sequences specific to HIV.

Signal-enhancer molecules encapsulated liposome as a valuable sensing and amplification platform combining the aptasensor for ultrasensitive ECL immunoassay.

PubMed

Mao, Li; Yuan, Ruo; Chai, Yaqin; Zhuo, Ying; Xiang, Yun

2011-06-15

An innovatory ECL immunoassay strategy was proposed to detect the newly developing heart failure biomarker N-terminal pro-brain natriuretic peptide (NT-proBNP). Firstly, this strategy used small molecules encapsulated liposome as immune label to construct a sandwich immune sensing platform for NT-proBNP. Then the ECL aptasensor was prepared to collect and detect the small molecules released from the liposome. Finally, based on the ECL signal changes caused by the small molecules, the ECL signal indirectly reflected the level of NT-proBNP antigen. In this experiment, the cocaine was chosen as the proper small molecule that can act as signal-enhancer to enhance the ECL of Ru(bpy)(3)(2+). The cocaine-encapsulated liposomes were successfully characterized by TEM. The quantificational calculation proved the ∼5.3×10(3) cocaine molecules per liposome enough to perform the assignment of signal amplification. The cocaine-binding ECL aptasensor further promoted the work aimed at amplifying signal. The performance of NT-proBNP assay by the proposed strategy exhibited high sensitivity and high specificities with a linear relationship over 0.01-500 ng mL(-1) range, and a detection limit down to 0.77 pg mL(-1). Copyright © 2011 Elsevier B.V. All rights reserved.

Two-Way Gold Nanoparticle Label-Free Sensing of Specific Sequence and Small Molecule Targets Using Switchable Concatemers.

PubMed

Zhu, Longjiao; Shao, Xiangli; Luo, Yunbo; Huang, Kunlung; Xu, Wentao

2017-05-19

A two-way colorimetric biosensor based on unmodified gold nanoparticles (GNPs) and a switchable double-stranded DNA (dsDNA) concatemer have been demonstrated. Two hairpin probes (H1 and H2) were first designed that provided the fuels to assemble the dsDNA concatemers via hybridization chain reaction (HCR). A functional hairpin (FH) was rationally designed to recognize the target sequences. All the hairpins contained a single-stranded DNA (ssDNA) loop and sticky end to prevent GNPs from salt-induced aggregation. In the presence of target sequence, the capture probe blocked in the FH recognizes the target to form a duplex DNA, which causes the release of the initiator probe by FH conformational change. This process then starts the alternate-opening of H1 and H2 through HCR, and dsDNA concatemers grow from the target sequence. As a result, unmodified GNPs undergo salt-induced aggregation because the formed dsDNA concatemers are stiffer and provide less stabilization. A light purple-to-blue color variation was observed in the bulk solution, termed the light-off sensing way. Furthermore, H1 ingeniously inserted an aptamer sequence to generate dsDNA concatemers with multiple small molecule binding sites. In the presence of small molecule targets, concatemers can be disassembled into mixtures with ssDNA sticky ends. A blue-to-purple reverse color variation was observed due to the regeneration of the ssDNA, termed the light-on way. The two-way biosensor can detect both nucleic acids and small molecule targets with one sensing device. This switchable sensing element is label-free, enzyme-free, and sophisticated-instrumentation-free. The detection limits of both targets were below nanomolar.

The colibactin warhead crosslinks DNA

NASA Astrophysics Data System (ADS)

Vizcaino, Maria I.; Crawford, Jason M.

2015-05-01

Members of the human microbiota are increasingly being correlated to human health and disease states, but the majority of the underlying microbial metabolites that regulate host-microbe interactions remain largely unexplored. Select strains of Escherichia coli present in the human colon have been linked to the initiation of inflammation-induced colorectal cancer through an unknown small-molecule-mediated process. The responsible non-ribosomal peptide-polyketide hybrid pathway encodes ‘colibactin’, which belongs to a largely uncharacterized family of small molecules. Genotoxic small molecules from this pathway that are capable of initiating cancer formation have remained elusive due to their high instability. Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure. The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity. The data support unexpected models for both colibactin biosynthesis and its mode of action.

Interferometric Scattering Microscopy for the Study of Molecular Motors

PubMed Central

Andrecka, J.; Takagi, Y.; Mickolajczyk, K.J.; Lippert, L.G.; Sellers, J.R.; Hancock, W.O.; Goldman, Y.E.; Kukura, P.

2016-01-01

Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20 nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods. PMID:27793291

A Single-Molecule Barcoding System using Nanoslits for DNA Analysis

NASA Astrophysics Data System (ADS)

Jo, Kyubong; Schramm, Timothy M.; Schwartz, David C.

Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels, creating molecular barcodes, which are efficiently read using fluorescence resonance energy transfer techniques for minimizing noise from unincorporated labels. As such, our integrative approach for the realization of genomic analysis through nanoconfinement, named nanocoding, was demonstrated through the barcoding and mapping of bacterial artificial chromosomal molecules, thereby providing the basis for a high-throughput platform competent for whole genome investigations.

Labeling proteins inside living cells using external fluorophores for microscopy.

PubMed

Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

2016-12-09

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

New Dioxaborolane Chemistry Enables [18F]-Positron-Emitting, Fluorescent [18F]-Multimodality Biomolecule Generation from the Solid Phase

PubMed Central

Crisp, Jessica L.; Vera, David R.; Tsien, Roger Y.; Ting, Richard

2016-01-01

New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates antibody immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled antibody release from the solid phase, separated from unlabeled antibody, and creates [18F]-trifluoroborate-antibody for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. This dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [18F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody (mAb) that labels prostate tumors and Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb (FDA approved) that labels lung adenocarcinoma tumors. Colocalized, tumor-specific NIRF and PET imaging confirm utility of the new technology. The described chemistry should allow labeling of many commercial systems, diabodies, nanoparticles, and small molecules for dual modality imaging of many diseases. PMID:27064381

New Dioxaborolane Chemistry Enables [(18)F]-Positron-Emitting, Fluorescent [(18)F]-Multimodality Biomolecule Generation from the Solid Phase.

PubMed

Rodriguez, Erik A; Wang, Ye; Crisp, Jessica L; Vera, David R; Tsien, Roger Y; Ting, Richard

2016-05-18

New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates antibody immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled antibody release from the solid phase, separated from unlabeled antibody, and creates [(18)F]-trifluoroborate-antibody for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. This dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [(18)F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody (mAb) that labels prostate tumors and Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb (FDA approved) that labels lung adenocarcinoma tumors. Colocalized, tumor-specific NIRF and PET imaging confirm utility of the new technology. The described chemistry should allow labeling of many commercial systems, diabodies, nanoparticles, and small molecules for dual modality imaging of many diseases.

In Situ Visualization of Lipid Raft Domains by Fluorescent Glycol Chitosan Derivatives.

PubMed

Jiang, Yao-Wen; Guo, Hao-Yue; Chen, Zhan; Yu, Zhi-Wu; Wang, Zhifei; Wu, Fu-Gen

2016-07-05

Lipid rafts are highly ordered small microdomains mainly composed of glycosphingolipids, cholesterol, and protein receptors. Optically distinguishing lipid raft domains in cell membranes would greatly facilitate the investigations on the structure and dynamics of raft-related cellular behaviors, such as signal transduction, membrane transport (endocytosis), adhesion, and motility. However, current strategies about the visualization of lipid raft domains usually suffer from the low biocompatibility of the probes, invasive detection, or ex situ observation. At the same time, naturally derived biomacromolecules have been extensively used in biomedical field and their interaction with cells remains a long-standing topic since it is closely related to various fundamental studies and potential applications. Herein, noninvasive visualization of lipid raft domains in model lipid bilayers (supported lipid bilayers and giant unilamellar vesicles) and live cells was successfully realized in situ using fluorescent biomacromolecules: the fluorescein isothiocyanate (FITC)-labeled glycol chitosan molecules. We found that the lipid raft domains in model or real membranes could be specifically stained by the FITC-labeled glycol chitosan molecules, which could be attributed to the electrostatic attractive interaction and/or hydrophobic interaction between the probes and the lipid raft domains. Since the FITC-labeled glycol chitosan molecules do not need to completely insert into the lipid bilayer and will not disturb the organization of lipids, they can more accurately visualize the raft domains as compared with other fluorescent dyes that need to be premixed with the various lipid molecules prior to the fabrication of model membranes. Furthermore, the FITC-labeled glycol chitosan molecules were found to be able to resist cellular internalization and could successfully visualize rafts in live cells. The present work provides a new way to achieve the imaging of lipid rafts and also sheds new light on the interaction between biomacromolecules and lipid membranes.

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

PubMed

Peng, Tao; Hang, Howard C

2016-11-02

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Optical Fluorescent Imaging to Monitor Temporal Effects of Microbubble-Mediated Ultrasound Therapy

PubMed Central

Sorace, Anna G.; Saini, Reshu; Rosenthal, Eben; Warram, Jason M.; Zinn, Kurt R.; Hoyt, Kenneth

2013-01-01

Microbubble-mediated ultrasound therapy can noninvasively enhance drug delivery to localized regions in the body. This technique can be beneficial in cancer therapy, but currently there are limitations to tracking the therapeutic effects. The purpose of this experiment was to investigate the potential of fluorescent imaging for monitoring the temporal effects of microbubble-mediated ultrasound therapy. Mice were implanted with 2LMP breast cancer cells. The animals underwent microbubble-mediated ultrasound therapy in the presence of Cy5.5 fluorescent-labeled IgG antibody (large molecule) or Cy5.5 dye (small molecule) and microbubble contrast agents. Control animals were administered fluorescent molecules only. Animals were transiently imaged in vivo at 1, 10, 30, and 60 min post therapy using a small animal optical imaging system. Tumors were excised and analyzed ex vivo. Tumors were homogenized and emulsion imaged for Cy5.5 fluorescence. Monitoring in vivo results showed significant influx of dye into the tumor (p < 0.05) using the small molecule, but not in the large molecule group (p > 0.05). However, after tumor emulsion, significantly higher dye concentration was detected in therapy group tumors for both small and large molecule groups in comparison to their control counterparts (p < 0.01). This paper explores a noninvasive optical imaging method for monitoring the effects of microbubble-mediated ultrasound therapy in a cancer model. It provides temporal information following the process of increasing extravasation of molecules into target tumors. PMID:23357902

Optical fluorescent imaging to monitor temporal effects of microbubble-mediated ultrasound therapy.

PubMed

Sorace, Anna G; Saini, Reshu; Rosenthal, Eben; Warram, Jason M; Zinn, Kurt R; Hoyt, Kenneth

2013-02-01

Microbubble-mediated ultrasound therapy can noninvasively enhance drug delivery to localized regions in the body. This technique can be beneficial in cancer therapy, but currently there are limitations to tracking the therapeutic effects. The purpose of this experiment was to investigate the potential of fluorescent imaging for monitoring the temporal effects of microbubble-mediated ultrasound therapy. Mice were implanted with 2LMP breast cancer cells. The animals underwent microbubble-mediated ultrasound therapy in the presence of Cy5.5 fluorescent-labeled IgG antibody (large molecule) or Cy5.5 dye (small molecule) and microbubble contrast agents. Control animals were administered fluorescent molecules only. Animals were transiently imaged in vivo at 1, 10, 30, and 60 min post therapy using a small animal optical imaging system. Tumors were excised and analyzed ex vivo. Tumors were homogenized and emulsion imaged for Cy5.5 fluorescence. Monitoring in vivo results showed significant influx of dye into the tumor (p < 0.05) using the small molecule, but not in the large molecule group (p > 0.05). However, after tumor emulsion, significantly higher dye concentration was detected in therapy group tumors for both small and large molecule groups in comparison to their control counterparts (p <0.01). This paper explores a noninvasive optical imaging method for monitoring the effects of microbubble-mediated ultrasound therapy in a cancer model. It provides temporal information following the process of increasing extravasation of molecules into target tumors.

Relative quantification of biomarkers using mixed-isotope labeling coupled with MS

PubMed Central

Chapman, Heidi M; Schutt, Katherine L; Dieter, Emily M; Lamos, Shane M

2013-01-01

The identification and quantification of important biomarkers is a critical first step in the elucidation of biological systems. Biomarkers take many forms as cellular responses to stimuli and can be manifested during transcription, translation, and/or metabolic processing. Increasingly, researchers have relied upon mixed-isotope labeling (MIL) coupled with MS to perform relative quantification of biomarkers between two or more biological samples. MIL effectively tags biomarkers of interest for ease of identification and quantification within the mass spectrometer by using isotopic labels that introduce a heavy and light form of the tag. In addition to MIL coupled with MS, a number of other approaches have been used to quantify biomarkers including protein gel staining, enzymatic labeling, metabolic labeling, and several label-free approaches that generate quantitative data from the MS signal response. This review focuses on MIL techniques coupled with MS for the quantification of protein and small-molecule biomarkers. PMID:23157360

Labeling Biomolecules with Radiorhenium - a Review of the Bifunctional Chelators

PubMed Central

Liu, Guozheng; Hnatowich, Donald J.

2007-01-01

For radiotherapy, biomolecules such as intact antibodies, antibody fragments, peptides, DNAs and other oligomers have all been labeled with radiorhenium (186Re and 188Re). Three different approaches have been employed that may be referred to as direct, indirect and integral labeling. Direct labeling applies to proteins and involves the initial reduction of endogenous disulfide bridges to provide chelation sites. Indirect labeling can apply to most biomolecules and involves the initial attachment of an exogenous chelator. Finally, integral labeling is a special case applying only to small molecules in which the metallic radionuclide serves to link two parts of a biomolecule together in forming the labeled complex. While the number of varieties for the direct and integral radiolabeling approaches is rather limited, a fairly large and diverse number of chelators have been reported in the case of indirect labeling. Our objective herein is to provide an overview of the various chelators that have been used in the indirect labeling of biomolecules with radiorhenium, including details on the labeling procedures, the stability of the radiolabel and, where possible, the influence of the label on biological properties. PMID:17504162

Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET

NASA Astrophysics Data System (ADS)

Heitkamp, Thomas; Grisshammer, Reinhard; Börsch, Michael

2018-02-01

Neurotensin receptor 1 (NTSR1) is a G protein-coupled receptor that is important for signaling in the brain and the gut. Its agonist ligand neurotensin (NTS), a 13-amino-acid peptide, binds with nanomolar affinity from the extracellular side to NTSR1 and induces conformational changes that trigger intracellular signaling processes. Our goal is to monitor the conformational dynamics of single fluorescently labeled NTSR1. For this, we fused the fluorescent protein mNeonGreen to the C terminus of NTSR1, purified the receptor fusion protein from E. coli membranes, and reconstituted NTSR1 into liposomes with E. coli polar lipids. Using single-molecule anisotropy measurements, NTSR1 was found to be monomeric in liposomes, with a small fraction being dimeric and oligomeric, showing homoFRET. Similar results were obtained for NTSR1 in detergent solution. Furthermore, we demonstrated agonist binding to NTSR1 by time-resolved single-molecule Förster resonance energy transfer (smFRET), using neurotensin labeled with the fluorophore ATTO594.

Accelerator mass spectrometry in the biomedical sciences: applications in low-exposure biomedical and environmental dosimetry

NASA Astrophysics Data System (ADS)

Felton, J. S.; Turteltaub, K. W.; Vogel, J. S.; Balhorn, R.; Gledhill, B. L.; Southon, J. R.; Caffee, M. W.; Finkel, R. C.; Nelson, D. E.; Proctor, I. D.; Davis, J. C.

1990-12-01

We are utilizing accelerator mass spectrometry as a sensitive detector for tracking the disposition of radioisotopically labeled molecules in the biomedical sciences. These applications have shown the effectiveness of AMS as a tool to quantify biologically important molecules at extremely low levels. For example, AMS is being used to determine the amount of carcinogen covalently bound to animal DNA (DNA adduct) at levels relevent to human exposure. Detection sensitivities are 1 carcinogen molecule bound in 1011 to 1012 DNA bases, depending on the specific activity of the radiolabeled carcinogen. Studies have been undertaken in our laboratory utilizing heterocyclic amine food-borne carcinogens and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent environmental carcinogen, to study the metabolism of carcinogens at low doses. In addition, AMS is being used to detect the presence of rare proteins (mutant forms of protamine) in human sperm. Approximately l per 106 sperm analyzed contain the rare form of the protamine. Protamine isolated from this small number of cells is being analyzed by AMS, following 14C labeling. Thus, AMS can be used to verify the identity of an extremely small amount of biological material. Furthermore, an additional improvement of 2 orders of magnitude in the sensitivity of biomédical tracer studies is suggested by preliminary work with bacterial hosts depleted in radiocarbon. Other problems in the life sciences where detection sensitivity or sample sizes are limitations should also benefit from AMS. Studies are underway to measure the molecular targeting of cancer chemotherapeutics in human tissue and to pursue applications for receptor biology. We are also applying other candidate isotopes, such as 3H (double labeling with 14C) and 41Ca (bone absorption) to problems in biology. The detection of 36Cl and 26Al have applications for determination of human neutron exposure and understanding neurological toxicity, respectively. The results described here with 14C-labeled molecules coupled with new isotope applications clearly show AMS technology to be an important new tool for the biomedical sciences community.

End labeling procedures: an overview.

PubMed

Hilario, Elena

2004-09-01

There are two ways to label a DNA molecular; by the ends or all along the molecule. End labeling can be performed at the 3'- or 5'-end. Labeling at the 3' end is performed by filling 3'-end recessed ends with a mixture or labeled and unlabeled dNTPs using Klenow or T4 DNA polymerases. Both reactions are template dependent. Terminal deoxynucleotide transferase incorporates dNTPs at the 3' end of any kind of DNA molecule or RNA. Labels incorporated at the 3'-end of the DNA molecule prevent any further extension or ligation to any other molecule, but this can be overcome by labeling the 5'-end of the desired DNA molecule. 5'-end labeling is performed by enzymatic methods (T4 polynucleotide kinase exchange and forward reactions), by chemical modification of sensitized oligonucleotides with phosphoroamidite, or by combined methods. Probe cleanup is recommended when high background problems occur, but caution should be taken not to damage the attached probe with harsh chemicals or by light exposure.

Synthetic-Molecule/Protein Hybrid Probe with Fluorogenic Switch for Live-Cell Imaging of DNA Methylation.

PubMed

Hori, Yuichiro; Otomura, Norimichi; Nishida, Ayuko; Nishiura, Miyako; Umeno, Maho; Suetake, Isao; Kikuchi, Kazuya

2018-02-07

Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.

Subclass-specific labeling of protein-reactive natural products with customized nucleophilic probes.

PubMed

Rudolf, Georg C; Koch, Maximilian F; Mandl, Franziska A M; Sieber, Stephan A

2015-02-23

Natural products represent a rich source of bioactive compounds that constitute a large fraction of approved drugs. Among those are molecules with electrophilic scaffolds, such as Michael acceptors, β-lactams, and epoxides that irreversibly inhibit essential enzymes based on their catalytic mechanism. In the search for novel bioactive molecules, current methods are challenged by the frequent rediscovery of known chemical entities. Herein small nucleophilic probes that attack electrophilic natural products and enhance their detection by HPLC-UV and HPLC-MS are introduced. A screen of diverse probe designs revealed one compound with a desired selectivity for epoxide- and maleimide-based antibiotics. Correspondingly, the natural products showdomycin and phosphomycin could be selectively targeted in extracts of their natural producing organism, in which the probe-modified molecules exhibited superior retention and MS detection relative to their unmodified counterparts. This method may thus help to discover small, electrophilic molecules that might otherwise easily elude detection in complex samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Black phosphorus-assisted laser desorption ionization mass spectrometry for the determination of low-molecular-weight compounds in biofluids.

PubMed

He, Xiao-Mei; Ding, Jun; Yu, Lei; Hussain, Dilshad; Feng, Yu-Qi

2016-09-01

Quantitative analysis of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been a challenging task due to matrix-derived interferences in low m/z region and poor reproducibility of MS signal response. In this study, we developed an approach by applying black phosphorus (BP) as a matrix-assisted laser desorption ionization (MALDI) matrix for the quantitative analysis of small molecules for the first time. Black phosphorus-assisted laser desorption/ionization mass spectrometry (BP/ALDI-MS) showed clear background and exhibited superior detection sensitivity toward quaternary ammonium compounds compared to carbon-based materials. By combining stable isotope labeling (SIL) strategy with BP/ALDI-MS (SIL-BP/ALDI-MS), a variety of analytes labeled with quaternary ammonium group were sensitively detected. Moreover, the isotope-labeled forms of analytes also served as internal standards, which broadened the analyte coverage of BP/ALDI-MS and improved the reproducibility of MS signals. Based on these advantages, a reliable method for quantitative analysis of aldehydes from complex biological samples (saliva, urine, and serum) was successfully established. Good linearities were obtained for five aldehydes in the range of 0.1-20.0 μM with correlation coefficients (R (2)) larger than 0.9928. The LODs were found to be 20 to 100 nM. Reproducibility of the method was obtained with intra-day and inter-day relative standard deviations (RSDs) less than 10.4 %, and the recoveries in saliva samples ranged from 91.4 to 117.1 %. Taken together, the proposed SIL-BP/ALDI-MS strategy has proved to be a reliable tool for quantitative analysis of aldehydes from complex samples. Graphical Abstract An approach for the determination of small molecules was developed by using black phosphorus (BP) as a matrix-assisted laser desorption ionization (MALDI) matrix.

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

PubMed Central

Price, Allen C.; Pilkiewicz, Kevin R.; Graham, Thomas G.W.; Song, Dan; Eaves, Joel D.; Loparo, Joseph J.

2015-01-01

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length. PMID:25992731

Identification of Antibody and Small Molecule Antagonists of Ferroportin-Hepcidin Interaction

PubMed Central

Ross, Sandra L.; Biswas, Kaustav; Rottman, James; Allen, Jennifer R.; Long, Jason; Miranda, Les P.; Winters, Aaron; Arvedson, Tara L.

2017-01-01

The iron exporter ferroportin and its ligand, the hormone hepcidin, control fluxes of stored and recycled iron for use in a variety of essential biochemical processes. Inflammatory disorders and malignancies are often associated with high hepcidin levels, leading to ferroportin down-regulation, iron sequestration in tissue macrophages and subsequent anemia. The objective of this research was to develop reagents to characterize the expression of ferroportin, the interaction between ferroportin and hepcidin, as well as to identify novel ferroportin antagonists capable of maintaining iron export in the presence of hepcidin. Development of investigative tools that enabled cell-based screening assays is described in detail, including specific and sensitive monoclonal antibodies that detect endogenously-expressed human and mouse ferroportin and fluorescently-labeled chemically-synthesized human hepcidin. Large and small molecule antagonists inhibiting hepcidin-mediated ferroportin internalization were identified, and unique insights into the requirements for interaction between these two key iron homeostasis molecules are provided. PMID:29209212

Comparison of /sup 125/I-labeled and /sup 14/C-Labeled peptides of the major outer membrane protein of Chlamydia Trachomatis Strain L2/434 separated by high-performance liquid chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Judd, R.C.; Caldwell, H.D.

1985-01-01

The objective of this study was to determine if in-gel chloramine-T radioiodination adequately labels OM proteins to allow for accurate and precise structural comparison of these molecules. Therefore, intrinsically /sup 14/C-amino acid labeled proteins and /sup 125/I-labeled proteins were cleaved with two endopeptidic reagents and the peptide fragments separated by HPLC. A comparison of retention times of the fragments, as determined by differential radiation counting, thus indicated whether /sup 125/Ilabeling identified of all the peptide peaks seen in the /sup 14/Clabeled proteins. Results demonstrated that radioiodination yields complete and accurate information about the primary structure of outer membrane proteins. Inmore » addition, it permits the use of extremely small amounts of protein allowing for method optimization and multiple separations to insure reproducibility.« less

Design and Application of Synthetic Receptors for Recognition of Methylated Lysine and Supramolecular Affinity Labeling

NASA Astrophysics Data System (ADS)

Gober, Isaiah Nathaniel

This dissertation involves the design and synthesis of new synthetic receptors and their application in the molecular recognition of methylated lysine and their use as tools for chemical biology. The dissertation is divided into four parts. The first section focuses on the development of a novel labeling method that is based on ligand-directed affinity labeling principles. In this labeling method, a synthetic receptor that binds to trimethyl lysine (Kme3) is attached through a linker to an electrophilic tag group that can react with a nucleophilic amine in a histone peptide. This affinity labeling probe, which we called CX4-ONBD, is equipped with an electrophilic tag that allows for turn-on fluorescence labeling of Kme3 histone peitdes. We show that the probe gives a pronounced turn-on fluorescence response when it is incubated with a histone peptide that contains Kme3 and a nearby reactive lysine. This probe also displays >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. This represents the first time a synthetic receptor has been used for affinity labeling purposes, and it also expands on the chemical toolkit that is available for sensing PTMs like lysine methylation. In the second section, the supramolecular affinity labeling method that was optimized using CX4-ONBD was applied to the development of a real-time assay for measuring enzymatic activity. More specifically, the probe was used to create a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Most commercial kits for HDAC activity have limited substrate scope, and other common methods used for characterizing enzymatic activity often require chromatographic separation and are therefore not high-throughput. This small molecule receptor-mediated affinity labeling strategy allowed for facile readout of HDAC activity and inhibition. Overall, this application of supramolecular affinity labeling expands on the possible ways for detecting PTMs and may find use in the development of new assays for enzymes that lack robust methods for measuring their activity. The third section explores the development of new small molecule receptors capable of selectively binding hydrophilic guests in water, such as the lower methylation states of lysine. We identified a receptor, A2I, that has improved binding affinity and selectivity for dimethyllysine (Kme2). The receptor was discovered and synthesized by using dynamic combinatorial chemistry (DCC) to redesign a small molecule receptor (A2B ) that preferentially binds trimethyllysine (Kme3). Incorporating a biphenyl monomer with ortho-di-substituted carboxylates into the receptor lead to the formation of a salt bridge interaction with Kme2. These favorable electrostatic and hydrogen bonding interactions produced a receptor with 32-fold tighter binding to Kme2, which is the highest affinity synthetic receptor for Kme2 in the context of a peptide that has been reported. This work provides insight into effective strategies for binding hydrophilic, cationic guests in water and is an encouraging result toward a synthetic receptor that selectively binds Kme2 over other methylation states of lysine. In the final section, a small molecule receptor for Kme3 (A 2B) was redesigned using DCC to incorporate either aromatic or acidic amino acids into the receptor. We proposed that the incorporation of amino acids could introduce additional non-covalent interactions (such as cation-pi, electrostatic, and hydrogen bonding) with a guest bound inside the pocket of the receptor. However, selective non-covalent interactions between the amino acid side chain on the modified receptor and the bound methylated lysine guest could not be achieved. This is most likely due to the conformational flexibility of the amino acid-functionalized receptors. Furthermore, attaching amino acids to the receptor seemed to increase non-specific electrostatic interactions, resulting in tighter binding to the unmethylated lysine peptide (compared to A2B). Ultimately, this highlights the importance of incorporating monomers with less conformational flexibility that can rigidly place functional groups into the binding pocket.

Application of ex situ dynamic nuclear polarization in studying small molecules.

PubMed

Ludwig, Christian; Marin-Montesinos, Ildefonso; Saunders, Martin G; Emwas, Abdul-Hamid; Pikramenou, Zoe; Hammond, Stephen P; Günther, Ulrich L

2010-06-14

Dynamic nuclear polarization (DNP) has become an attractive technique to boost the sensitivity of NMR experiments. In the case of ex situ polarizations two-dimensional (2D) spectra are limited by the short lifetime of the polarization after dissolution and sample transfer to a high field NMR magnet. This limitation can be overcome by various approaches. Here we show how the use of (13)C-labelled acetyl tags can help to obtain 2D-HMQC spectra for many small molecules, owing to a nuclear Overhauser enhancement between (13)C spins originating from the long-lived carbonyl carbon, which extends the lifetimes of other (13)C spins with shorter longitudinal relaxation times. We also show an alternative approach of using an optimized polarization matrix.

Imaging of platelet-derived growth factor receptor β expression in glioblastoma xenografts using affibody molecule 111In-DOTA-Z09591.

PubMed

Tolmachev, Vladimir; Varasteh, Zohreh; Honarvar, Hadis; Hosseinimehr, Seyed Jalal; Eriksson, Olof; Jonasson, Per; Frejd, Fredrik Y; Abrahmsen, Lars; Orlova, Anna

2014-02-01

The overexpression and excessive signaling of platelet-derived growth factor receptor β (PDGFRβ) has been detected in cancers, atherosclerosis, and a variety of fibrotic diseases. Radionuclide in vivo visualization of PDGFRβ expression might help to select PDGFRβ targeting treatment for these diseases. The goal of this study was to evaluate the feasibility of in vivo radionuclide imaging of PDGFRβ expression using an Affibody molecule, a small nonimmunoglobulin affinity protein. The PDGFRβ-binding Z09591 Affibody molecule was site-specifically conjugated with a maleimido derivative of DOTA and labeled with (111)In. Targeting of the PDGFRβ-expressing U-87 MG glioblastoma cell line using (111)In-DOTA-Z09591 was evaluated in vitro and in vivo. DOTA-Z09591 was stably labeled with (111)In with preserved specific binding to PDGFRβ-expressing cells in vitro. The dissociation constant for (111)In-DOTA-Z09591 binding to U-87 MG cells was determined to be 92 ± 10 pM. In mice bearing U-87 MG xenografts, the tumor uptake of (111)In-DOTA-Z09591 was 7.2 ± 2.4 percentage injected dose per gram and the tumor-to-blood ratio was 28 ± 14 at 2 h after injection. In vivo receptor saturation experiments demonstrated that targeting of U-87 MG xenografts in mice was PDGFRβ-specific. U-87 MG xenografts were clearly visualized using small-animal SPECT/CT at 3 h after injection. This study demonstrates the feasibility of in vivo visualization of PDGFRβ-expressing xenografts using an Affibody molecule. Further development of radiolabeled Affibody molecules might provide a useful clinical imaging tool for PDGFRβ expression during various pathologic conditions.

Gating capacitive field-effect sensors by the charge of nanoparticle/molecule hybrids.

PubMed

Poghossian, Arshak; Bäcker, Matthias; Mayer, Dirk; Schöning, Michael J

2015-01-21

The semiconductor field-effect platform is a powerful tool for chemical and biological sensing with direct electrical readout. In this work, the field-effect capacitive electrolyte-insulator-semiconductor (EIS) structure - the simplest field-effect (bio-)chemical sensor - modified with citrate-capped gold nanoparticles (AuNPs) has been applied for a label-free electrostatic detection of charged molecules by their intrinsic molecular charge. The EIS sensor detects the charge changes in AuNP/molecule inorganic/organic hybrids induced by the molecular adsorption or binding events. The feasibility of the proposed detection scheme has been exemplarily demonstrated by realizing capacitive EIS sensors consisting of an Al-p-Si-SiO2-silane-AuNP structure for the label-free detection of positively charged cytochrome c and poly-d-lysine molecules as well as for monitoring the layer-by-layer formation of polyelectrolyte multilayers of poly(allylamine hydrochloride)/poly(sodium 4-styrene sulfonate), representing typical model examples of detecting small proteins and macromolecules and the consecutive adsorption of positively/negatively charged polyelectrolytes, respectively. For comparison, EIS sensors without AuNPs have been investigated, too. The adsorption of molecules on the surface of AuNPs has been verified via the X-ray photoelectron spectroscopy method. In addition, a theoretical model of the functioning of the capacitive field-effect EIS sensor functionalized with AuNP/charged-molecule hybrids has been discussed.

Ultrasmall visible-to-near-infrared emitting silver-sulfide quantum dots for cancer detection and imaging

NASA Astrophysics Data System (ADS)

Tang, Rui; Xu, Baogang; Shen, Duanwen; Sudlow, Gail; Achilefu, Samuel

2018-02-01

The large size of many near infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. We developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from visible to near-infrared in spectrum and a QD core diameter between less than 5 nm. Further functionalization of these Ag2S QDs with different type of molecules such as targeting peptides, retains monodisperse, relatively small water soluble QDs without loss of the functionality of the peptide's high binding affinity to cancerous tumor. Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and non-cytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.

NUTS and BOLTS: Applications of Fluorescence Detected Sedimentation

PubMed Central

Kroe, Rachel R.; Laue, Thomas M.

2008-01-01

Analytical ultracentrifugation is a widely used method for characterizing the solution behavior of macromolecules. However, the two commonly used detectors (absorbance and interference) impose some fundamental restrictions on the concentrations and complexity of the solutions that can be analyzed. The recent addition of a fluorescence detector for the XL-I analytical ultracentrifuge (AU-FDS) enables two different types of sedimentation experiments. First, the AU-FDS can detect picomolar concentrations of labeled solutes allowing the characterization of very dilute solutions of macromolecules, applications we call Normal Use Tracer Sedimentation (NUTS). The great sensitivity of NUTS analysis allows the characterization of small quantities of materials and high affinity interactions. Second, AU-FDS allows characterization of trace quantities of labeled molecules in solutions containing high concentrations and complex mixtures of unlabeled molecules, applications we call Biological On Line Tracer Sedimentation (BOLTS). The discrimination of BOLTS enables the size distribution of a labeled macromolecule to be determined in biological milieu such as cell lysates and serum. Examples are presented that embody features of both NUTS and BOLTS applications, along with our observations on these applications. PMID:19103145

Surface diffusion of CO on silica-supported Ru particles: 13C nuclear magnetic resonance studies

NASA Astrophysics Data System (ADS)

Duncan, T. M.; Thayer, A. M.; Root, T. W.

1990-02-01

Portions of CO adsorbed on Ru particles, selected by the orientation of the C-O bond relative to an external magnetic field, are labeled by inversion of the 13C nuclear magnetic dipole. Changes in the orientation of the CO bond of these labeled molecules are then observed with 13C NMR spectroscopy. The temperature dependence and rate of reorientation are consistent with surface diffusion on Ru particles with small numbers of flat faces. The insensitivity to CO pressure in the range 0.5-100 Torr discounts stimulated desorption by gas-phase CO.

Observing the conformation of individual SNARE proteins inside live cells

NASA Astrophysics Data System (ADS)

Weninger, Keith

2010-10-01

Protein conformational dynamics are directly linked to function in many instances. Within living cells, protein dynamics are rarely synchronized so observing ensemble-averaged behaviors can hide details of signaling pathways. Here we present an approach using single molecule fluorescence resonance energy transfer (FRET) to observe the conformation of individual SNARE proteins as they fold to enter the SNARE complex in living cells. Proteins were recombinantly expressed, labeled with small-molecule fluorescent dyes and microinjected for in vivo imaging and tracking using total internal reflection microscopy. Observing single molecules avoids the difficulties of averaging over unsynchronized ensembles. Our approach is easily generalized to a wide variety of proteins in many cellular signaling pathways.

Laser desorption ionization mass spectrometry: Recent progress in matrix-free and label-assisted techniques.

PubMed

Mandal, Arundhoti; Singha, Monisha; Addy, Partha Sarathi; Basak, Amit

2017-10-13

The MALDI-based mass spectrometry, over the last three decades, has become an important analytical tool. It is a gentle ionization technique, usually applicable to detect and characterize analytes with high molecular weights like proteins and other macromolecules. The earlier difficulty of detection of analytes with low molecular weights like small organic molecules and metal ion complexes with this technique arose due to the cluster of peaks in the low molecular weight region generated from the matrix. To detect such molecules and metal ion complexes, a four-prong strategy has been developed. These include use of alternate matrix materials, employment of new surface materials that require no matrix, use of metabolites that directly absorb the laser light, and the laser-absorbing label-assisted LDI-MS (popularly known as LALDI-MS). This review will highlight the developments with all these strategies with a special emphasis on LALDI-MS. © 2017 Wiley Periodicals, Inc.

Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction

PubMed Central

Ban, Hitoshi; Nagano, Masanobu; Gavrilyuk, Julia; Hakamata, Wataru; Inokuma, Tsubasa; Barbas, Carlos F.

2013-01-01

The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective tri-functionalization of proteins is readily achieved. In particular cases, we noted PTAD decomposition resulted in formation of a putative isocyanate by-product that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene) glycol chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions. PMID:23534985

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stavis, Samuel M; Edel, Joshua B; Samiee, Kevan T

A nanofluidic channel fabricated in fused silica with an approximately 500 nm square cross section was used to isolate, detect and identify individual quantum dot conjugates. The channel enables the rapid detection of every fluorescent entity in solution. A laser of selected wavelength was used to excite multiple species of quantum dots and organic molecules, and the emission spectra were resolved without significant signal rejection. Quantum dots were then conjugated with organic molecules and detected to demonstrate efficient multicolor detection. PCH was used to analyze coincident detection and to characterize the degree of binding. The use of a small fluidicmore » channel to detect quantum dots as fluorescent labels was shown to be an efficient technique for multiplexed single molecule studies. Detection of single molecule binding events has a variety of applications including high throughput immunoassays.« less

A Method for Identifying Small-Molecule Aggregators Using Photonic Crystal Biosensor Microplates

PubMed Central

Chan, Leo L.; Lidstone, Erich A.; Finch, Kristin E.; Heeres, James T.; Hergenrother, Paul J.; Cunningham, Brian T.

2010-01-01

Small molecules identified through high-throughput screens are an essential element in pharmaceutical discovery programs. It is now recognized that a substantial fraction of small molecules exhibit aggregating behavior leading to false positive results in many screening assays, typically due to nonspecific attachment to target proteins. Therefore, the ability to efficiently identify compounds within a screening library that aggregate can streamline the screening process by eliminating unsuitable molecules from further consideration. In this work, we show that photonic crystal (PC) optical biosensor microplate technology can be used to identify and quantify small-molecule aggregation. A group of aggregators and nonaggregators were tested using the PC technology, and measurements were compared with those gathered by three alternative methods: dynamic light scattering (DLS), an α-chymotrypsin colorimetric assay, and scanning electron microscopy (SEM). The PC biosensor measurements of aggregation were confirmed by visual observation using SEM, and were in general agreement with the α-chymotrypsin assay. DLS measurements, in contrast, demonstrated inconsistent readings for many compounds that are found to form aggregates in shapes, very different from the classical spherical particles assumed in DLS modeling. As a label-free detection method, the PC biosensor aggregation assay is simple to implement and provides a quantitative direct measurement of the mass density of material adsorbed to the transducer surface, whereas the microplate-based sensor format enables compatibility with high-throughput automated liquid-handling methods used in pharmaceutical screening. PMID:20930952

Application of fluorescently labeled tracer technique for detection of natural active macromolecules in Chinese medicine.

PubMed

Zeng, Qiao-Hui; Zhang, Xue-Wu; Xu, Kai-Peng; Jiang, Jian-Guo

2014-02-01

Active substances in traditional Chinese Medicine (TCM) contain not only a variety of small molecules, but also many other macromolecules (TCMMs), such as proteins, peptides and polysaccharides. Active TCMM can achieve good therapeutic effects by regulating the body's overall function with lower side effects. This review summarized the literatures published in recent years on the application of fluorescently labeled tracer technique for detection of natural active macromolecules in TCM. Classified by fluorescent markers, applications of fluorescein, rhodamine, and quantum dots (QDs) in TCMM active tracer are reviewed, and the methods and principles of TCMM fluorescent marker are illustrated. Studies on active TCMMs and their action mechanism are quite difficult due to a multitarget, multicomponent, and multipath system of TCM. However, the development of fluorescently labeled active tracer technique (FLATT) provides this research with new tools. Traditional fluorescent markers have many deficiencies, such as easily quenched, short luminous cycle, and intrinsic toxicity. Relatively, FLATT has many obvious advantages, and its application in TCMM is still at the early stage. In order to improve the overall level of fluorescence labeling in TCMM active tracer, the improvement on FLATT's detection sensitivity and biological affinity is urgent and critical to allow study of these interesting molecules.

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site | Center for Cancer Research

Cancer.gov

The lowest energy-binding conformation of an inhibitor bound to the dimeric interface of HIV-1 integrase core domain. The yellow region represents a unique allosteric binding site identified by affinity labeling and mass spectrometry and validated through mutagenesis. This site can provide a potential platform for the rational design of inhibitors selective for disruption of

A universal aptameric biosensor: Multiplexed detection of small analytes via aggregated perylene-based broad-spectrum quencher.

PubMed

Hu, Rong; Zhang, Xi; Xu, Qiang; Lu, Dan-Qing; Yang, Yun-Hui; Xu, Quan-Qing; Ruan, Qiong; Mo, Liu-Ting; Zhang, Xiao-Bing

2017-06-15

A universal aptameric system based on the taking advantage of double-stranded DNA/perylene diimide (dsDNA/PDI) as the signal probe was developed for multiplexed detection of small molecules. Aptamers are single-stranded DNA or RNA oligonucleotides which are selected in vitro by a process known as systematic evolution of ligands by exponential enrichment. In this work, we synthesized a new kind of PDI and reported this aggregated PDI could quench the double-stranded DNA (dsDNA)-labeled fluorophores with a high quenching efficiency. The quenching efficiencies on the fluorescence of FAM, TAMRA and Cy5 could reach to 98.3%±0.9%, 97.2%±0.6% and 98.1%±1.1%, respectively. This broad-spectrum quencher was then adopted to construct a multicolor biosensor via a label-free approach. A structure-switching-triggered enzymatic recycling amplification was employed for signal amplification. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity towards small analytes. For other targets, changing the corresponding aptamer can achieve the goal. The quencher did not interfere with the catalytic activity of nuclease. The biosensor could be manipulated with similar sensitivity no matter in pre-addition or post-addition manner. Moreover, simultaneous and multiplexed analysis of several small molecules in homogeneous solution was achieved, demonstrating its potential application in the rapid screening of multiple biotargets. Copyright © 2017 Elsevier B.V. All rights reserved.

A Freeze Substitution Fixation-Based Gold Enlarging Technique for EM Studies of Endocytosed Nanogold-Labeled Molecules

PubMed Central

He, Wanzhong; Kivork, Christine; Machinani, Suman; Morphew, Mary K.; Gail, Anna M.; Tesar, Devin B.; Tiangco, Noreen E.; McIntosh, J. Richard; Bjorkman, Pamela J.

2007-01-01

We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand-receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically-fixed samples that reduced autonucleation, and a new pre-embedding gold-enlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies. PMID:17723309

Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip.

PubMed

Wang, Chen; Ouyang, Jun; Ye, De-Kai; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

2012-08-07

Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency.

Shrink-wrap Vesicles

PubMed Central

Fujikawa, Shelly M.; Chen, Irene A.; Szostak, Jack W.

2008-01-01

We describe a simple approach to the controlled removal of molecules from the membrane of large unilamellar vesicles made of fatty acids. Such vesicles shrink dramatically upon mixing with micelles composed of a mixture of fatty acid and phospholipid (POPC), as fatty acid molecules leave the vesicle membrane and accumulate within the mixed micelles. Vesicle shrinkage was confirmed by dynamic light scattering, fluorescence recovery after photobleaching of labeled vesicles, and fluorescence resonance energy transfer between lipid dyes incorporated into the vesicle membrane. Most of the encapsulated impermeable solute is retained during shrinkage, becoming concentrated by a factor of at least 50-fold in the final small vesicles. This unprecedented combination of vesicle shrinkage with retention of contents allows for the preparation of small vesicles containing high solute concentrations, and may find applications in liposomal drug delivery. PMID:16342983

Small molecule inhibitors of ERCC1-XPF protein-protein interaction synergize alkylating agents in cancer cells.

PubMed

Jordheim, Lars Petter; Barakat, Khaled H; Heinrich-Balard, Laurence; Matera, Eva-Laure; Cros-Perrial, Emeline; Bouledrak, Karima; El Sabeh, Rana; Perez-Pineiro, Rolando; Wishart, David S; Cohen, Richard; Tuszynski, Jack; Dumontet, Charles

2013-07-01

The benefit of cancer chemotherapy based on alkylating agents is limited because of the action of DNA repair enzymes, which mitigate the damage induced by these agents. The interaction between the proteins ERCC1 and XPF involves two major components of the nucleotide excision repair pathway. Here, novel inhibitors of this interaction were identified by virtual screening based on available structures with use of the National Cancer Institute diversity set and a panel of DrugBank small molecules. Subsequently, experimental validation of the in silico screening was undertaken. Top hits were evaluated on A549 and HCT116 cancer cells. In particular, the compound labeled NSC 130813 [4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2-[(4-methyl-1-piperazinyl)methyl

Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay

PubMed Central

2013-01-01

Background Uncoating of the HIV-1 core plays a critical role during early post-fusion stages of infection but is poorly understood. Microscopy-based assays are unable to easily distinguish between intact and partially uncoated viral cores. Results In this study, we used 5-ethynyl uridine (EU) to label viral-associated RNA during HIV production. At early time points after infection with EU-labeled virions, the viral-associated RNA was stained with an EU-specific dye and was detected by confocal microscopy together with viral proteins. We observed that detection of the viral-associated RNA was specific for EU-labeled virions, was detected only after viral fusion with target cells, and occurred after an initial opening of the core. In vitro staining of cores showed that the opening of the core allowed the small molecule dye, but not RNase A or antibodies, inside. Also, staining of the viral-associated RNA, which is co-localized with nucleocapsid, decays over time after viral infection. The decay rate of RNA staining is dependent on capsid (CA) stability, which was altered by CA mutations or a small molecule inducer of HIV-1 uncoating. While the staining of EU-labeled RNA was not affected by inhibition of reverse transcription, the kinetics of core opening of different CA mutants correlated with initiation of reverse transcription. Analysis of the E45A CA mutant suggests that initial core opening is independent of complete capsid disassembly. Conclusions Taken together, our results establish a novel RNA accessibility-based assay that detects an early event in HIV-1 uncoating and can be used to further define this process. PMID:23835323

MALDI Mass Spectrometry Imaging for Visualizing In Situ Metabolism of Endogenous Metabolites and Dietary Phytochemicals

PubMed Central

Fujimura, Yoshinori; Miura, Daisuke

2014-01-01

Understanding the spatial distribution of bioactive small molecules is indispensable for elucidating their biological or pharmaceutical roles. Mass spectrometry imaging (MSI) enables determination of the distribution of ionizable molecules present in tissue sections of whole-body or single heterogeneous organ samples by direct ionization and detection. This emerging technique is now widely used for in situ label-free molecular imaging of endogenous or exogenous small molecules. MSI allows the simultaneous visualization of many types of molecules including a parent molecule and its metabolites. Thus, MSI has received much attention as a potential tool for pathological analysis, understanding pharmaceutical mechanisms, and biomarker discovery. On the other hand, several issues regarding the technical limitations of MSI are as of yet still unresolved. In this review, we describe the capabilities of the latest matrix-assisted laser desorption/ionization (MALDI)-MSI technology for visualizing in situ metabolism of endogenous metabolites or dietary phytochemicals (food factors), and also discuss the technical problems and new challenges, including MALDI matrix selection and metabolite identification, that need to be addressed for effective and widespread application of MSI in the diverse fields of biological, biomedical, and nutraceutical (food functionality) research. PMID:24957029

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy.

PubMed

Lemcke, Heiko; Voronina, Natalia; Steinhoff, Gustav; David, Robert

2017-06-19

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Creating and virtually screening databases of fluorescently-labelled compounds for the discovery of target-specific molecular probes

NASA Astrophysics Data System (ADS)

Kamstra, Rhiannon L.; Dadgar, Saedeh; Wigg, John; Chowdhury, Morshed A.; Phenix, Christopher P.; Floriano, Wely B.

2014-11-01

Our group has recently demonstrated that virtual screening is a useful technique for the identification of target-specific molecular probes. In this paper, we discuss some of our proof-of-concept results involving two biologically relevant target proteins, and report the development of a computational script to generate large databases of fluorescence-labelled compounds for computer-assisted molecular design. The virtual screening of a small library of 1,153 fluorescently-labelled compounds against two targets, and the experimental testing of selected hits reveal that this approach is efficient at identifying molecular probes, and that the screening of a labelled library is preferred over the screening of base compounds followed by conjugation of confirmed hits. The automated script for library generation explores the known reactivity of commercially available dyes, such as NHS-esters, to create large virtual databases of fluorescence-tagged small molecules that can be easily synthesized in a laboratory. A database of 14,862 compounds, each tagged with the ATTO680 fluorophore was generated with the automated script reported here. This library is available for downloading and it is suitable for virtual ligand screening aiming at the identification of target-specific fluorescent molecular probes.

Communication: Alamethicin can capture lipid-like molecules in the membrane

NASA Astrophysics Data System (ADS)

Afanasyeva, Ekaterina F.; Syryamina, Victoria N.; Dzuba, Sergei A.

2017-01-01

Alamethicin (Alm) is a 19-mer antimicrobial peptide produced by fungus Trichoderma viride. Above a threshold concentration, Alm forms pores across the membrane, providing a mechanism of its antimicrobial action. Here we show that at a small concentration which is below the threshold value, Alm participates in formation of nanoscale lipid-mediated clusters of guest lipid-like molecules in the membrane. These results are obtained by electron spin echo (ESE) technique—a pulsed version of electron paramagnetic resonance—on spin-labeled stearic acid in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer with Alm added at 1/200 peptide-to-lipid ratio. ESE decay measurements are interpreted assuming that stearic acid molecules in the membrane are assembling around the Alm molecule. One may suggest that this Alm capturing effect on the guest lipid-like molecules could be important for the peptide antimicrobial action.

Small-molecule-based protein-labeling technology in live cell studies: probe-design concepts and applications.

PubMed

Mizukami, Shin; Hori, Yuichiro; Kikuchi, Kazuya

2014-01-21

The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention. Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed. First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of β-lactamase, which forms specific covalent bonds with β-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic β-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In the future, we expect that protein-labeling systems with finely designed probes will lead to novel methodologies that allow researchers to image biomolecules and to perturb protein functions.

A competitive immunoassay for sensitive detection of small molecules chloramphenicol based on luminol functionalized silver nanoprobe.

PubMed

Yu, Xiuxia; He, Yi; Jiang, Jie; Cui, Hua

2014-02-17

Chloramphenicol (CHL) as a broad-spectrum antibiotic has a broad action spectrum against Gram-positive and Gram-negative bacteria, as well as anaerobes. The use of CHL is strictly restricted in poultry because of its toxic effect. However, CHL is still illegally used in animal farming because of its accessibility and low cost. Therefore, sensitive methods are highly desired for the determination of CHL in foodstuffs. The immunoassays based on labeling as an important tool have been reported for the detection of CHL residues in food-producing animals. However, most of the labeling procedures require multi-step reactions and purifications and thus they are complicated and time-consuming. Recently, in our previous work, luminol functionalized silver nanoparticles have been successfully synthesized, which exhibits higher CL efficiency than luminol functionalized gold nanoparticles. In this work, the new luminol functionalized silver nanoparticles have been used for the labeling of small molecules CHL for the first time and a competitive chemiluminescent immunoassay has been developed for the detection of CHL. Owing to the amplification of silver nanoparticles, high sensitivity for CHL could be achieved with a low detection limit of 7.6×10(-9) g mL(-1) and a wide linear dynamic range of 1.0×10(-8)-1.0×10(-6) g mL(-1). This method has also been successfully applied to determine CHL in milk and honey samples with a good recoveries (92% and 102%, 99% and 107% respectively), indicating that the method is feasible for the determination of CHL in real milk and honey samples. The labeling procedure is simple, convenient and fast, superior to previously reported labeling procedures. The immunoassay is also simple, fast, sensitive and selective. It is of application potential for the determination of CHL in foodstuffs. Copyright © 2014 Elsevier B.V. All rights reserved.

Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling.

PubMed

Liu, Hui; Dong, Peng; Ioannou, Maria S; Li, Li; Shea, Jamien; Pasolli, H Amalia; Grimm, Jonathan B; Rivlin, Patricia K; Lavis, Luke D; Koyama, Minoru; Liu, Zhe

2018-01-09

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic range of ∼10,000-fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by two orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially restricted subnuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

Decreasing photobleaching by silver island films: application to muscle⋆

PubMed Central

Muthu, P.; Gryczynski, I.; Gryczynski, Z.; Talent, J.; Akopova, I.; Jain, K.; Borejdo, J.

2007-01-01

Recently it has become possible to study interactions between proteins at the level of single molecules. This requires collecting data from an extremely small volume, small enough to contain one molecule—typically of the order of attoliters (10−18 L). Collection of data from such a small volume with sufficiently high signal-to-noise ratio requires that the rate of photon detection per molecule be high. This calls for a large illuminating light flux, which in turn leads to rapid photobleaching of the fluorophores that are labeling the proteins. To decrease photobleaching, we measured fluorescence from a sample placed on coverslips coated with silver island films (SIF). SIF reduce photobleaching because they enhance fluorescence brightness and significantly decrease fluorescence lifetime. Increase in the brightness effectively decreases photobleaching because illumination can be attenuated to obtain the same fluorescence intensity. Decrease of lifetime decreases photobleaching because short lifetime minimizes the probability of oxygen attack while the fluorophore is in the excited state. The decrease of photobleaching was demonstrated in skeletal muscle. Myofibrils were labeled lightly with rhodamine–phalloidin, placed on coverslips coated with SIF, illuminated by total internal reflection, and observed through a confocal aperture. We show that SIF causes the intensity of phalloidin fluorescence to increase 4- to 5- fold and its fluorescence lifetime to decrease on average 23-fold. As a consequence, the rate of photobleaching of four or five molecules of actin of a myofibril on Olympus coverslips coated with SIF decreased at least 30-fold in comparison with photobleaching on an uncoated coverslip. Significant decrease of photobleaching makes the measurement of signal from a single cross-bridge of contracting muscle feasible. PMID:17531183

Surface stress sensor based on MEMS Fabry-Perot interferometer with high wavelength selectivity for label-free biosensing

NASA Astrophysics Data System (ADS)

Takahashi, Toshiaki; Hizawa, Takeshi; Misawa, Nobuo; Taki, Miki; Sawada, Kazuaki; Takahashi, Kazuhiro

2018-05-01

We have developed a surface stress sensor based on a microelectromechanical Fabry-Perot interferometer with high wavelength selectivity by using Au half-mirrors, for highly sensitive label-free biosensing. When the target molecule is adsorbed by the antigen-antibody reaction onto a movable membrane with a thin Au film, which acts as an upper mirror of the optical interferometer, the amount of deflection of the movable membrane deflected by the change in surface stress can be detected with high sensitivity. To improve the signal at the small membrane deflection region of this biosensor resulting in detection of low concentration molecules, by integrating 50 nm-thick Au half-mirrors, the wavelength selectivity of the optical interferometer has been successfully improved 6.6 times. Furthermore, the peak shift in the reflection spectrum due to the adsorption of bovine serum albumin (BSA) antigen with a concentration of 10 ng ml-l by the antigen-antibody reaction was spectroscopically measured on the fabricated optical interferometer, and the deflection amount of the movable membrane after 10 min treatment was 2.4 times larger than that of nonspecific adsorption with the avidin molecules. This result indicated that the proposed sensor can be used for selective detection of low-concentration target antigen molecules.

DNA motion capture reveals the mechanical properties of DNA at the mesoscale.

PubMed

Price, Allen C; Pilkiewicz, Kevin R; Graham, Thomas G W; Song, Dan; Eaves, Joel D; Loparo, Joseph J

2015-05-19

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Quantum dot conjugates in a sub-micrometer fluidic channel

DOEpatents

Stavis, Samuel M.; Edel, Joshua B.; Samiee, Kevan T.; Craighead, Harold G.

2010-04-13

A nanofluidic channel fabricated in fused silica with an approximately 500 nm square cross section was used to isolate, detect and identify individual quantum dot conjugates. The channel enables the rapid detection of every fluorescent entity in solution. A laser of selected wavelength was used to excite multiple species of quantum dots and organic molecules, and the emission spectra were resolved without significant signal rejection. Quantum dots were then conjugated with organic molecules and detected to demonstrate efficient multicolor detection. PCH was used to analyze coincident detection and to characterize the degree of binding. The use of a small fluidic channel to detect quantum dots as fluorescent labels was shown to be an efficient technique for multiplexed single molecule studies. Detection of single molecule binding events has a variety of applications including high throughput immunoassays.

Quantum dot conjugates in a sub-micrometer fluidic channel

DOEpatents

Stavis, Samuel M [Ithaca, NY; Edel, Joshua B [Brookline, MA; Samiee, Kevan T [Ithaca, NY; Craighead, Harold G [Ithaca, NY

2008-07-29

A nanofluidic channel fabricated in fused silica with an approximately 500 nm square cross section was used to isolate, detect and identify individual quantum dot conjugates. The channel enables the rapid detection of every fluorescent entity in solution. A laser of selected wavelength was used to excite multiple species of quantum dots and organic molecules, and the emission spectra were resolved without significant signal rejection. Quantum dots were then conjugated with organic molecules and detected to demonstrate efficient multicolor detection. PCH was used to analyze coincident detection and to characterize the degree of binding. The use of a small fluidic channel to detect quantum dots as fluorescent labels was shown to be an efficient technique for multiplexed single molecule studies. Detection of single molecule binding events has a variety of applications including high throughput immunoassays.

Development of a PET Prostate-Specific Membrane Antigen Imaging Agent: Preclinical Translation for Future Clinical Application

DTIC Science & Technology

2016-10-01

small-molecule peptidomimetic imaging agents labeled with positron emitting fluorine- 18 . These data will enable the filing of an exploratory IND...outcome. 15. SUBJECT TERMS Prostate Cancer, Prostate Specific Membrane Antigen (PSMA), Fluorine- 18 , Molecular Imaging, Radiotracer, Automated...Synthesis, Phosphoramidate, Inhibitor, Peptide Mimic, Peptidomimetic 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 . NUMBER OF PAGES 19a

Emerging applications of label-free optical biosensors

NASA Astrophysics Data System (ADS)

Zanchetta, Giuliano; Lanfranco, Roberta; Giavazzi, Fabio; Bellini, Tommaso; Buscaglia, Marco

2017-01-01

Innovative technical solutions to realize optical biosensors with improved performance are continuously proposed. Progress in material fabrication enables developing novel substrates with enhanced optical responses. At the same time, the increased spectrum of available biomolecular tools, ranging from highly specific receptors to engineered bioconjugated polymers, facilitates the preparation of sensing surfaces with controlled functionality. What remains often unclear is to which extent this continuous innovation provides effective breakthroughs for specific applications. In this review, we address this challenging question for the class of label-free optical biosensors, which can provide a direct signal upon molecular binding without using secondary probes. Label-free biosensors have become a consolidated approach for the characterization and screening of molecular interactions in research laboratories. However, in the last decade, several examples of other applications with high potential impact have been proposed. We review the recent advances in label-free optical biosensing technology by focusing on the potential competitive advantage provided in selected emerging applications, grouped on the basis of the target type. In particular, direct and real-time detection allows the development of simpler, compact, and rapid analytical methods for different kinds of targets, from proteins to DNA and viruses. The lack of secondary interactions facilitates the binding of small-molecule targets and minimizes the perturbation in single-molecule detection. Moreover, the intrinsic versatility of label-free sensing makes it an ideal platform to be integrated with biomolecular machinery with innovative functionality, as in case of the molecular tools provided by DNA nanotechnology.

Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins.

PubMed

Venkataramani, Varun; Kardorff, Markus; Herrmannsdörfer, Frank; Wieneke, Ralph; Klein, Alina; Tampé, Robert; Heilemann, Mike; Kuner, Thomas

2018-04-03

With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the 'labeling barrier' and to bypass photobleaching in multi-plane, whole-cell 3D experiments.

quenched-smFISH: Counting small RNA in Pathogenic Bacteria

NASA Astrophysics Data System (ADS)

Shepherd, Douglas; Li, Nan; Micheva-Viteva, Sofiya; Munsky, Brian; Hong-Geller, Elizabeth; Werner, James

2014-03-01

Here, we present a modification to single-molecule fluorescence in situ hybridization, quenched smFISH (q-smFISH), that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short nucleic acid targets can be detected when the background of unbound singly dye-labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. Exploiting an automated, multi-color wide-field microscope and GPU-accelerated data analysis package, we analyzed the statistics of sRNA expression in thousands of individual Yersinia pseudotuberculosis and Yersinia pestis bacteria before and during a simulated infection. Before infection, we find only a small fraction of either bacteria express the small RNAs YSR35 or YSP8. The copy numbers of these RNA are increased during simulated infection, suggesting a role in pathogenesis. The ability to directly quantify expression level changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in bacterial regulatory networks.

Resonant cavity enhanced multi-analyte sensing

NASA Astrophysics Data System (ADS)

Bergstein, David Alan

Biological research and medicine increasingly depend on interrogating binding interactions among small segments of DNA, RNA, protein, and bio-specific small molecules. Microarray technology, which senses the affinity for target molecules in solution for a multiplicity of capturing agents fixed to a surface, has been used in biological research for gene expression profiling and in medicine for molecular biomarker detection. Label-free affinity sensing is preferable as it avoids fluorescent labeling of the target molecules, reducing test cost and variability. The Resonant Cavity Imaging Biosensor (RCIB) is a label-free optical inference based technique introduced that scales readily to high throughput and employs an optical resonant cavity to enhance sensitivity by a factor of 100 or more. Near-infrared light centered at 1512.5 nm couples resonantly through a cavity constructed from Si/SiO2 Bragg reflectors, one of which serves as the binding surface. As the wavelength is swept 5 nm, an Indium-Gallium-Arsenide digital camera monitors cavity transmittance at each pixel with resolution 128 x 128. A wavelength shift in the local resonant response of the optical cavity indicates binding. Positioning the sensing surface with respect to the standing wave pattern of the electric field within the cavity, one can control the sensitivity of the measurement to the presence of bound molecules thereby enhancing or suppressing sensitivity where appropriate. Transmitted intensity at thousands of pixel locations are recorded simultaneously in a 10 s, 5 nm scan. An initial proof-of-principle setup was constructed. A sample was fabricated with 25, 100 mum wide square regions, each with a different density of 1 mum square depressions etched 12 nm into the S1O 2 surface. The average depth of each etched region was found with 0.05 nm RMS precision when the sample remains loaded in the setup and 0.3 nm RMS precision when the sample is removed and replaced. Selective binding of the protein avidin to biotin conjugated bovine serum albumin was demonstrated with 50 pg/mm2 sensitivity. Analysis and discussion of these results provides a path toward improved performance.

Elucidation of the Hsp90 C-terminal Inhibitor Binding Site

PubMed Central

Matts, Robert L.; Dixit, Anshuman; Peterson, Laura B.; Sun, Liang; Voruganti, Sudhakar; Kalyanaraman, Palgunan; Hartson, Steve D.; Verkhivker, Gennady M.; Blagg, Brian S. J.

2011-01-01

The Hsp90 chaperone machine is required for the folding, activation and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains, however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein. PMID:21548602

Attachment of second harmonic-active moiety to molecules for detection of molecules at interfaces

DOEpatents

Salafsky, Joshua S.; Eisenthal, Kenneth B.

2005-10-11

This invention provides methods of detecting molecules at an interface, which comprise labeling the molecules with a second harmonic-active moiety and detecting the labeled molecules at the interface using a surface selective technique. The invention also provides methods for detecting a molecule in a medium and for determining the orientation of a molecular species within a planar surface using a second harmonic-active moiety and a surface selective technique.

STUDIES ON THE ORIGIN OF RIBOSOMES IN AMOEBA PROTEUS

PubMed Central

Craig, Nessly; Goldstein, Lester

1969-01-01

The origin of cytoplasmic RNA and ribosomes was studied in Amoeba proteus by transplantation of a radioactive nucleus into an unlabeled cell followed by examination of the cytoplasm of the recipient for the presence of label. When a RNA-labeled nucleus was used, label appeared in the ribosomes, ribosomal RNA, and soluble RNA. Since the kinetics of appearance of labeled RNA indicates that the nucleus was not injured during the transfer, and since the transferred nuclear pool of labeled acid-soluble RNA precursors is inadequate to account for the amount of cytoplasmic RNA label, it is concluded that cytoplasmic ribosomal RNA is derived from acid-insoluble nuclear RNA and is probably transported as an intact molecule. Likewise, cytoplasmic soluble RNA probably originated in the nucleus, although labeling by terminal exchange in the cytoplasm is also possible. The results were completely different when a protein-labeled nucleus was grafted into an unlabeled host. In this case, label was found only in soluble proteins in the host cell cytoplasm, and there were no (or very few) radioactive ribosomes. This suggests that the nuclear pool of ribosomal protein and ribosomal protein precursors is relatively small and perhaps nonexistent (and, furthermore, shows that there was no cytoplasmic ribosomal contamination of the transferred nucleus). PMID:5765758

Plasmonic nanoparticles-decorated diatomite biosilica: extending the horizon of on-chip chromatography and label-free biosensing.

PubMed

Kong, Xianming; Li, Erwen; Squire, Kenny; Liu, Ye; Wu, Bo; Cheng, Li-Jing; Wang, Alan X

2017-11-01

Diatomite consists of fossilized remains of ancient diatoms and is a type of naturally abundant photonic crystal biosilica with multiple unique physical and chemical functionalities. In this paper, we explored the fluidic properties of diatomite as the matrix for on-chip chromatography and, simultaneously, the photonic crystal effects to enhance the plasmonic resonances of metallic nanoparticles for surface-enhanced Raman scattering (SERS) biosensing. The plasmonic nanoparticle-decorated diatomite biosilica provides a lab-on-a-chip capability to separate and detect small molecules from mixture samples with ultra-high detection sensitivity down to 1 ppm. We demonstrate the significant potential for biomedical applications by screening toxins in real biofluid, achieving simultaneous label-free biosensing of phenethylamine and miR21cDNA in human plasma with unprecedented sensitivity and specificity. To the best of our knowledge, this is the first time demonstration to detect target molecules from real biofluids by on-chip chromatography-SERS techniques. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aggregated silver nanoparticles based surface-enhanced Raman scattering enzyme-linked immunosorbent assay for ultrasensitive detection of protein biomarkers and small molecules.

PubMed

Liang, Jiajie; Liu, Hongwu; Huang, Caihong; Yao, Cuize; Fu, Qiangqiang; Li, Xiuqing; Cao, Donglin; Luo, Zhi; Tang, Yong

2015-06-02

Lowering the detection limit is critical to the design of bioassays required for medical diagnostics, environmental monitoring, and food safety regulations. The current sensitivity of standard color-based analyte detection limits the further use of enzyme-linked immunosorbent assays (ELISAs) in research and clinical diagnoses. Here, we demonstrate a novel method that uses the Raman signal as the signal-generating system of an ELISA and combines surface-enhanced Raman scattering (SERS) with silver nanoparticles aggregation for ultrasensitive analyte detection. The enzyme label of the ELISA controls the dissolution of Raman reporter-labeled silver nanoparticles through hydrogen peroxide and generates a strong Raman signal when the analyte is present. Using this assay, prostate-specific antigen (PSA) and the adrenal stimulant ractopamine (Rac) were detected in whole serum and urine at the ultralow concentrations of 10(-9) and 10(-6) ng/mL, respectively. The methodology proposed here could potentially be applied to other molecules detection as well as PSA and Rac.

Functions and Mechanisms of Sleep in Flies and Mammals

DTIC Science & Technology

2007-02-01

serotonin receptor likely to mediate the known interaction between the serotonergic Raphe nucleus and the LC (Htr1d). We have also confirmed the prior... Chemistry . His research focuses on mass spectrometry, a technique that will augment research on the mechanisms of sleep and complement microarray gene...labeling (ICAT, ITRAQ, etc); 8) MALDI and electrospray FTMS for the identification of small molecule structure ; 9) Gas phase reactions within the FTMS

A Highly Specific Gold Nanoprobe for Live-Cell Single-Molecule Imaging

NASA Astrophysics Data System (ADS)

Leduc, Cécile; Si, Satyabrata; Gautier, Jérémie; Soto-Ribeiro, Martinho; Wehrle-Haller, Bernhard; Gautreau, Alexis; Giannone, Grégory; Cognet, Laurent; Lounis, Brahim

2013-04-01

Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Due to these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5-nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used GFPs with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.

Spectral density mapping at multiple magnetic fields suitable for 13C NMR relaxation studies

NASA Astrophysics Data System (ADS)

Kadeřávek, Pavel; Zapletal, Vojtěch; Fiala, Radovan; Srb, Pavel; Padrta, Petr; Přecechtělová, Jana Pavlíková; Šoltésová, Mária; Kowalewski, Jozef; Widmalm, Göran; Chmelík, Josef; Sklenář, Vladimír; Žídek, Lukáš

2016-05-01

Standard spectral density mapping protocols, well suited for the analysis of 15N relaxation rates, introduce significant systematic errors when applied to 13C relaxation data, especially if the dynamics is dominated by motions with short correlation times (small molecules, dynamic residues of macromolecules). A possibility to improve the accuracy by employing cross-correlated relaxation rates and on measurements taken at several magnetic fields has been examined. A suite of protocols for analyzing such data has been developed and their performance tested. Applicability of the proposed protocols is documented in two case studies, spectral density mapping of a uniformly labeled RNA hairpin and of a selectively labeled disaccharide exhibiting highly anisotropic tumbling. Combination of auto- and cross-correlated relaxation data acquired at three magnetic fields was applied in the former case in order to separate effects of fast motions and conformational or chemical exchange. An approach using auto-correlated relaxation rates acquired at five magnetic fields, applicable to anisotropically moving molecules, was used in the latter case. The results were compared with a more advanced analysis of data obtained by interpolation of auto-correlated relaxation rates measured at seven magnetic fields, and with the spectral density mapping of cross-correlated relaxation rates. The results showed that sufficiently accurate values of auto- and cross-correlated spectral density functions at zero and 13C frequencies can be obtained from data acquired at three magnetic fields for uniformly 13C -labeled molecules with a moderate anisotropy of the rotational diffusion tensor. Analysis of auto-correlated relaxation rates at five magnetic fields represents an alternative for molecules undergoing highly anisotropic motions.

Intranucleus Single-Molecule Imaging in Living Cells.

PubMed

Shao, Shipeng; Xue, Boxin; Sun, Yujie

2018-06-01

Many critical processes occurring in mammalian cells are stochastic and can be directly observed at the single-molecule level within their physiological environment, which would otherwise be obscured in an ensemble measurement. There are various fundamental processes in the nucleus, such as transcription, replication, and DNA repair, the study of which can greatly benefit from intranuclear single-molecule imaging. However, the number of such studies is relatively small mainly because of lack of proper labeling and imaging methods. In the past decade, tremendous efforts have been devoted to developing tools for intranuclear imaging. Here, we mainly describe the recent methodological developments of single-molecule imaging and their emerging applications in the live nucleus. We also discuss the remaining issues and provide a perspective on future developments and applications of this field. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Saturation recovery EPR and ELDOR at W-band for spin labels

PubMed Central

Froncisz, Wojciech; Camenisch, Theodore G.; Ratke, Joseph J.; Anderson, James R.; Subczynski, Witold K.; Strangeway, Robert A.; Sidabras, Jason W.; Hyde, James S.

2008-01-01

A reference-arm W-band (94 GHz) microwave bridge with two sample-irradiation arms for saturation recovery (SR) EPR and ELDOR experiments is described. Frequencies in each arm are derived from 2 GHz synthesizers that have a common time-base and are translated to 94 GHz in steps of 33 and 59 GHz. Intended applications are to nitroxide radical spin labels and spin probes in the liquid phase. An enabling technology is the use of a W-band loop-gap resonator (LGR) (J.W. Sidabras et al., Rev. Sci. Instrum. 78 (2007) 034701). The high efficiency parameter (8.2 GW−1/2 with sample) permits the saturating pump pulse level to be just 5 mW or less. Applications of SR EPR and ELDOR to the hydrophilic spin labels 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl (CTPO) and 2,2,6,6,-tetramethyl-4-piperidone-1-oxyl (TEMPONE) are described in detail. In the SR ELDOR experiment, nitrogen nuclear relaxation as well as Heisenberg exchange transfer saturation from pumped to observed hyperfine transitions. SR ELDOR was found to be an essential method for measurements of saturation transfer rates for small molecules such as TEMPONE. Free induction decay (FID) signals for small nitroxides at W-band are also reported. Results are compared with multifrequency measurements of T1e previously reported for these molecules in the range of 2 to 35 GHz (J.S. Hyde et al., J. Phys. Chem. B 108 (2004) 9524–9529). The values of T1e decrease at 94 GHz relative to values at 35 GHz. PMID:18547848

Photonic-plasmonic hybrid single-molecule nanosensor measures the effect of fluorescent labels on DNA-protein dynamics

PubMed Central

Liang, Feng; Guo, Yuzheng; Hou, Shaocong; Quan, Qimin

2017-01-01

Current methods to study molecular interactions require labeling the subject molecules with fluorescent reporters. However, the effect of the fluorescent reporters on molecular dynamics has not been quantified because of a lack of alternative methods. We develop a hybrid photonic-plasmonic antenna-in-a-nanocavity single-molecule biosensor to study DNA-protein dynamics without using fluorescent labels. Our results indicate that the fluorescein and fluorescent protein labels decrease the interaction between a single DNA and a protein due to weakened electrostatic interaction. Although the study is performed on the DNA-XPA system, the conclusion has a general implication that the traditional fluorescent labeling methods might be misestimating the molecular interactions. PMID:28560341

A two-step strategy to visually identify molecularly imprinted polymers for tagged proteins.

PubMed

Brandis, Alexander; Partouche, Eran; Yechezkel, Tamar; Salitra, Yoseph; Shkoulev, Vladimir; Scherz, Avigdor; Grynszpan, Flavio

2017-08-01

A practical and relatively simple method to identify molecularly imprinted polymers capable of binding proteins via the molecular tagging (epitope-like) approach has been developed. In our two-step method, we first challenge a previously obtained anti-tag molecularly imprinted polymer with a small molecule including the said tag of choice (a biotin derivative as shown here or other) connected to a linker bound to a second biotin moiety. An avidin molecule partially decorated with fluorescent labels is then allowed to bind the available biotin derivative associated with the polymer matrix. At the end of this simple process, and after washing off all the low-affinity binding molecules from the polymer matrix, only suitable molecularly imprinted polymers binding avidin through its previously acquired small molecule tag (or epitope-like probe, in a general case) will remain fluorescent. For confirmation, we tested the selective performance of the anti-biotin molecularly imprinted polymer binding it to biotinylated alkaline phosphatase. Residual chemical activity of the enzyme on the molecularly imprinted polymer solid support was observed. In all cases, the corresponding nonimprinted polymer controls were inactive. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computational Approaches for Designing Protein/Inhibitor Complexes and Membrane Protein Variants

NASA Astrophysics Data System (ADS)

Vijayendran, Krishna Gajan

Drug discovery of small-molecule protein inhibitors is a vast enterprise that involves several scientific disciplines (i.e. genomics, cell biology, x-ray crystallography, chemistry, computer science, statistics), with each discipline focusing on a particular aspect of the process. In this thesis, I use computational and experimental approaches to explore the most fundamental aspect of drug discovery: the molecular interactions of small-molecules inhibitors with proteins. In Part I (Chapters I and II), I describe how computational docking approaches can be used to identify structurally diverse molecules that can inhibit multiple protein targets in the brain. I illustrate this approach using the examples of microtubule-stabilizing agents and inhibitors of cyclooxygenase(COX)-I and 5-lipoxygenase (5-LOX). In Part II (Chapters III and IV), I focus on membrane proteins, which are notoriously difficult to work with due to their low natural abundances, low yields for heterologous over expression, and propensities toward aggregation. I describe a general approach for designing water-soluble variants of membrane proteins, for the purpose of developing cell-free, label-free, detergent-free, solution-phase studies of protein structure and small-molecule binding. I illustrate this approach through the design of a water-soluble variant of the membrane protein Smoothened, wsSMO. This wsSMO stands to serve as a first-step towards developing membrane protein analogs of this important signaling protein and drug target.

Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

PubMed

Portal, Christophe F; Seifert, Jan-Marcus; Buehler, Christof; Meisner-Kober, Nicole-Claudia; Auer, Manfred

2014-07-16

We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence fluctuation analysis techniques working at single molecule resolution, like fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity diffusion analysis (FIDA), etc., it became important to work with homogeneously labeled target proteins. Each molecule participating in a binding equilibrium should be detectable when it freely fluctuates through the confocal focus of a microscope. The measured photon burst for each transition contains information about the size and the stoichiometry of a protein complex. Therefore, it is important to work with reagents that contain an exact number of tracers per protein at identical positions. The ideal fluorescent tracer-protein complex stoichiometry is 1:1. While genetic tags such as fluorescent proteins (FPs) are widely used to detect proteins, FPs have several limitations compared to chemical tags. For example, FPs cannot easily compete with organic dyes in the flexibility of modification and spectral range; moreover, FPs have disadvantages in brightness and photostability and are therefore not ideal for most biochemical single molecule studies. We present the synthesis of a series of exemplaric toolbox reagents and labeling results on three target proteins which were needed for high throughput screening experiments using fluorescence fluctuation analysis at single molecule resolution. On one target, Hu-antigen R (HuR), we demonstrated the activity of the 1:1 labeled protein in ribonucleic acid (RNA) binding, and the ease of resolving the stoichiometry of an RNA-HuR complex using the same dye on protein and RNA by Fluorescence Intensity Multiple Distribution Analysis (FIMDA) detection.

Heterogeneous distribution of dye-labelled biomineralizaiton proteins in calcite crystals

NASA Astrophysics Data System (ADS)

Liu, Chuang; Xie, Liping; Zhang, Rongqing

2015-12-01

Biominerals are highly ordered crystals mediated by organic matters especially proteins in organisms. However, how specific proteins are distributed inside biominerals are not well understood. In the present study, we use fluorescein isothiocyanate (FITC) to label extracted proteins from the shells of bivalve Pinctada fucata. By confocal laser scanning microscopy (CLSM), we observe a heterogeneous distribution of dye-labelled proteins inside synthetic calcite at the microscale. Proteins from the prismatic calcite layers accumulate at the edge of crystals while proteins from the nacreous aragonite layers accumulate at the center of crystals. Raman and X-ray powder diffraction show that both the proteins cannot alter the crystal phase. Scanning electron microscope demonstrates both proteins are able to affect the crystal morphology. This study may provide a direct approach for the visualization of protein distributions in crystals by small-molecule dye-labelled proteins as the additives in the crystallization process and improve our understanding of intracrystalline proteins distribution in biogenic calcites.

N-Sulfonyl-β-lactam hapten as an effective labeling reagent for aldolase mAb.

PubMed

Inokuma, Tsubasa; Fuller, Roberta P; Barbas, Carlos F

2015-04-15

Utilization of chemically programmed antibodies (cpAbs) is regarded to be one of the most efficient methods for the development of therapeutic systems. cpAbs can extend the half-life of programming reagents, activate immune systems via the Fc region of antibodies and achieve universal vaccination by attaching varieties of small, programmed molecules. In the current study, we aimed to develop a novel labeling reagent for the preparation of cpAbs and found that N-sulfonyl-β-lactams (NSBLs) were optimal. NSBL can be synthesized from readily available 4-(bromomethyl)benzenesulfonyl chloride via few simple manipulations and can label the aldolase monoclonal antibody (mAb) 84G3, which could not be labeled effectively by the conventional labeling reagent, N-acyl-β-lactam (NABL). We also demonstrated that the conjugate, which consists of mAb 84G3 and an NSBL bearing a biotin moiety, maintained strong binding activity to streptavidin. In addition, the stability assay of NSBL revealed that NSBLs can tolerate aqueous media without significant decomposition over 24h. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ultrasound-microbubble-mediated intercellular adhesion molecule-1 small interfering ribonucleic acid transfection attenuates neointimal formation after arterial injury in mice.

PubMed

Suzuki, Jun-ichi; Ogawa, Masahito; Takayama, Kiyoshi; Taniyama, Yoshiaki; Morishita, Ryuichi; Hirata, Yasunobu; Nagai, Ryozo; Isobe, Mitsuaki

2010-03-02

The purpose of this study was to investigate the efficiency of small interfering ribonucleic acid (siRNA) in murine arteries. We transfected it using a nonviral ultrasound-microbubble-mediated in vivo gene delivery system. siRNA is an effective methodology to suppress gene function. The siRNA can be synthesized easily; however, a major obstacle in the use of siRNA as therapeutics is the difficulty involved in effective in vivo delivery. To investigate the efficiency of nonviral ultrasound-microbubble-mediated in vivo siRNA delivery, we used a fluorescein-labeled siRNA, green fluorescent protein (GFP) siRNA, and intercellular adhesion molecule (ICAM)-1 siRNA in murine arteries. Murine femoral arteries were injured using flexible wires to establish arterial injury. The fluorescein-labeled siRNA and GFP siRNA showed that this nonviral approach could deliver siRNA into target arteries effectively without any tissue damage and systemic adverse effects. ICAM-1 siRNA transfection into murine injured arteries significantly suppressed the development of neointimal formation in comparison to those in the control group. Immunohistochemistry revealed that accumulation of T cells and adhesion molecule positive cells was observed in nontreated injured arteries, whereas siRNA suppressed accumulation. The nonviral ultrasound-microbubble delivery of siRNA ensures effective transfection into target arteries. ICAM-1 siRNA has the potential to suppress arterial neointimal formation. Transfection of siRNA can be beneficial for the clinical treatment of cardiovascular and other inflammatory diseases. Copyright 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

PubMed Central

Provost, Christopher R.; Sun, Luo

2010-01-01

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells. PMID:20485262

A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure

PubMed Central

Hauser, Christina T.; Tsien, Roger Y.

2007-01-01

Site-specific fluorescent labeling of proteins in vivo remains one of the most powerful techniques for imaging complex processes in live cells. Although fluorescent proteins in many colors are useful tools for tracking expression and localization of fusion proteins in cells, these relatively large tags (>220 aa) can perturb protein folding, trafficking and function. Much smaller genetically encodable domains (<15 aa) offer complementary advantages. We introduce a small fluorescent chelator whose membrane-impermeant complex with nontoxic Zn2+ ions binds tightly but reversibly to hexahistidine (His6) motifs on surface-exposed proteins. This live-cell label helps to resolve a current controversy concerning externalization of the stromal interaction molecule STIM1 upon depletion of Ca2+ from the endoplasmic reticulum. Whereas N-terminal fluorescent protein fusions interfere with surface exposure of STIM1, short His6 tags are accessible to the dye or antibodies, demonstrating externalization. PMID:17360414

Quantitation of Membrane-Ligand Interactions Using Backscattering Interferometry

PubMed Central

Baksh, Michael M.; Kussrow, Amanda K.; Mileni, Mauro; Finn, M.G.; Bornhop, Darryl J.

2011-01-01

Though membrane-associated proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show backscattering interferometry (BSI) to be a viable technique for quantifying ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic- and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity, and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest. PMID:21399645

Osmoregulation in Methanogens (and Other Interesting Organisms)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roberts, Mary Fedarko

2014-12-03

Our research has been aimed at (i) identifying, (ii) determining mode of regulation, and (iii) understanding how different classes of compatible solutes (also termed osmolytes) affect macromolecular stability in response to osmotic and thermal stress. For solutes we have identified (e.g., di-inositol-1,1’-phosphate (DIP)), we used NMR to elucidate biosynthetic pathways and then cloned suspected enzymes in the pathway to explore how they are regulated. Compatible solutes are thought to protect proteins from thermal and osmotic stresses by being excluded from the surface, allowing critical water molecules to interact with the protein. This implies there are no specific binding interactions betweenmore » osmolytes and proteins. However, we and others have often observed very specific solute effects for proteins that suggest a more direct interaction between solute and protein is likely can occur. Measuring such a weak interaction is extremely difficult. We have developed a solution NMR method, high-resolution field cycling relaxometry, that can measure spin-lattice relaxation rates as a function of magnetic field from 11.7 (the field of a 500 MHz spectrometer) to 0.003 T. The methodology is ideal for nuclei in small molecules with moderately long relaxation times at high fields – phosphate groups (31P), enriched carbonyls (13C), or methyl groups (1H). The protein of interest is spin-labeled to introduce a large dipole on it that will dominate the relaxation of nuclei on any small molecules that bind transiently. The key is to measure relaxation below 1-2 T (and extract nuclei-spin label distances in the bound complex) where the small molecule relaxation will be dominated by dipolar mechanisms with a correlation time indicative of the large protein complex. Our explorations of an inositol monophosphatase (the last step in DIP generation) localized four discrete binding sides for the thermoprotectant α-glutamate. This is a novel approach, and while the work did not fully explain how this solute protected the IMPase from thermal denaturation, it did showcase a new and exciting method to monitor weak binding in biological systems.« less

99mTc: Labeling Chemistry and Labeled Compounds

NASA Astrophysics Data System (ADS)

Alberto, R.; Abram, U.

This chapter reviews the radiopharmaceutical chemistry of technetium related to the synthesis of perfusion agents and to the labeling of receptor-binding biomolecules. To understand the limitations of technetium chemistry imposed by future application of the complexes in nuclear medicine, an introductory section analyzes the compulsory requirements to be considered when facing the incentive of introducing a novel radiopharmaceutical into the market. Requirements from chemistry, routine application, and market are discussed. In a subsequent section, commercially available 99mTc-based radiopharmaceuticals are treated. It covers the complexes in use for imaging the most important target organs such as heart, brain, or kidney. The commercially available radiopharmaceuticals fulfill the requirements outlined earlier and are discussed with this background. In a following section, the properties and perspectives of the different generations of radiopharmaceuticals are described in a general way, covering characteristics for perfusion agents and for receptor-specific molecules. Technetium chemistry for the synthesis of perfusion agents and the different labeling approaches for target-specific biomolecules are summarized. The review comprises a general introduction to the common approaches currently in use, employing the N x S4-x , [3+1] and 2-hydrazino-nicotinicacid (HYNIC) method as well as more recent strategies such as the carbonyl and the TcN approach. Direct labeling without the need of a bifunctional chelator is briefly reviewed as well. More particularly, recent developments in the labeling of concrete targeting molecules, the second generation of radiopharmaceuticals, is then discussed and prominent examples with antibodies/peptides, neuroreceptor targeting small molecules, myocardial imaging agents, vitamins, thymidine, and complexes relevant to multidrug resistance are given. In addition, a new approach toward peptide drug development is described. The section has a focus on coordination and labeling chemistry, but biological results are briefly summarized as well. The last (and shortest) section finally intends to give a (subjective) outlook for the future role of 99mTc-based radiopharmaceuticals. Critical comments are spread over the whole article but are concentrated in this section. Despite the increasing competition of diagnostic radiopharmacy by other commonly applied methods in medicine such as magnetic resonance imaging (MRI) or ultrasound, the authors are convinced that 99mTc will play a key role also in future if novel approaches are added and the requirements from chemistry biology and the market considered in research to a stronger extent.

Butter feeding enhances TNF-alpha production from macrophages and lymphocyte adherence in murine small intestinal microvessels.

PubMed

Fujiyama, Yoichi; Hokari, Ryota; Miura, Soichiro; Watanabe, Chikako; Komoto, Shunsuke; Oyama, Tokushige; Kurihara, Chie; Nagata, Hiroshi; Hibi, Toshifumi

2007-11-01

Dietary fat is known to modulate immune functions. Intake of an animal fat-rich diet has been linked to increased risk of inflammation; however, little is known about how animal fat ingestion directly affects intestinal immune function. The objective of this study was to assess the effect of butter feeding on lymphocyte migration in intestinal mucosa and the changes in adhesion molecules and cytokines involved in this effect. T-lymphocytes isolated from the spleen were fluorescence-labeled and injected into recipient mice. Butter was administered into the duodenum, and villus microvessels of the small intestinal mucosa were observed under an intravital microscope. mRNA expression of adhesion molecules and cytokines in the intestinal mucosa were determined by quantitative PCR. The effect of butter feeding on tumor necrosis factor (TNF)-alpha mRNA expression of intestinal macrophages was also determined. Intraluminal butter administration significantly increased lymphocyte adherence to intestinal microvessels accompanied by increases in expression levels of adhesion molecules ICAM-1, MAdCAM-1 and VCAM-1. This accumulation was significantly attenuated by anti-MAdCAM-1 and anti-ICAM-1 antibodies. Butter administration significantly increased TNF-alpha in the lamina proprial macrophages but not interleukin-6. Anti-TNF-alpha treatment attenuated the enhanced expression of adhesion molecules induced by butter administration. T-lymphocyte adherence to microvessels of the small intestinal mucosa was significantly enhanced after butter ingestion. This enhancement is due to increase in expression levels of adhesion molecules of the intestinal mucosa, which is mediated by TNF-alpha from macrophages in the intestinal lamina propria.

Nanodiamonds as multi-purpose labels for microscopy.

PubMed

Hemelaar, S R; de Boer, P; Chipaux, M; Zuidema, W; Hamoh, T; Martinez, F Perona; Nagl, A; Hoogenboom, J P; Giepmans, B N G; Schirhagl, R

2017-04-07

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.

Innovations in biomedical nanoengineering: nanowell array biosensor.

PubMed

Seo, YoungTae; Jeong, Sunil; Lee, JuKyung; Choi, Hak Soo; Kim, Jonghan; Lee, HeaYeon

2018-01-01

Nanostructured biosensors have pioneered biomedical engineering by providing highly sensitive analyses of biomolecules. The nanowell array (NWA)-based biosensing platform is particularly innovative, where the small size of NWs within the array permits extremely profound sensing of a small quantity of biomolecules. Undoubtedly, the NWA geometry of a gently-sloped vertical wall is critical for selective docking of specific proteins without capillary resistances, and nanoprocessing has contributed to the fabrication of NWA electrodes on gold substrate such as molding process, e-beam lithography, and krypton-fluoride (KrF) stepper semiconductor method. The Lee group at the Mara Nanotech has established this NW-based biosensing technology during the past two decades by engineering highly sensitive electrochemical sensors and providing a broad range of detection methods from large molecules (e.g., cells or proteins) to small molecules (e.g., DNA and RNA). Nanosized gold dots in the NWA enhance the detection of electrochemical biosensing to the range of zeptomoles in precision against the complementary target DNA molecules. In this review, we discuss recent innovations in biomedical nanoengineering with a specific focus on novel NWA-based biosensors. We also describe our continuous efforts in achieving a label-free detection without non-specific binding while maintaining the activity and stability of immobilized biomolecules. This research can lay the foundation of a new platform for biomedical nanoengineering systems.

Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform

PubMed Central

Wagner, Tobias; Greschik, Holger; Burgahn, Teresa; Schmidtkunz, Karin; Schott, Anne-Kathrin; McMillan, Joel; Baranauskienė, Lina; Xiong, Yan; Fedorov, Oleg; Jin, Jian; Oppermann, Udo; Matulis, Daumantas; Schüle, Roland; Jung, Manfred

2016-01-01

Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein–protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader. PMID:26893353

Innovations in biomedical nanoengineering: nanowell array biosensor

NASA Astrophysics Data System (ADS)

Seo, YoungTae; Jeong, Sunil; Lee, JuKyung; Choi, Hak Soo; Kim, Jonghan; Lee, HeaYeon

2018-04-01

Nanostructured biosensors have pioneered biomedical engineering by providing highly sensitive analyses of biomolecules. The nanowell array (NWA)-based biosensing platform is particularly innovative, where the small size of NWs within the array permits extremely profound sensing of a small quantity of biomolecules. Undoubtedly, the NWA geometry of a gently-sloped vertical wall is critical for selective docking of specific proteins without capillary resistances, and nanoprocessing has contributed to the fabrication of NWA electrodes on gold substrate such as molding process, e-beam lithography, and krypton-fluoride (KrF) stepper semiconductor method. The Lee group at the Mara Nanotech has established this NW-based biosensing technology during the past two decades by engineering highly sensitive electrochemical sensors and providing a broad range of detection methods from large molecules (e.g., cells or proteins) to small molecules (e.g., DNA and RNA). Nanosized gold dots in the NWA enhance the detection of electrochemical biosensing to the range of zeptomoles in precision against the complementary target DNA molecules. In this review, we discuss recent innovations in biomedical nanoengineering with a specific focus on novel NWA-based biosensors. We also describe our continuous efforts in achieving a label-free detection without non-specific binding while maintaining the activity and stability of immobilized biomolecules. This research can lay the foundation of a new platform for biomedical nanoengineering systems.

MICROSCOPIC USES OF NANOGOLD.

DOE Office of Scientific and Technical Information (OSTI.GOV)

HAINFELD,J.F.POWELL,R.D.FURUYA,F.R.

2003-04-17

Gold has been used for immunocytochemistry since 1971 when Faulk and Taylor discovered adsorption of antibodies to colloidal gold. It is an ideal label for electron microscopy (EM) due to its high atomic number, which scatters electrons efficiently, and the fact that preparative methods have been developed to make uniform particles in the appropriate size range of 5 to 30 nm. Use in light microscopy (LM) generally requires silver enhancement (autometallography; AMG) of these small gold particles. Significant advances in this field since that time have included a better understanding of the conditions for best antibody adsorption, more regular goldmore » size production, adsorption of other useful molecules, like protein A, and advances in silver enhancement. Many studies have also been accomplished showing the usefulness of these techniques to cell biology and biomedical research. A further advance in this field was the development of Nanogold{trademark}, a 1.4 nm gold cluster. A significant difference from colloidal gold is that Nanogold is actually a coordination compound containing a gold core covalently linked to surface organic groups. These in turn may be covalently attached to antibodies. This approach to immunolabeling has several advantages compared to colloidal gold such as vastly better penetration into tissues, generally greater sensitivity, and higher density of labeling. Since Nanogold is covalently coupled to antibodies, it may also be directly coupled to almost any protein, peptide, carbohydrate, or molecule of interest, including molecules which do not adsorb to colloidal gold. This increases the range of probes possible, and expands the applications of gold labeling.« less

A Metal-Free Method for Producing MRI Contrast at Amyloid-Beta

PubMed Central

Hilt, Silvia; Tang, Tang; Walton, Jeffrey H.; Budamagunta, Madhu; Maezawa, Izumi; Kálai, Tamás; Hideg, Kálmán; Singh, Vikrant; Wulff, Heike; Gong, Qizhi; Jin, Lee-Way; Louie, Angelique; Voss, John C.

2017-01-01

Alzheimer’s disease (AD) is characterized by depositions of the amyloid-β (Aβ) peptide in the brain. The disease process develops over decades, with substantial neurological loss occurring before a clinical diagnosis of dementia can be rendered. It is therefore imperative to develop methods that permit early detection and monitoring of disease progression. In addition, the multifactorial pathogenesis of AD has identified several potential avenues for AD intervention. Thus, evaluation of therapeutic candidates over lengthy trial periods also demands a practical, noninvasive method for measuring Aβ in the brain. Magnetic resonance imaging (MRI) is the obvious choice for such measurements, but contrast enhancement for Aβ has only been achieved using Gd(III)-based agents. There is great interest in gadolinium-free methods to image the brain. In this study, we provide the first demonstration that a nitroxide-based small-molecule produces MRI contrast in brain specimens with elevated levels of Aβ. The molecule is comprised of a fluorene (a molecule with high affinity for Aβ) and a nitroxide spin label (a paramagnetic MRI contrast species). Labeling of brain specimens with the spin-labeled fluorene produces negative contrast in samples from AD model mice whereas no negative contrast is seen in specimens harvested from wild-type mice. Injection of SLF into live mice resulted in good brain penetration, with the compound able to generate contrast 24-hr post injection. These results provide a proof of concept method that can be used for early, noninvasive, gadolinium-free detection of amyloid plaques by magnetic resonance imaging (MRI). PMID:27911291

Carbon nanofibers arrays: A novel tool for microdelivery of biomolecules to plants

DOE PAGES

Davern, Sandra M.; McKnight, Timothy E.; Kalluri, Udaya C.; ...

2016-04-27

Effective methods for delivering bioprobes into the cells of intact plants are essential for investigating diverse biological processes. Increasing research on trees, such as Populus spp., for bioenergy applications is driving the need for techniques that work well with tree species. This report introduces vertically aligned carbon nanofiber (VACNF) arrays as a new tool for microdelivery of labeled molecules to Populus leaf tissue and whole plants. We demonstrated that VACNFs penetrate the leaf surface to deliver sub-microliter quantities of solution containing fluorescent or radiolabeled molecules into Populus leaf cells. Importantly, VACNFs proved to be gentler than abrasion with carborundum, amore » common way to introduce material into leaves. Unlike carborundum, VACNFs did not disrupt cell or tissue integrity, nor did they induce production of hydrogen peroxide, a typical wound response. We show that femtomole to picomole quantities of labeled molecules (fluorescent dyes, small proteins and dextran), ranging from 0.5–500 kDa, can be introduced by VACNFs, and we demonstrate the use of the approach to track delivered probes from their site of introduction on the leaf to distal plant regions. VACNF arrays thus offer an attractive microdelivery method for the introduction of biomolecules and other probes into trees and potentially other types of plants.« less

Carbon nanofibers arrays: A novel tool for microdelivery of biomolecules to plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davern, Sandra M.; McKnight, Timothy E.; Kalluri, Udaya C.

Effective methods for delivering bioprobes into the cells of intact plants are essential for investigating diverse biological processes. Increasing research on trees, such as Populus spp., for bioenergy applications is driving the need for techniques that work well with tree species. This report introduces vertically aligned carbon nanofiber (VACNF) arrays as a new tool for microdelivery of labeled molecules to Populus leaf tissue and whole plants. We demonstrated that VACNFs penetrate the leaf surface to deliver sub-microliter quantities of solution containing fluorescent or radiolabeled molecules into Populus leaf cells. Importantly, VACNFs proved to be gentler than abrasion with carborundum, amore » common way to introduce material into leaves. Unlike carborundum, VACNFs did not disrupt cell or tissue integrity, nor did they induce production of hydrogen peroxide, a typical wound response. We show that femtomole to picomole quantities of labeled molecules (fluorescent dyes, small proteins and dextran), ranging from 0.5–500 kDa, can be introduced by VACNFs, and we demonstrate the use of the approach to track delivered probes from their site of introduction on the leaf to distal plant regions. VACNF arrays thus offer an attractive microdelivery method for the introduction of biomolecules and other probes into trees and potentially other types of plants.« less

Carbon Nanofiber Arrays: A Novel Tool for Microdelivery of Biomolecules to Plants

PubMed Central

Davern, Sandra M.; McKnight, Timothy E.; Morrell-Falvey, Jennifer L.; Shpak, Elena D.; Kalluri, Udaya C.; Jelenska, Joanna; Greenberg, Jean T.; Mirzadeh, Saed

2016-01-01

Effective methods for delivering bioprobes into the cells of intact plants are essential for investigating diverse biological processes. Increasing research on trees, such as Populus spp., for bioenergy applications is driving the need for techniques that work well with tree species. This report introduces vertically aligned carbon nanofiber (VACNF) arrays as a new tool for microdelivery of labeled molecules to Populus leaf tissue and whole plants. We demonstrated that VACNFs penetrate the leaf surface to deliver sub-microliter quantities of solution containing fluorescent or radiolabeled molecules into Populus leaf cells. Importantly, VACNFs proved to be gentler than abrasion with carborundum, a common way to introduce material into leaves. Unlike carborundum, VACNFs did not disrupt cell or tissue integrity, nor did they induce production of hydrogen peroxide, a typical wound response. We show that femtomole to picomole quantities of labeled molecules (fluorescent dyes, small proteins and dextran), ranging from 0.5–500 kDa, can be introduced by VACNFs, and we demonstrate the use of the approach to track delivered probes from their site of introduction on the leaf to distal plant regions. VACNF arrays thus offer an attractive microdelivery method for the introduction of biomolecules and other probes into trees and potentially other types of plants. PMID:27119338

18F-FDG labeling of mesenchymal stem cells and multipotent adult progenitor cells for PET imaging: effects on ultrastructure and differentiation capacity.

PubMed

Wolfs, Esther; Struys, Tom; Notelaers, Tineke; Roberts, Scott J; Sohni, Abhishek; Bormans, Guy; Van Laere, Koen; Luyten, Frank P; Gheysens, Olivier; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2013-03-01

Because of their extended differentiation capacity, stem cells have gained great interest in the field of regenerative medicine. For the development of therapeutic strategies, more knowledge on the in vivo fate of these cells has to be acquired. Therefore, stem cells can be labeled with radioactive tracer molecules such as (18)F-FDG, a positron-emitting glucose analog that is taken up and metabolically trapped by the cells. The aim of this study was to optimize the radioactive labeling of mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs) in vitro with (18)F-FDG and to investigate the potential radiotoxic effects of this labeling procedure with a range of techniques, including transmission electron microscopy (TEM). Mouse MSCs and rat MAPCs were used for (18)F-FDG uptake kinetics and tracer retention studies. Cell metabolic activity, proliferation, differentiation and ultrastructural changes after labeling were evaluated using an Alamar Blue reagent, doubling time calculations and quantitative TEM, respectively. Additionally, mice were injected with MSCs and MAPCs prelabeled with (18)F-FDG, and stem cell biodistribution was investigated using small-animal PET. The optimal incubation period for (18)F-FDG uptake was 60 min. Significant early tracer washout was observed, with approximately 30%-40% of the tracer being retained inside the cells 3 h after labeling. Cell viability, proliferation, and differentiation capacity were not severely affected by (18)F-FDG labeling. No major changes at the ultrastructural level, considering mitochondrial length, lysosome size, the number of lysosomes, the number of vacuoles, and the average rough endoplasmic reticulum width, were observed with TEM. Small-animal PET experiments with radiolabeled MAPCs and MSCs injected intravenously in mice showed a predominant accumulation in the lungs and a substantial elution of (18)F-FDG from the cells. MSCs and MAPCs can be successfully labeled with (18)F-FDG for molecular imaging purposes. The main cellular properties are not rigorously affected. TEM confirmed that the cells' ultrastructural properties are not influenced by (18)F-FDG labeling. Small-animal PET studies confirmed the intracellular location of the tracer and the possibility of imaging injected prelabeled stem cell types in vivo. Therefore, direct labeling of MSCs and MAPCs with (18)F-FDG is a suitable technique to noninvasively assess cell delivery and early retention with PET.

Visualization of DNA molecules in time during electrophoresis

NASA Technical Reports Server (NTRS)

Lubega, Seth

1991-01-01

For several years individual DNA molecules have been observed and photographed during agarose gel electrophoresis. The DNA molecule is clearly the largest molecule known. Nevertheless, the largest molecule is still too small to be seen using a microscope. A technique developed by Morikawa and Yanagida has made it possible to visualize individual DNA molecules. When these long molecules are labeled with appropriate fluorescence dyes and observed under a fluorescence microscope, although it is not possible to directly visualize the local ultrastructure of the molecules, yet because they are long light emitting chains, their microscopic dynamical behavior can be observed. This visualization works in the same principle that enables one to observe a star through a telescope because it emits light against a dark background. The dynamics of individual DNA molecules migrating through agarose matrix during electrophoresis have been described by Smith et al. (1989), Schwartz and Koval (1989), and Bustamante et al. (1990). DNA molecules during agarose gel electrophoresis advance lengthwise thorough the gel in an extended configuration. They display an extension-contraction motion and tend to bunch up in their leading ends as the 'heads' find new pores through the gel. From time to time they get hooked on obstacles in the gel to form U-shaped configurations before they resume their linear configuration.

Surface plasmon resonance sensor for femtomolar detection of testosterone with water-compatible macroporous molecularly imprinted film.

PubMed

Zhang, Qingwen; Jing, Lijing; Zhang, Jinling; Ren, Yamin; Wang, Yang; Wang, Yi; Wei, Tianxin; Liedberg, Bo

2014-10-15

A novel water-compatible macroporous molecularly imprinted film (MIF) has been developed for rapid, sensitive, and label-free detection of small molecule testosterone in urine. The MIF was synthesized by photo copolymerization of monomers (methacrylic acid [MAA] and 2-hydroxyethyl methacrylate [HEMA]), cross-linker (ethylene glycol dimethacrylate, EGDMA), and polystyrene nanoparticles (PS NPs) in combination with template testosterone molecules. The PS NPs and template molecules were subsequently removed to form an MIF with macroporous structures and the specific recognition sites of testosterone. Incubation of artificial urine and human urine on the MIF and the non-imprinted film (NIF), respectively, indicated undetectable nonspecific adsorption. Accordingly, the MIF was applied on a surface plasmon resonance (SPR) sensor for the detection of testosterone in phosphate-buffered saline (PBS) and artificial urine with a limit of detection (LOD) down to 10(-15)g/ml. To the best of our knowledge, the LOD is considered as one of the lowest among the SPR sensors for the detection of small molecules. The control experiments performed with analogue molecules such as progesterone and estradiol demonstrated the good selectivity of this MIF for sensing testosterone. Furthermore, this MIF-based SPR sensor shows high stability and reproducibility over 8months of storage at room temperature, which is more robust than protein-based biosensors. Copyright © 2014 Elsevier Inc. All rights reserved.

Syringolin A selectively labels the 20 S proteasome in murine EL4 and wild-type and bortezomib-adapted leukaemic cell lines.

PubMed

Clerc, Jérôme; Florea, Bogdan I; Kraus, Marianne; Groll, Michael; Huber, Robert; Bachmann, André S; Dudler, Robert; Driessen, Christoph; Overkleeft, Herman S; Kaiser, Markus

2009-11-02

The natural product syringolin A (SylA) is a potent proteasome inhibitor with promising anticancer activities. To further investigate its potential as a lead structure, selectivity profiling with cell lysates was performed. At therapeutic concentrations, a rhodamine-tagged SylA derivative selectively bound to the 20 S proteasome active sites without detectable off-target labelling. Additional profiling with lysates of wild-type and bortezomib-adapted leukaemic cell lines demonstrated the retention of this proteasome target and subsite selectivity as well as potency even in clinically relevant cell lines. Our studies, therefore, propose that further development of SylA might indeed result in an improved small molecule for the treatment of leukaemia.

Elastin Shapes Small Molecule Distribution in Lymph Node Conduits.

PubMed

Lin, Yujia; Louie, Dante; Ganguly, Anutosh; Wu, Dequan; Huang, Peng; Liao, Shan

2018-05-01

The spatial and temporal Ag distribution determines the subsequent T cell and B cell activation at the distinct anatomical locations in the lymph node (LN). It is well known that LN conduits facilitate small Ag distribution in the LN, but the mechanism of how Ags travel along LN conduits remains poorly understood. In C57BL/6J mice, using FITC as a fluorescent tracer to study lymph distribution in the LN, we found that FITC preferentially colocalized with LN capsule-associated (LNC) conduits. Images generated using a transmission electron microscope showed that LNC conduits are composed of solid collagen fibers and are wrapped with fibroblastic cells. Superresolution images revealed that high-intensity FITC is typically colocalized with elastin fibers inside the LNC conduits. Whereas tetramethylrhodamine isothiocyanate appears to enter LNC conduits as effectively as FITC, fluorescently-labeled Alexa-555-conjugated OVA labels significantly fewer LNC conduits. Importantly, injection of Alexa-555-conjugated OVA with LPS substantially increases OVA distribution along elastin fibers in LNC conduits, indicating immune stimulation is required for effective OVA traveling along elastin in LN conduits. Finally, elastin fibers preferentially surround lymphatic vessels in the skin and likely guide fluid flow to the lymphatic vessels. Our studies demonstrate that fluid or small molecules are preferentially colocalized with elastin fibers. Although the exact mechanism of how elastin fibers regulate Ag trafficking remains to be explored, our results suggest that elastin can be a potentially new target to direct Ag distribution in the LN during vaccine design. Copyright © 2018 by The American Association of Immunologists, Inc.

Small-amplitude backbone motions of the spin-labeled lipopeptide trichogin GA IV in a lipid membrane as revealed by electron spin echo.

PubMed

Syryamina, Victoria N; Isaev, Nikolay P; Peggion, Cristina; Formaggio, Fernando; Toniolo, Claudio; Raap, Jan; Dzuba, Sergei A

2010-09-30

Trichogin GA IV is a lipopeptide antibiotic of fungal origin, which is known to be able to modify the membrane permeability. TOAC nitroxide spin-labeled analogues of this membrane active peptide were investigated in hydrated bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) by electron spin echo (ESE) spectroscopy. Because the TOAC nitroxide spin label is rigidly attached to the peptide backbone, it may report on the backbone orientational dynamics. The ESE signal in this system is observed below ∼150 K. Previously, three-pulse stimulated ESE was found to be sensitive to two types of orientational motion of spin-labeled POPC lipid bilayers at these temperatures. The first type is fast stochastic librations, with a correlation time on the nanosecond scale (which also manifests itself in a two-pulse primary ESE experiment). The second type is slow millisecond inertial rotations. In the present work, we find that at low molar peptide to lipid ratio (1:200), where the individual peptide molecules are randomly distributed at the membrane surface, the spin labels show only a fast type of motion. At the high molar peptide to lipid ratio (1:20), a slow motion is also observed. Because at this high concentration trichogin GA IV is known to change its orientation from the in-plane topology to the transmembrane disposition, the observed onset of a slow motion may be safely attributed to the dynamics of peptides, which are elongated along the lipid molecules of the membrane. The possible interrelation between this backbone rotational motion of the peptide antibiotic and the membrane leakage is discussed.

Consistent assignment of the vibrations of symmetric and asymmetric para-disubstituted benzene molecules

NASA Astrophysics Data System (ADS)

Andrejeva, Anna; Gardner, Adrian M.; Tuttle, William D.; Wright, Timothy G.

2016-03-01

We give a description of the phenyl-ring-localized vibrational modes of the ground states of the para-disubstituted benzene molecules including both symmetric and asymmetric cases. In line with others, we quickly conclude that the use of Wilson mode labels is misleading and ambiguous; we conclude the same regarding the related ones of Varsányi. Instead we label the modes consistently based upon the Mulliken (Herzberg) method for the modes of para-difluorobenzene (pDFB). Since we wish the labelling scheme to cover both symmetrically- and asymmetrically-substituted molecules, we apply the Mulliken labelling under C2v symmetry. By studying the variation of the vibrational wavenumbers with mass of the substituent, we are able to identify the corresponding modes across a wide range of molecules and hence provide consistent assignments. Particularly interesting are pairs of vibrations that evolve from in- and out-of-phase motions in pDFB to more localized modes in asymmetric molecules. We consider the para isomers of the following: the symmetric dihalobenzenes, xylene, hydroquinone, the asymmetric dihalobenzenes, halotoluenes, halophenols and cresol.

Combination of Mass Signal Amplification and Isotope-Labeled Alkanethiols for the Multiplexed Detection of miRNAs.

PubMed

Kang, Hyunook; Hong, Seol-Hye; Sung, Jiha; Yeo, Woon-Seok

2017-08-04

We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope-labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am-tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am-tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am-tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO 4 concentrations. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis and preliminary PET imaging of 11C and 18F isotopologues of the ROS1/ALK inhibitor lorlatinib

NASA Astrophysics Data System (ADS)

Collier, Thomas Lee; Normandin, Marc D.; Stephenson, Nickeisha A.; Livni, Eli; Liang, Steven H.; Wooten, Dustin W.; Esfahani, Shadi A.; Stabin, Michael G.; Mahmood, Umar; Chen, Jianqing; Wang, Wei; Maresca, Kevin; Waterhouse, Rikki N.; El Fakhri, Georges; Richardson, Paul; Vasdev, Neil

2017-06-01

Lorlatinib (PF-06463922) is a next-generation small-molecule inhibitor of the orphan receptor tyrosine kinase c-ros oncogene 1 (ROS1), which has a kinase domain that is physiologically related to anaplastic lymphoma kinase (ALK), and is undergoing Phase I/II clinical trial investigations for non-small cell lung cancers. An early goal is to measure the concentrations of this drug in brain tumour lesions of lung cancer patients, as penetration of the blood-brain barrier is important for optimal therapeutic outcomes. Here we prepare both 11C- and 18F-isotopologues of lorlatinib to determine the biodistribution and whole-body dosimetry assessments by positron emission tomography (PET). Non-traditional radiolabelling strategies are employed to enable an automated multistep 11C-labelling process and an iodonium ylide-based radiofluorination. Carbon-11-labelled lorlatinib is routinely prepared with good radiochemical yields and shows reasonable tumour uptake in rodents. PET imaging in non-human primates confirms that this radiotracer has high brain permeability.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

NASA Astrophysics Data System (ADS)

Duman, M.; Pfleger, M.; Zhu, R.; Rankl, C.; Chtcheglova, L. A.; Neundlinger, I.; Bozna, B. L.; Mayer, B.; Salio, M.; Shepherd, D.; Polzella, P.; Moertelmaier, M.; Kada, G.; Ebner, A.; Dieudonne, M.; Schütz, G. J.; Cerundolo, V.; Kienberger, F.; Hinterdorfer, P.

2010-03-01

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ~ 25 to ~ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

PubMed

Duman, M; Pfleger, M; Zhu, R; Rankl, C; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Mayer, B; Salio, M; Shepherd, D; Polzella, P; Moertelmaier, M; Kada, G; Ebner, A; Dieudonne, M; Schütz, G J; Cerundolo, V; Kienberger, F; Hinterdorfer, P

2010-03-19

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Superresolution microscopy with transient binding.

PubMed

Molle, Julia; Raab, Mario; Holzmeister, Susanne; Schmitt-Monreal, Daniel; Grohmann, Dina; He, Zhike; Tinnefeld, Philip

2016-06-01

For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Synthesis of a Far-Red Photoactivatable Silicon-Containing Rhodamine for Super-Resolution Microscopy.

PubMed

Grimm, Jonathan B; Klein, Teresa; Kopek, Benjamin G; Shtengel, Gleb; Hess, Harald F; Sauer, Markus; Lavis, Luke D

2016-01-26

The rhodamine system is a flexible framework for building small-molecule fluorescent probes. Changing N-substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si-containing analogue of Q-rhodamine. This probe is the first example of a "caged" Si-rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red-shifted to allow multicolor imaging. The dye is a useful label for super-resolution imaging and constitutes a new scaffold for far-red fluorogenic molecules. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

One-to-one quantum dot-labeled single long DNA probes.

PubMed

He, Shibin; Huang, Bi-Hai; Tan, Junjun; Luo, Qing-Ying; Lin, Yi; Li, Jun; Hu, Yong; Zhang, Lu; Yan, Shihan; Zhang, Qi; Pang, Dai-Wen; Li, Lijia

2011-08-01

Quantum dots (QDs) have been received most attention due to their unique properties. Constructing QDs conjugated with certain number of biomolecules is considered as one of the most important research goals in nanobiotechnology. In this study, we report polymerase chain reaction (PCR) amplification of primer oligonucleotides bound to QDs, termed as QD-based PCR. Characterization of QD-based PCR products by gel electrophoresis and atomic force microscopy showed that QD-labeled long DNA strands were synthesized and only a single long DNA strand was conjugated with a QD. The QD-based PCR products still kept fluorescence properties. Moreover, the one-to-one QD-labeled long DNA conjugates as probes could detect a single-copy gene on maize chromosomes by fluorescence in situ hybridization. Labeling a single QD to a single long DNA will make detection of small single-copy DNA fragments, quantitative detection and single molecule imaging come true by nanotechnology, and it will promote medical diagnosis and basic biological research as well as nano-material fabrication. Copyright © 2011 Elsevier Ltd. All rights reserved.

Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling.

PubMed

Hoffmann, Celine; Leroy-Dudal, Johanne; Patel, Salima; Gallet, Olivier; Pauthe, Emmanuel

2008-01-01

Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.

DNA-length-dependent quenching of fluorescently labeled iron oxide nanoparticles with gold, graphene oxide and MoS2 nanostructures.

PubMed

Balcioglu, Mustafa; Rana, Muhit; Robertson, Neil; Yigit, Mehmet V

2014-08-13

We controlled the fluorescence emission of a fluorescently labeled iron oxide nanoparticle using three different nanomaterials with ultraefficient quenching capabilities. The control over the fluorescence emission was investigated via spacing introduced by the surface-functionalized single-stranded DNA molecules. DNA molecules were conjugated on different templates, either on the surface of the fluorescently labeled iron oxide nanoparticles or gold and nanographene oxide. The efficiency of the quenching was determined and compared with various fluorescently labeled iron oxide nanoparticle and nanoquencher combinations using DNA molecules with three different lengths. We have found that the template for DNA conjugation plays significant role on quenching the fluorescence emission of the fluorescently labeled iron oxide nanoparticles. We have observed that the size of the DNA controls the quenching efficiency when conjugated only on the fluorescently labeled iron oxide nanoparticles by setting a spacer between the surfaces and resulting change in the hydrodynamic size. The quenching efficiency with 12mer, 23mer and 36mer oligonucleotides decreased to 56%, 54% and 53% with gold nanoparticles, 58%, 38% and 32% with nanographene oxide, 46%, 38% and 35% with MoS2, respectively. On the other hand, the presence, not the size, of the DNA molecules on the other surfaces quenched the fluorescence significantly with different degrees. To understand the effect of the mobility of the DNA molecules on the nanoparticle surface, DNA molecules were attached to the surface with two different approaches. Covalently immobilized oligonucleotides decreased the quenching efficiency of nanographene oxide and gold nanoparticles to ∼22% and ∼21%, respectively, whereas noncovalently adsorbed oligonucleotides decreased it to ∼25% and ∼55%, respectively. As a result, we have found that each nanoquencher has a powerful quenching capability against a fluorescent nanoparticle, which can be tuned with surface functionalized DNA molecules.

The multi-state energy landscape of the SAM-I riboswitch: A single-molecule Förster resonance energy transfer spectroscopy study

NASA Astrophysics Data System (ADS)

Manz, Christoph; Kobitski, Andrei Yu.; Samanta, Ayan; Jäschke, Andres; Nienhaus, G. Ulrich

2018-03-01

RNA (ribonucleic acid) molecules are highly flexible biopolymers fluctuating at physiological temperatures among many different conformations that are represented by minima in a hierarchical conformational free energy landscape. Here we have employed single-molecule FRET (smFRET) to explore the energy landscape of the B. subtilis yitJ SAM-I riboswitch (RS). In this small RNA molecule, specific binding of an S-adenosyl-L-methionine (SAM) ligand in the aptamer domain regulates gene expression by inducing structural changes in another domain, the expression platform, causing transcription termination by the RNA polymerase. We have measured smFRET histograms over wide ranges of Mg2+ concentration for three RS variants that were specifically labeled with fluorescent dyes on different sites. In the analysis, different conformations are associated with discrete Gaussian model distributions, which are typically fairly broad on the FRET efficiency scale and thus can be extremely challenging to unravel due to their mutual overlap. Our earlier work on two SAM-I RS variants revealed four major conformations. By introducing a global fitting procedure which models both the Mg2+ concentration dependencies of the fractional populations and the average FRET efficiencies of the individual FRET distributions according to Mg2+ binding isotherms, we were able to consistently describe the histogram data of both variants at all studied Mg2+ concentrations. With the third FRET-labeled variant, however, we found significant deviations when applying the four-state model to the data. This can arise because the different FRET labeling of the new variant allows two states to be distinguished that were previously not separable due to overlap. Indeed, the resulting five-state model presented here consistently describes the smFRET histograms of all three variants as well as their variations with Mg2+ concentration. We also performed a triangulation of the donor position for two of the constructs to explore how the expression platform is oriented with respect to the aptamer.

Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein-Protein Interaction.

PubMed

Liu, Degang; Xu, David; Liu, Min; Knabe, William Eric; Yuan, Cai; Zhou, Donghui; Huang, Mingdong; Meroueh, Samy O

2017-03-28

Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.

The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy.

PubMed

Drabik, Dominik; Przybyło, Magda; Sikorski, Aleksander; Langner, Marek

2016-03-01

Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or βBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.

Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

PubMed

Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

2016-04-01

Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

Evaluation of genotoxicity testing of FDA approved large molecule therapeutics.

PubMed

Sawant, Satin G; Fielden, Mark R; Black, Kurt A

2014-10-01

Large molecule therapeutics (MW>1000daltons) are not expected to enter the cell and thus have reduced potential to interact directly with DNA or related physiological processes. Genotoxicity studies are therefore not relevant and typically not required for large molecule therapeutic candidates. Regulatory guidance supports this approach; however there are examples of marketed large molecule therapeutics where sponsors have conducted genotoxicity studies. A retrospective analysis was performed on genotoxicity studies of United States FDA approved large molecule therapeutics since 1998 identified through the Drugs@FDA website. This information was used to provide a data-driven rationale for genotoxicity evaluations of large molecule therapeutics. Fifty-three of the 99 therapeutics identified were tested for genotoxic potential. None of the therapeutics tested showed a positive outcome in any study except the peptide glucagon (GlucaGen®) showing equivocal in vitro results, as stated in the product labeling. Scientific rationale and data from this review indicate that testing of a majority of large molecule modalities do not add value to risk assessment and support current regulatory guidance. Similarly, the data do not support testing of peptides containing only natural amino acids. Peptides containing non-natural amino acids and small molecules in conjugated products may need to be tested. Copyright © 2014 Elsevier Inc. All rights reserved.

A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells.

PubMed

Geng, Yijie; Feng, Bradley

2016-07-01

The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen, we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure, while cell surface marker analyses revealed a VE-cadherin(+)CD31(+)CD34(+)KDR(+)CD43(-) putative endothelial progenitor population. Furthermore, molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL(+) multi-cellular modules and a VEGFR3(+) sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation.

A Phytochemical-Sensing Strategy Based on Mass Spectrometry Imaging and Metabolic Profiling for Understanding the Functionality of the Medicinal Herb Green Tea.

PubMed

Fujimura, Yoshinori; Miura, Daisuke; Tachibana, Hirofumi

2017-09-27

Low-molecular-weight phytochemicals have health benefits and reduce the risk of diseases, but the mechanisms underlying their activities have remained elusive because of the lack of a methodology that can easily visualize the exact behavior of such small molecules. Recently, we developed an in situ label-free imaging technique, called mass spectrometry imaging, for visualizing spatially-resolved biotransformations based on simultaneous mapping of the major bioactive green tea polyphenol and its phase II metabolites. In addition, we established a mass spectrometry-based metabolic profiling technique capable of evaluating the bioactivities of diverse green tea extracts, which contain multiple phytochemicals, by focusing on their compositional balances. This methodology allowed us to simultaneously evaluate the relative contributions of the multiple compounds present in a multicomponent system to its bioactivity. This review highlights small molecule-sensing techniques for visualizing the complex behaviors of herbal components and linking such information to an enhanced understanding of the functionalities of multicomponent medicinal herbs.

Porous photonic crystal external cavity laser biosensor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Qinglan; Peh, Jessie; Hergenrother, Paul J.

2016-08-15

We report the design, fabrication, and testing of a photonic crystal (PC) biosensor structure that incorporates a porous high refractive index TiO{sub 2} dielectric film that enables immobilization of capture proteins within an enhanced surface-area volume that spatially overlaps with the regions of resonant electromagnetic fields where biomolecular binding can produce the greatest shifts in photonic crystal resonant wavelength. Despite the nanoscale porosity of the sensor structure, the PC slab exhibits narrowband and high efficiency resonant reflection, enabling the structure to serve as a wavelength-tunable element of an external cavity laser. In the context of sensing small molecule interactions withmore » much larger immobilized proteins, we demonstrate that the porous structure provides 3.7× larger biosensor signals than an equivalent nonporous structure, while the external cavity laser (ECL) detection method provides capability for sensing picometer-scale shifts in the PC resonant wavelength caused by small molecule binding. The porous ECL achieves a record high figure of merit for label-free optical biosensors.« less

Fab fragment labeled with ICG-derivative for detecting digestive tract cancer.

PubMed

Yano, Hiromi; Muguruma, Naoki; Ito, Susumu; Aoyagi, Eriko; Kimura, Tetsuo; Imoto, Yoshitaka; Cao, Jianxin; Inoue, Shohei; Sano, Shigeki; Nagao, Yoshimitsu; Kido, Hiroshi

2006-09-01

In previous studies, we generated infrared ray fluorescence-labeled monoclonal antibodies and developed an infrared ray fluorescence endoscope capable of detecting the monoclonal antibodies to establish a novel diagnostic technique for gastrointestinal cancer. Although the whole IgG molecule has commonly been used for preparation of labeled antibodies, labeled IgG displays insufficient sensitivity and specificity, probably resulting from non-specific binding of the Fc fragment to target cells or interference between fluorochromes on the identical labeled antibody, which might be caused by molecular structure. In this in vitro study, we characterized an Fc-free fluorescence-labeled Fab fragment, which was expected to yield more specific binding to target cells than the whole IgG molecule. An anti-mucin antibody and ICG-ATT, an ICG derivative, were used as the labeled antibody and labeling compound, respectively. Paraffin sections of excised gastric cancer tissues were subjected to staining. The labeled whole IgG molecule (ICG-ATT-labeled IgG) and the labeled Fab fragment (ICG-ATT-labeled Fab) were prepared according to a previous report, and the fluorescence properties, antibody activities, and features of fluorescence microscope images obtained from paraffin sections were compared. Both ICG-ATT-labeled Fab and ICG-ATT-labeled IgG were excited by a near infrared ray of 766nm, and maximum emission occurred at 804nm. Antibody activities of ICG-ATT-labeled Fab were shown to be similar to those of unlabeled anti-MUC1 antibody. The fluorescence intensity obtained from paraffin sections of excised gastric cancer tissues revealed a tendency to be greater with ICG-ATT-labeled Fab than with ICG-ATT-labeled IgG. The infrared ray fluorescence-labeled Fab fragment was likely to be more specific than the conventionally labeled antibodies. Fragmentation of antibodies is considered to contribute to improved sensitivity and specificity of labeled antibodies for detection of micro gastrointestinal cancers.

Incorporation of functionalized gold nanoparticles into nanofibers for enhanced attachment and differentiation of mammalian cells

PubMed Central

2012-01-01

Background Electrospun nanofibers have been widely used as substrata for mammalian cell culture owing to their structural similarity to natural extracellular matrices. Structurally consistent electrospun nanofibers can be produced with synthetic polymers but require chemical modification to graft cell-adhesive molecules to make the nanofibers functional. Development of a facile method of grafting functional molecules on the nanofibers will contribute to the production of diverse cell type-specific nanofiber substrata. Results Small molecules, peptides, and functionalized gold nanoparticles were successfully incorporated with polymethylglutarimide (PMGI) nanofibers through electrospinning. The PMGI nanofibers functionalized by the grafted AuNPs, which were labeled with cell-adhesive peptides, enhanced HeLa cell attachment and potentiated cardiomyocyte differentiation of human pluripotent stem cells. Conclusions PMGI nanofibers can be functionalized simply by co-electrospinning with the grafting materials. In addition, grafting functionalized AuNPs enable high-density localization of the cell-adhesive peptides on the nanofiber. The results of the present study suggest that more cell type-specific synthetic substrata can be fabricated with molecule-doped nanofibers, in which diverse functional molecules are grafted alone or in combination with other molecules at different concentrations. PMID:22686683

Rapid Characterization of Magnetic Moment of Cells for Magnetic Separation

PubMed Central

Ooi, Chinchun; Earhart, Christopher M.; Wilson, Robert J.; Wang, Shan X.

2014-01-01

NCI-H1650 lung cancer cell lines labeled with magnetic nanoparticles via the Epithelial Cell Adhesion Molecule (EpCAM) antigen were previously shown to be captured at high efficiencies by a microfabricated magnetic sifter. If fine control and optimization of the magnetic separation process is to be achieved, it is vital to be able to characterize the labeled cells’ magnetic moment rapidly. We have thus adapted a rapid prototyping method to obtain the saturation magnetic moment of these cells. This method utilizes a cross-correlation algorithm to analyze the cells’ motion in a simple fluidic channel to obtain their magnetophoretic velocity, and is effective even when the magnetic moments of cells are small. This rapid characterization is proven useful in optimizing our microfabricated magnetic sifter procedures for magnetic cell capture. PMID:24771946

Flow cytometry of HEK 293T cells interacting with polyelectrolyte multilayer capsules containing fluorescein-labeled poly(acrylic acid) as a pH sensor.

PubMed

Reibetanz, Uta; Halozan, David; Brumen, Milan; Donath, Edwin

2007-06-01

Polyelectrolyte multilayer sensor capsules, 5 microm in diameter, which contained fluorescein-labeled poly(acrylic acid) (PAAAF) as pH-sensitive reporter molecules, were fabricated and employed to explore their endocytotic uptake into HEK 293T cells by flow cytometry. The percentage of capsules residing in the endolysosomal compartment was estimated from the fluorescence intensity decrease caused by acidification. Capsules attached to the extracellular surface of the plasma membrane were identified by trypan blue quenching. The number of capsules in the cytoplasm was rather small, being below the detection limit of the method. The advantages of polyelectrolyte multilayer capsules are that the fluorophore is protected from interaction with cellular compartments and that the multilayer can be equipped with additional functions.

The separation between the 5'-3' ends in long RNA molecules is short and nearly constant.

PubMed

Leija-Martínez, Nehemías; Casas-Flores, Sergio; Cadena-Nava, Rubén D; Roca, Joan A; Mendez-Cabañas, José A; Gomez, Eduardo; Ruiz-Garcia, Jaime

2014-12-16

RNA molecules play different roles in coding, decoding and gene expression regulation. Such roles are often associated to the RNA secondary or tertiary structures. The folding dynamics lead to multiple secondary structures of long RNA molecules, since an RNA molecule might fold into multiple distinct native states. Despite an ensemble of different structures, it has been theoretically proposed that the separation between the 5' and 3' ends of long single-stranded RNA molecules (ssRNA) remains constant, independent of their base content and length. Here, we present the first experimental measurements of the end-to-end separation in long ssRNA molecules. To determine this separation, we use single molecule Fluorescence Resonance Energy Transfer of fluorescently end-labeled ssRNA molecules ranging from 500 to 5500 nucleotides in length, obtained from two viruses and a fungus. We found that the end-to-end separation is indeed short, within 5-9 nm. It is remarkable that the separation of the ends of all RNA molecules studied remains small and similar, despite the origin, length and differences in their secondary structure. This implies that the ssRNA molecules are 'effectively circularized' something that might be a general feature of RNAs, and could result in fine-tuning for translation and gene expression regulation. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality

PubMed Central

Yu, Haixiang; Yang, Weijuan; Alkhamis, Obtin; Canoura, Juan; Yang, Kyung-Ae; Xiao, Yi

2018-01-01

Abstract Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dye. We first demonstrate that diethylthiatricarbocyanine (Cy7) can stack into DNA three-way junctions (TWJs) in a sequence-independent fashion, greatly altering the dye's absorbance spectrum. We then design a TWJ-containing structured library and isolate an aptamer against 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone that is an emerging drug of abuse. This aptamer intrinsically binds Cy7 within its TWJ domain, but MDPV efficiently displaces the dye, resulting in a change in absorbance within seconds. This assay is label-free, and detects nanomolar concentrations of MDPV. It also recognizes other synthetic cathinones, offering the potential to detect newly-emerging designer drugs, but does not detect structurally-similar non-cathinone compounds or common cutting agents. Moreover, we demonstrate that the Cy7-displacement colorimetric assay is more sensitive than a conventional strand-displacement fluorescence assay. We believe our strategy offers an effective generalized approach for the development of sensitive dye-displacement colorimetric assays for other small-molecule targets. PMID:29361056

Fluorophore Labeled Kinase Detects Ligands That Bind within the MAPK Insert of p38α Kinase

PubMed Central

Termathe, Martin; Grütter, Christian; Rabiller, Matthias; van Otterlo, Willem A. L.; Rauh, Daniel

2012-01-01

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway. PMID:22768308

Development of a miRNA surface-enhanced Raman scattering assay using benchtop and handheld Raman systems

NASA Astrophysics Data System (ADS)

Schechinger, Monika; Marks, Haley; Locke, Andrea; Choudhury, Mahua; Cote, Gerard

2018-01-01

DNA-functionalized nanoparticles, when paired with surface-enhanced Raman spectroscopy (SERS), can rapidly detect microRNA. However, widespread use of this approach is hindered by drawbacks associated with large and expensive benchtop Raman microscopes. MicroRNA-17 (miRNA-17) has emerged as a potential epigenetic indicator of preeclampsia, a condition that occurs during pregnancy. Biomarker detection using an SERS point-of-care device could enable prompt diagnosis and prevention as early as the first trimester. Recently, strides have been made in developing portable Raman systems for field applications. An SERS assay for miRNA-17 was assessed and translated from traditional benchtop Raman microscopes to a handheld system. Three different photoactive molecules were compared as potential Raman reporter molecules: a chromophore, malachite green isothiocyanate (MGITC), a fluorophore, tetramethylrhodamine isothiocyanate, and a polarizable small molecule 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). For the benchtop Raman microscope, the DTNB-labeled assay yielded the greatest sensitivity under 532-nm laser excitation, but the MGITC-labeled assay prevailed at 785 nm. Conversely, DTNB was preferable for the miniaturized 785-nm Raman system. This comparison showed significant SERS enhancement variation in response to 1-nM miRNA-17, implying that the sensitivity of the assay may be more heavily dependent on the excitation wavelength, instrumentation, and Raman reporter chosen than on the plasmonic coupling from DNA/miRNA-mediated nanoparticle assemblies.

Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides.

PubMed

Zhao, Qiang; Lv, Qin; Wang, Hailin

2015-08-15

We previously reported a fluorescence anisotropy (FA) approach for small molecules using tetramethylrhodamine (TMR) labeled aptamer. It relies on target-binding induced change of intramolecular interaction between TMR and guanine (G) base. TMR-labeling sites are crucial for this approach. Only terminal ends and thymine (T) bases could be tested for TMR labeling in our previous work, possibly causing limitation in analysis of different targets with this FA strategy. Here, taking the analysis of adenosine triphosphate (ATP) as an example, we demonstrated a success of conjugating TMR on other bases of aptamer adenine (A) or cytosine (C) bases and an achievement of full mapping various labeling sites of aptamers. We successfully constructed aptamer fluorescence anisotropy (FA) sensors for adenosine triphosphate (ATP). We conjugated single TMR on adenine (A), cytosine (C), or thymine (T) bases or terminals of a 25-mer aptamer against ATP and tested FA responses of 14 TMR-labeled aptamer to ATP. The aptamers having TMR labeled on the 16th base C or 23rd base A were screened out and exhibited significant FA-decreasing or FA-increasing responses upon ATP, respectively. These two favorable TMR-labeled aptamers enabled direct FA sensing ATP with a detection limit of 1 µM and the analysis of ATP in diluted serum. The comprehensive screening various TMR labeling sites of aptamers facilitates the successful construction of FA sensors using TMR-labeled aptamers. It will expand application of TMR-G interaction based aptamer FA strategy to a variety of targets. Copyright © 2015 Elsevier B.V. All rights reserved.

Cationized Magnetoferritin Enables Rapid Labeling and Concentration of Gram-Positive and Gram-Negative Bacteria in Magnetic Cell Separation Columns

PubMed Central

Spencer, J.; Schwarzacher, W.

2016-01-01

ABSTRACT In order to identify pathogens rapidly and reliably, bacterial capture and concentration from large sample volumes into smaller ones are often required. Magnetic labeling and capture of bacteria using a magnetic field hold great promise for achieving this goal, but the current protocols have poor capture efficiency. Here, we present a rapid and highly efficient approach to magnetic labeling and capture of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria using cationized magnetoferritin (cat-MF). Magnetic labeling was achieved within a 1-min incubation period with cat-MF, and 99.97% of the labeled bacteria were immobilized in commercially available magnetic cell separation (MACS) columns. Longer incubation times led to more efficient capture, with S. aureus being immobilized to a greater extent than E. coli. Finally, low numbers of magnetically labeled E. coli bacteria (<100 CFU per ml) were immobilized with 100% efficiency and concentrated 7-fold within 15 min. Therefore, our study provides a novel protocol for rapid and highly efficient magnetic labeling, capture, and concentration of both Gram-positive and Gram-negative bacteria. IMPORTANCE Antimicrobial resistance (AMR) is a significant global challenge. Rapid identification of pathogens will retard the spread of AMR by enabling targeted treatment with suitable agents and by reducing inappropriate antimicrobial use. Rapid detection methods based on microfluidic devices require that bacteria are concentrated from large volumes into much smaller ones. Concentration of bacteria is also important to detect low numbers of pathogens with confidence. Here, we demonstrate that magnetic separation columns capture small amounts of bacteria with 100% efficiency. Rapid magnetization was achieved by exposing bacteria to cationic magnetic nanoparticles, and magnetized bacteria were concentrated 7-fold inside the column. Thus, bacterial capture and concentration were achieved within 15 min. This approach could be extended to encompass the capture and concentration of specific pathogens, for example, by functionalizing magnetic nanoparticles with antibodies or small molecule probes. PMID:27060124

Cationized Magnetoferritin Enables Rapid Labeling and Concentration of Gram-Positive and Gram-Negative Bacteria in Magnetic Cell Separation Columns.

PubMed

Correia Carreira, S; Spencer, J; Schwarzacher, W; Seddon, A M

2016-06-15

In order to identify pathogens rapidly and reliably, bacterial capture and concentration from large sample volumes into smaller ones are often required. Magnetic labeling and capture of bacteria using a magnetic field hold great promise for achieving this goal, but the current protocols have poor capture efficiency. Here, we present a rapid and highly efficient approach to magnetic labeling and capture of both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria using cationized magnetoferritin (cat-MF). Magnetic labeling was achieved within a 1-min incubation period with cat-MF, and 99.97% of the labeled bacteria were immobilized in commercially available magnetic cell separation (MACS) columns. Longer incubation times led to more efficient capture, with S. aureus being immobilized to a greater extent than E. coli Finally, low numbers of magnetically labeled E. coli bacteria (<100 CFU per ml) were immobilized with 100% efficiency and concentrated 7-fold within 15 min. Therefore, our study provides a novel protocol for rapid and highly efficient magnetic labeling, capture, and concentration of both Gram-positive and Gram-negative bacteria. Antimicrobial resistance (AMR) is a significant global challenge. Rapid identification of pathogens will retard the spread of AMR by enabling targeted treatment with suitable agents and by reducing inappropriate antimicrobial use. Rapid detection methods based on microfluidic devices require that bacteria are concentrated from large volumes into much smaller ones. Concentration of bacteria is also important to detect low numbers of pathogens with confidence. Here, we demonstrate that magnetic separation columns capture small amounts of bacteria with 100% efficiency. Rapid magnetization was achieved by exposing bacteria to cationic magnetic nanoparticles, and magnetized bacteria were concentrated 7-fold inside the column. Thus, bacterial capture and concentration were achieved within 15 min. This approach could be extended to encompass the capture and concentration of specific pathogens, for example, by functionalizing magnetic nanoparticles with antibodies or small molecule probes. Copyright © 2016 Correia Carreira et al.

Probes labelled with energy transfer coupled dyes

DOEpatents

Mathies, R.A.; Glazer, A.; Ju, J.

1997-11-18

Compositions are provided comprising sets of fluorescent labels carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids. 7 figs.

Fluorescent labels and their use in separations

DOEpatents

Mathies, Richard A.; Glazer, Alexander; Ju, Jingyue

1997-01-01

Compositions are provided comprising sets of fluorescent labels carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids.

Probes labelled with energy transfer coupled dyes

DOEpatents

Mathies, Richard A.; Glazer, Alexander; Ju, Jingyue

1997-01-01

Compositions are provided comprising sets of fluorescent labels carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids.

Enzymatic production of single-molecule FISH and RNA capture probes.

PubMed

Gaspar, Imre; Wippich, Frank; Ephrussi, Anne

2017-10-01

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed. © 2017 Gaspar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Methods for the selective detection of alkyne-presenting molecules and related compositions and systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valdez, Carlos A.; Vu, Alexander K.

Provided herein are methods for selectively detecting an alkyne-presenting molecule in a sample and related detection reagents, compositions, methods and systems. The methods include contacting a detection reagent with the sample for a time and under a condition to allow binding of the detection reagent to the one or more alkyne-presenting molecules possibly present in the matrix to the detection reagent. The detection reagent includes an organic label moiety presenting an azide group. The binding of the azide group to the alkyne-presenting molecules results in emission of a signal from the organic label moiety.

Concentration methods for high-resolution THz spectroscopy of nucleic-acid biomolecules and crystals

NASA Astrophysics Data System (ADS)

Brown, E. R.; Zhang, W.; Mendoza, E. A.; Kuznetsova, Y.; Brueck, S. R. J.; Rahman, M.; Norton, M. L.

2012-03-01

Biomolecules can exhibit low-lying vibrational modes in the THz region which are detectable in transmission given a strong molecular dipole moment and optical depth, and a spectrometer of adequate sensitivity. The nucleic acids are particularly interesting because of applications such as label-free gene assay, bio-agent detection, etc. However for nucleic acids, sample preparation and THz coupling are of paramount importance because of the strong absorption by liquid water and the small concentration of molecules present in physiological solutions. Concentration methods become necessary to make the THz vibrational modes detectable, either by concentrating the nucleic-acid sample itself in a small volume but large area, or by concentrating the THz radiation down to the volume of the sample. This paper summarizes one type of the first method: nanofluidic channel arrays for biological nucleic acids; and two types of the second method: (1) a circular-waveguide pinhole, and (2) a circular-waveguide, conical-horn coupling structure, both for DNA crystals. The first method has been demonstrated on a very short artificial nucleic acid [small-interfering (si) RNA (17-to-25 bp)] and a much longer, biological molecule [Lambda-phage DNA (48.5 kbp)]. The second method has been demonstrated on small (~100 micron) single crystals of DNA grown by the sitting-drop method.

Single-molecule imaging at high fluorophore concentrations by local activation of dye

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geertsema, Hylkje J.; Mangel, Walter F.; Schulte, Aartje C.

Single-molecule fluorescence microscopy is a powerful approach to observe biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual, labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Then, making use ofmore » short-distance energy-transfer mechanisms, the fluorescence from only those proteins bound to their substrate are selectively activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 (IFI16) with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease (pVIc-AVP) on DNA in the presence of a background of hundreds of nM Cy5 fluorophore.« less

Single-molecule imaging at high fluorophore concentrations by local activation of dye

DOE PAGES

Geertsema, Hylkje J.; Mangel, Walter F.; Schulte, Aartje C.; ...

2015-02-17

Single-molecule fluorescence microscopy is a powerful approach to observe biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual, labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Then, making use ofmore » short-distance energy-transfer mechanisms, the fluorescence from only those proteins bound to their substrate are selectively activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 (IFI16) with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease (pVIc-AVP) on DNA in the presence of a background of hundreds of nM Cy5 fluorophore.« less

Bioorthogonal Chemical Imaging for Biomedicine

NASA Astrophysics Data System (ADS)

Min, Wei

2017-06-01

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because relatively bulky fluorescent labels could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, we have developed a bioorthogonal chemical imaging platform. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes, nitriles and stable isotopes including 2H and 13C), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, multiplicity and biocompatibility for imaging small biomolecules in live systems including tissues and organisms. Exciting biomedical applications such as imaging fatty acid metabolism related to lipotoxicity, glucose uptake and metabolism, drug trafficking, protein synthesis, DNA replication, protein degradation, RNA synthesis and tumor metabolism will be presented. This bioorthogonal chemical imaging platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, further chemical and spectroscopic strategies allow for multicolor bioorthogonal chemical imaging, a valuable technique in the era of "omics". We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species, bringing small molecules under the illumination of modern light microscopy.

Agonist-Directed Desensitization of the β2-Adrenergic Receptor

PubMed Central

Goral, Vasiliy; Jin, Yan; Sun, Haiyan; Ferrie, Ann M.; Wu, Qi; Fang, Ye

2011-01-01

The β2-adrenergic receptor (β2AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the β2AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective β2AR desensitization at the whole cell level. PMID:21541288

A protocol for a systematic review to identify allergenic tree nuts and the molecules responsible for their allergenic properties.

PubMed

Javed, Bushra; Padfield, Philip; Sperrin, Matthew; Simpson, Angela; Mills, E N Clare

2017-08-01

Food regulations require that tree nuts and derived ingredients are included on food labels in order to help individuals with IgE-mediated allergies to avoid them. However, there is no consensus regarding which tree nut species should be included in this definition and specified on food labels. Allergen detection methods used for monitoring foods target allergen molecules, but it not clear which are the most relevant molecules to choose. A modified population-exposure-comparators-outcome (PECO) approach has been developed to systematically review the evidence regarding (1) which allergenic tree nuts should be included in food allergen labelling lists and (2) which are the clinically relevant allergens which should be used as analytical targets. A search strategy and criteria against which the evidence will be evaluated have been developed. The resulting evidence will be used to rank tree nuts with regards their ability to cause IgE-mediated allergies, and allergen molecules regarding their capacity to elicit an allergic reaction. The results of the systematic review will enable risk assessors and managers to identify tree nut species that should be included in food allergen labelling lists and ensure analytical methods for determination of allergens in foods are targeting appropriate molecules. Copyright © 2017. Published by Elsevier Ltd.

Single-Molecule Plasmon Sensing: Current Status and Future Prospects

PubMed Central

2017-01-01

Single-molecule detection has long relied on fluorescent labeling with high quantum-yield fluorophores. Plasmon-enhanced detection circumvents the need for labeling by allowing direct optical detection of weakly emitting and completely nonfluorescent species. This review focuses on recent advances in single molecule detection using plasmonic metal nanostructures as a sensing platform, particularly using a single particle–single molecule approach. In the past decade two mechanisms for plasmon-enhanced single-molecule detection have been demonstrated: (1) by plasmonically enhancing the emission of weakly fluorescent biomolecules, or (2) by monitoring shifts of the plasmon resonance induced by single-molecule interactions. We begin with a motivation regarding the importance of single molecule detection, and advantages plasmonic detection offers. We describe both detection mechanisms and discuss challenges and potential solutions. We finalize by highlighting the exciting possibilities in analytical chemistry and medical diagnostics. PMID:28762723

Immunoglobulin subunits of murine B lymphocytes: structure and associations with other membrane proteins.

PubMed Central

Vogel, L; Haustein, D

1989-01-01

The Ig subunit structure of murine B lymphocytes was studied by employing different radiolabelling techniques in combination with chemical cross-linking. The main membrane structure of IgM was a half molecule that was disulphide-linked to proteins with MW 30,000, 45,000 and 55,000, respectively. Small amounts of mu 2L2, microL disulphide-linked to a protein with MW 50,000, and free microL were also detected. The main IgD structures were half molecules disulphide-linked to two proteins with MW 14,000 and two proteins with MW 16,000. Furthermore, IgD half molecules disulphide-linked to a protein with MW 16,000 and free half molecules could be demonstrated. Labelling with hydrophobic reagents showed that all Ig molecules and the protein with MW 50,000, linked to microL, penetrated the lipid bilayer, whereas the other IgM- and IgD-linked proteins probably did not. Additional proteins which were associated exclusively with IgM were detected by chemical cross-linking. These findings offer new possibilities for the investigation of the function(s) of antigen receptors on B cells. Images Figure 1 Figure 2 Figure 4 Figure 5 PMID:2787780

Methods and compositions for efficient nucleic acid sequencing

DOEpatents

Drmanac, Radoje

2006-07-04

Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

Methods and compositions for efficient nucleic acid sequencing

DOEpatents

Drmanac, Radoje

2002-01-01

Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

Biopharmaceutical production: Applications of surface plasmon resonance biosensors.

PubMed

Thillaivinayagalingam, Pranavan; Gommeaux, Julien; McLoughlin, Michael; Collins, David; Newcombe, Anthony R

2010-01-15

Surface plasmon resonance (SPR) permits the quantitative analysis of therapeutic antibody concentrations and impurities including bacteria, Protein A, Protein G and small molecule ligands leached from chromatography media. The use of surface plasmon resonance has gained popularity within the biopharmaceutical industry due to the automated, label free, real time interaction that may be exploited when using this method. The application areas to assess protein interactions and develop analytical methods for biopharmaceutical downstream process development, quality control, and in-process monitoring are reviewed. 2009 Elsevier B.V. All rights reserved.

Colloidal gold-labeled insulin complex. Characterization and binding to adipocytes.

PubMed

Moll, U M; Thun, C; Pfeiffer, E F

1986-01-01

Biologically active insulin gold complex was used as an ultrastructural marker to study insulin binding sites, uptake, and internalization in isolated rat adipocytes. The preparation conditions for monodispersed particles, ca. 16 nm in diameter and loaded with approximately 100 insulin molecules, are reported. The complex is stable for at least six weeks. Single particles or small clusters were scattered across the cell membrane. The distribution of unbound receptors seemed to be independent of the extensive system of pre-existing surface connected vesicles in adipocytes. The uptake of particles took place predominantly via non-coated pinocytotic invaginations; clathrin-coated pits did not seem to be important for this process. Lysosome-like structures contained aggregates of 10-15 particles. These data suggest that insulin gold complex is a useful marker for the specific labeling of insulin binding sites.

The whispering gallery mode biosensor: label-free detection from virus to single protein

NASA Astrophysics Data System (ADS)

Holler, S.; Dantham, V. R.; Keng, D.; Kolchenko, V.; Arnold, S.; Mulroe, Brigid; Paspaley-Grbavac, M.

2014-08-01

The whispering gallery mode (WGM) biosensor is a micro-optical platform capable of sensitive label-free detection of biological particles. Described by the reactive sensing principle (RSP), this analytic formulation quantifies the response of the system to the adsorption of bioparticles. Guided by the RSP, the WGM biosensor enabling from detection of virus (e.g., Human Papillomavirus, HPV) to the ultimate goal of single protein detection. The latter was derived from insights into the RSP, which resulted in the development of a hybrid plasmonic WGM biosensor, which has recently demonstrated detection of individual protein cancer markers. Enhancements from bound gold nanoparticles provide the sensitivity to detect single protein molecules (66 kDa) with good signal-to-noise (S/N > 10), and project that detection of proteins as small as 5 kDa.

2D-IR Spectroscopy of an AHA Labeled Photoswitchable PDZ2 Domain.

PubMed

Stucki-Buchli, Brigitte; Johnson, Philip J M; Bozovic, Olga; Zanobini, Claudio; Koziol, Klemens L; Hamm, Peter; Gulzar, Adnan; Wolf, Steffen; Buchenberg, Sebastian; Stock, Gerhard

2017-12-14

We explore the capability of the non-natural amino acid azidohomoalanine (AHA) as an IR label to sense relatively small structural changes in proteins with the help of 2D IR difference spectroscopy. To that end, we AHA-labeled an allosteric protein (the PDZ2 domain from human tyrosine-phosphatase 1E) and furthermore covalently linked it to an azobenzene-derived photoswitch as to mimic its conformational transition upon ligand binding. To determine the strengths and limitations of the AHA label, in total six mutants have been investigated with the label at sites with varying properties. Only one mutant revealed a measurable 2D IR difference signal. In contrast to the commonly observed frequency shifts that report on the degree of solvation, in this case we observe an intensity change. To understand this spectral response, we performed classical MD simulations, evaluating local contacts of the AHA labels to water molecules and protein side chains and calculating the vibrational frequency on the basis of an electrostatic model. Although these simulations revealed in part significant and complex changes of the number of intraprotein and water contacts upon trans-cis photoisomerization, they could not provide a clear explanation of why this one label would stick out. Subsequent quantum-chemistry calculations suggest that the response is the result of an electronic interaction involving charge transfer of the azido group with sulfonate groups from the photoswitch. To the best of our knowledge, such an effect has not been described before.

Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions

PubMed Central

Nierth, Alexander; Jäschke, Andres

2011-01-01

Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope 32P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the 32P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups. PMID:21731729

Production of isotopically labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies.

PubMed

Gómez-Cortés, Pilar; Brenna, J Thomas; Sacks, Gavin L

2012-06-19

Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-α-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to use of a single standard.

Surface chemistry and morphology in single particle optical imaging

NASA Astrophysics Data System (ADS)

Ekiz-Kanik, Fulya; Sevenler, Derin Deniz; Ünlü, Neşe Lortlar; Chiari, Marcella; Ünlü, M. Selim

2017-05-01

Biological nanoparticles such as viruses and exosomes are important biomarkers for a range of medical conditions, from infectious diseases to cancer. Biological sensors that detect whole viruses and exosomes with high specificity, yet without additional labeling, are promising because they reduce the complexity of sample preparation and may improve measurement quality by retaining information about nanoscale physical structure of the bio-nanoparticle (BNP). Towards this end, a variety of BNP biosensor technologies have been developed, several of which are capable of enumerating the precise number of detected viruses or exosomes and analyzing physical properties of each individual particle. Optical imaging techniques are promising candidates among broad range of label-free nanoparticle detectors. These imaging BNP sensors detect the binding of single nanoparticles on a flat surface functionalized with a specific capture molecule or an array of multiplexed capture probes. The functionalization step confers all molecular specificity for the sensor's target but can introduce an unforeseen problem; a rough and inhomogeneous surface coating can be a source of noise, as these sensors detect small local changes in optical refractive index. In this paper, we review several optical technologies for label-free BNP detectors with a focus on imaging systems. We compare the surface-imaging methods including dark-field, surface plasmon resonance imaging and interference reflectance imaging. We discuss the importance of ensuring consistently uniform and smooth surface coatings of capture molecules for these types of biosensors and finally summarize several methods that have been developed towards addressing this challenge.

Hairpin assembly circuit-based fluorescence cooperative amplification strategy for enzyme-free and label-free detection of small molecule.

PubMed

Feng, Chunjing; Zhu, Jing; Sun, Jiewei; Jiang, Wei; Wang, Lei

2015-10-01

Here, we developed an enzyme-free, label-free, and sensitive fluorescence cooperative amplification strategy based on a hairpin assembly circuit which coupled catalytic hairpin assembly (CHA) with hybridization chain reaction (HCR) for small molecule adenosine. A double-strand DNA probe with aptamer-catalysis strand (Apt-C) and inhibit strand (Inh) was designed for adenosine recognition and signal transduction which was named as Apt-C/Inh. Hairpins H1 and H2 were employed for constructing the CHA, and hairpins H3 and H4 for the HCR. Through the binding of adenosine and the Apt-C, the Inh was released from the Apt-C/Inh. Then the free Apt-C initiated the CHA through successively opening H1 and H2, generating H1/H2 complex and recyclable Apt-C. Next, the released Apt-C entered another CHA cycle, and the H1/H2 complex further initiated the HCR of H3 and H4 which induced the formation of the concatemers of H3/H4 complex. Such a process brought the two ends of hairpins H3 into close proximity, yielding numerous integrated G-quadruplexes which were initially sequestered in the stem and two terminals of H3. Finally, N-methyl mesoporphyrin IX (NMM) was added to generate an enhanced fluorescence signal. In the proposed strategy, driven only by the energy from hybridization, one target could trigger multiple HCR events via CHA-based target-cycle, leading to a remarkable enzyme-free amplification for adenosine. The detection limit could achieve as low as 9.7 × 10(-7) mol L(-1). Furthermore, G-quadruplexes were applied to construct label-free hairpin assembly circuit, which made it more simple and cost-effective. The satisfactory recoveries were obtained when detecting adenosine in spiked human serum and urine samples, demonstrating the feasibility of this detection strategy in biological samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Spontaneous Fluctuations can Guide Drug Design Strategies for Structurally Disordered Proteins.

PubMed

Maity, Barun Kumar; Vishvakarma, Vicky; Surendran, Dayana; Rawat, Anoop; Das, Anirban; Pramanik, Shreya; Arfin, Najmul; Maiti, Sudipta

2018-06-21

Structure-based 'rational' drug-design strategies fail for diseases associated with intrinsically disordered proteins (IDPs). However, structural disorder allows large amplitude spontaneous intramolecular dynamics in a protein. We demonstrate a method that exploits this dynamics to provide quantitative information about the degree of interaction of an IDP with other molecules. A candidate ligand molecule may not bind strongly, but even momentary interactions can be expected to perturb the fluctuations. We measure the amplitude and frequency of the equilibrium fluctuations of fluorescently labeled small oligomers of hIAPP (an IDP associated with Type II diabetes) in a physiological solution, using nanosecond fluorescence cross-correlation spectroscopy. We show that the inter-terminal distance fluctuates at a characteristic timescale of 134 ± 10 ns, and 6.4 ± 0.2 % of the population is in the 'closed' (quenched) state at equilibrium. These fluctuations are affected in a dose-dependent manner by a series of small molecules known to reduce the toxicity of various amyloid peptides. The degree of interaction shows the following order: resveratrol < epicatechin ~ quercetin < congo red < epigallocatechin-3-gallate. Such ordering can provide a direction for exploring the chemical space for finding stronger-binding ligands. We test the biological relevance of these measurements by measuring the effect of these molecules on the affinity of hIAPP for lipid vesicles and cell membranes. We find that the ability of a molecule to modulate intramolecular fluctuations correlates well with its ability to lower membrane affinity. We conclude that structural disorder may provide new avenues for rational drug design for IDPs.

Single-Molecule Denaturation Mapping of Genomic DNA in Nanofluidic Channels

NASA Astrophysics Data System (ADS)

Reisner, Walter; Larsen, Niels; Kristensen, Anders; Tegenfeldt, Jonas O.; Flyvbjerg, Henrik

2009-03-01

We have developed a new DNA barcoding technique based on the partial denaturation of extended fluorescently labeled DNA molecules. We partially melt DNA extended in nanofluidic channels via a combination of local heating and added chemical denaturants. The melted molecules, imaged via a standard fluorescence videomicroscopy setup, exhibit a nonuniform fluorescence profile corresponding to a series of local dips and peaks in the intensity trace along the stretched molecule. We show that this barcode is consistent with the presence of locally melted regions and can be explained by calculations of sequence-dependent melting probability. We believe this melting mapping technology is the first optically based single molecule technique sensitive to genome wide sequence variation that does not require an additional enzymatic labeling or restriction scheme.

Positron Emission Tomography: Principles, Technology, and Recent Developments

NASA Astrophysics Data System (ADS)

Ziegler, Sibylle I.

2005-04-01

Positron emission tomography (PET) is a nuclear medical imaging technique for quantitative measurement of physiologic parameters in vivo (an overview of principles and applications can be found in [P.E. Valk, et al., eds. Positron Emission Tomography. Basic Science and Clinical Practice. 2003, Springer: Heidelberg]), based on the detection of small amounts of posi-tron-emitter-labelled biologic molecules. Various radiotracers are available for neuro-logical, cardiological, and oncological applications in the clinic and in research proto-cols. This overview describes the basic principles, technology, and recent develop-ments in PET, followed by a section on the development of a tomograph with ava-lanche photodiodes dedicated for small animal imaging as an example of efforts in the domain of high resolution tomographs.

Control of microtubule trajectory within an electric field by altering surface charge density

PubMed Central

Isozaki, Naoto; Ando, Suguru; Nakahara, Tasuku; Shintaku, Hirofumi; Kotera, Hidetoshi; Meyhöfer, Edgar; Yokokawa, Ryuji

2015-01-01

One of challenges for using microtubules (MTs) driven by kinesin motors in microfluidic environments is to control their direction of movement. Although applying physical biases to rectify MTs is prevalent, it has not been established as a design methodology in conjunction with microfluidic devices. In the future, the methodology is expected to achieve functional motor-driven nanosystems. Here, we propose a method to guide kinesin-propelled MTs in multiple directions under an electric field by designing a charged surface of MT minus ends labeled with dsDNA via a streptavidin-biotin interaction. MTs labeled with 20-bp or 50-bp dsDNA molecules showed significantly different trajectories according to the DNA length, which were in good agreement with values predicted from electrophoretic mobilities measured for their minus ends. Since the effective charge of labeled DNA molecules was equal to that of freely dispersed DNA molecules in a buffer solution, MT trajectory could be estimated by selecting labeling molecules with known charges. Moreover, the estimated trajectory enables to define geometrical sizes of a microfluidic device. This rational molecular design and prediction methodology allows MTs to be guided in multiple directions, demonstrating the feasibility of using molecular sorters driven by motor proteins. PMID:25567007

Control of microtubule trajectory within an electric field by altering surface charge density.

PubMed

Isozaki, Naoto; Ando, Suguru; Nakahara, Tasuku; Shintaku, Hirofumi; Kotera, Hidetoshi; Meyhöfer, Edgar; Yokokawa, Ryuji

2015-01-08

One of challenges for using microtubules (MTs) driven by kinesin motors in microfluidic environments is to control their direction of movement. Although applying physical biases to rectify MTs is prevalent, it has not been established as a design methodology in conjunction with microfluidic devices. In the future, the methodology is expected to achieve functional motor-driven nanosystems. Here, we propose a method to guide kinesin-propelled MTs in multiple directions under an electric field by designing a charged surface of MT minus ends labeled with dsDNA via a streptavidin-biotin interaction. MTs labeled with 20-bp or 50-bp dsDNA molecules showed significantly different trajectories according to the DNA length, which were in good agreement with values predicted from electrophoretic mobilities measured for their minus ends. Since the effective charge of labeled DNA molecules was equal to that of freely dispersed DNA molecules in a buffer solution, MT trajectory could be estimated by selecting labeling molecules with known charges. Moreover, the estimated trajectory enables to define geometrical sizes of a microfluidic device. This rational molecular design and prediction methodology allows MTs to be guided in multiple directions, demonstrating the feasibility of using molecular sorters driven by motor proteins.

Observation of giant conductance fluctuations in a protein

NASA Astrophysics Data System (ADS)

Zhang, Bintian; Song, Weisi; Pang, Pei; Zhao, Yanan; Zhang, Peiming; Csabai, István; Vattay, Gábor; Lindsay, Stuart

2017-12-01

Proteins are insulating molecular solids, yet even those containing easily reduced or oxidized centers can have single-molecule electronic conductances that are too large to account for with conventional transport theories. Here, we report the observation of remarkably high electronic conductance states in an electrochemically inactive protein, the ∼200 kD α V β 3 extracellular domain of human integrin. Large current pulses (up to nA) were observed for long durations (many ms, corresponding to many pC of charge transfer) at large gap (>5 nm) distances in an STM when the protein was bound specifically by a small peptide ligand attached to the electrodes. The effect is greatly reduced when a homologous, weakly binding protein (α 4 β 1) is used as a control. In order to overcome the limitations of the STM, the time- and voltage-dependence of the conductance were further explored using a fixed-gap (5 nm) tunneling junction device that was small enough to trap a single protein molecule at any one time. Transitions to a high conductance (∼nS) state were observed, the protein being ‘on’ for times from ms to tenths of a second. The high-conductance states only occur above ∼100 mV applied bias, and thus are not an equilibrium property of the protein. Nanoamp two-level signals indicate the specific capture of a single molecule in an electrode gap functionalized with the ligand. This offers a new approach to label-free electronic detection of single protein molecules. Electronic structure calculations yield a distribution of energy level spacings that is consistent with a recently proposed quantum-critical state for proteins, in which small fluctuations can drive transitions between localized and band-like electronic states.

Observation of Giant Conductance Fluctuations in a Protein

PubMed Central

Zhang, Bintian; Song, Weisi; Pang, Pei; Zhao, Yanan; Zhang, Peiming; Csabai, István; Vattay, Gábor; Lindsay, Stuart

2017-01-01

Proteins are insulating molecular solids, yet even those containing easily reduced or oxidized centers can have single-molecule electronic conductances that are too large to account for with conventional transport theories. Here, we report the observation of remarkably high electronic conductance states in an electrochemically-inactive protein, the ~200 kD αVβ3 extracelluar domain of human integrin. Large current pulses (up to nA) were observed for long durations (many ms, corresponding to many pC of charge transfer) at large gap (>5nm) distances in an STM when the protein was bound specifically by a small peptide ligand attached to the electrodes. The effect is greatly reduced when a homologous, weakly-binding protein (α4β1) is used as a control. In order to overcome the limitations of the STM, the time- and voltage-dependence of the conductance were further explored using a fixed-gap (5 nm) tunneling junction device that was small enough to trap a single protein molecule at any one time. Transitions to a high conductance (~ nS) state were observed, the protein being “on” for times from ms to tenths of a second. The high-conductance states only occur above ~ 100mV applied bias, and thus are not an equilibrium property of the protein. Nanoamp two-level signals indicate the specific capture of a single molecule in an electrode gap functionalized with the ligand. This offers a new approach to label-free electronic detection of single protein molecules. Electronic structure calculations yield a distribution of energy level spacings that is consistent with a recently proposed quantum-critical state for proteins, in which small fluctuations can drive transitions between localized and band-like electronic states. PMID:29552645

Development of a miRNA surface-enhanced Raman scattering assay using benchtop and handheld Raman systems.

PubMed

Schechinger, Monika; Marks, Haley; Locke, Andrea; Choudhury, Mahua; Cote, Gerard

2018-01-01

DNA-functionalized nanoparticles, when paired with surface-enhanced Raman spectroscopy (SERS), can rapidly detect microRNA. However, widespread use of this approach is hindered by drawbacks associated with large and expensive benchtop Raman microscopes. MicroRNA-17 (miRNA-17) has emerged as a potential epigenetic indicator of preeclampsia, a condition that occurs during pregnancy. Biomarker detection using an SERS point-of-care device could enable prompt diagnosis and prevention as early as the first trimester. Recently, strides have been made in developing portable Raman systems for field applications. An SERS assay for miRNA-17 was assessed and translated from traditional benchtop Raman microscopes to a handheld system. Three different photoactive molecules were compared as potential Raman reporter molecules: a chromophore, malachite green isothiocyanate (MGITC), a fluorophore, tetramethylrhodamine isothiocyanate, and a polarizable small molecule 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). For the benchtop Raman microscope, the DTNB-labeled assay yielded the greatest sensitivity under 532-nm laser excitation, but the MGITC-labeled assay prevailed at 785 nm. Conversely, DTNB was preferable for the miniaturized 785-nm Raman system. This comparison showed significant SERS enhancement variation in response to 1-nM miRNA-17, implying that the sensitivity of the assay may be more heavily dependent on the excitation wavelength, instrumentation, and Raman reporter chosen than on the plasmonic coupling from DNA/miRNA-mediated nanoparticle assemblies. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

PubMed Central

Jacquier, V.; Prummer, M.; Segura, J.-M.; Pick, H.; Vogel, H.

2006-01-01

Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of ≈190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception. PMID:16980412

Method of Stamping Surface-Enhance Raman Spectroscopy for Label-Free, Multiplexed, Molecular Sensing and Imaging

NASA Technical Reports Server (NTRS)

Shih, Wei-Chuan (Inventor)

2017-01-01

The present disclosure relates the use of a stamping surface enhanced Raman scattering (S-SERS) technique with nanoporous gold disk (NPGD) plasmonic substrates to produce a label-free, multiplexed molecular sensing and imaging technique. A NPGD SERS substrate is stamped onto a surface containing one or more target molecules, followed by SERS measurement of the target molecules located between the surface and SERS substrate. The target molecules may be deposited on the surface, which may be a carrier substrate such as polydimethylsiloxane (PDMS).

In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines.

PubMed

Custer, Thomas C; Walter, Nils G

2017-07-01

RNA plays a fundamental, ubiquitous role as either substrate or functional component of many large cellular complexes-"molecular machines"-used to maintain and control the readout of genetic information, a functional landscape that we are only beginning to understand. The cellular mechanisms for the spatiotemporal organization of the plethora of RNAs involved in gene expression are particularly poorly understood. Intracellular single-molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent mechanistic parameters that govern cellular RNA functions, including those of protein coding messenger RNAs (mRNAs). Progress has been hampered, however, by the scarcity of efficient high-yield methods to fluorescently label RNA molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ T7 RNA polymerase to body label an RNA with a cyanine dye, as well as yeast poly(A) polymerase to strategically place multiple 2'-azido-modifications for subsequent fluorophore labeling either between the body and tail or randomly throughout the tail. Using a combination of biochemical and single-molecule fluorescence microscopy approaches, we demonstrate that both yeast poly(A) polymerase labeling strategies result in fully functional mRNA, whereas protein coding is severely diminished in the case of body labeling. © 2016 The Protein Society.

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

PubMed

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Use of the interior cavity of the P22 capsid for site-specific initiation of atom-transfer radical polymerization with high-density cargo loading

NASA Astrophysics Data System (ADS)

Lucon, Janice; Qazi, Shefah; Uchida, Masaki; Bedwell, Gregory J.; Lafrance, Ben; Prevelige, Peter E.; Douglas, Trevor

2012-10-01

Virus-like particles (VLPs) have emerged as important and versatile architectures for chemical manipulation in the development of functional hybrid nanostructures. Here we demonstrate a successful site-selective initiation of atom-transfer radical polymerization reactions to form an addressable polymer constrained within the interior cavity of a VLP. Potentially, this protein-polymer hybrid of P22 and cross-linked poly(2-aminoethyl methacrylate) could be useful as a new high-density delivery vehicle for the encapsulation and delivery of small-molecule cargos. In particular, the encapsulated polymer can act as a scaffold for the attachment of small functional molecules, such as fluorescein dye or the magnetic resonance imaging (MRI) contrast agent Gd-diethylenetriaminepentacetate, through reactions with its pendant primary amine groups. Using this approach, a significant increase in the labelling density of the VLP, compared to that of previous modifications of VLPs, can be achieved. These results highlight the use of multimeric protein-polymer conjugates for their potential utility in the development of VLP-based MRI contrast agents with the possibility of loading other cargos.

Ultra-small dye-doped silica nanoparticles via modified sol-gel technique

NASA Astrophysics Data System (ADS)

Riccò, R.; Nizzero, S.; Penna, E.; Meneghello, A.; Cretaio, E.; Enrichi, F.

2018-05-01

In modern biosensing and imaging, fluorescence-based methods constitute the most diffused approach to achieve optimal detection of analytes, both in solution and on the single-particle level. Despite the huge progresses made in recent decades in the development of plasmonic biosensors and label-free sensing techniques, fluorescent molecules remain the most commonly used contrast agents to date for commercial imaging and detection methods. However, they exhibit low stability, can be difficult to functionalise, and often result in a low signal-to-noise ratio. Thus, embedding fluorescent probes into robust and bio-compatible materials, such as silica nanoparticles, can substantially enhance the detection limit and dramatically increase the sensitivity. In this work, ultra-small fluorescent silica nanoparticles (NPs) for optical biosensing applications were doped with a fluorescent dye, using simple water-based sol-gel approaches based on the classical Stöber procedure. By systematically modulating reaction parameters, controllable size tuning of particle diameters as low as 10 nm was achieved. Particles morphology and optical response were evaluated showing a possible single-molecule behaviour, without employing microemulsion methods to achieve similar results. [Figure not available: see fulltext.

Bioactive lipid coating of bone allografts directs engraftment and fate determination of bone marrow-derived cells in rat GFP chimeras

PubMed Central

Das, Anusuya; Segar, Claire E.; Chu, Yihsuan; Wang, Tiffany W.; Lin, Yong; Yang, Chunxi; Du, Xeujun; Ogle, Roy C.; Cui, Quanjun; Botchwey, Edward A.

2015-01-01

Bone grafting procedures are performed to treat wounds incurred during wartime trauma, accidents, and tumor resections. Endogenous mechanisms of repair are often insufficient to ensure integration between host and donor bone and subsequent restoration of function. We investigated the role that bone marrow-derived cells play in bone regeneration and sought to increase their contributions by functionalizing bone allografts with bioactive lipid coatings. Polymer-coated allografts were used to locally deliver the immunomodulatory small molecule FTY720 in tibial defects created in rat bone marrow chimeras containing genetically-labeled bone marrow for monitoring cell origin and fate. Donor bone marrow contributed significantly to both myeloid and osteogenic cells in remodeling tissue surrounding allografts. FTY720 coatings altered the phenotype of immune cells two weeks post-injury, which was associated with increased vascularization and bone formation surrounding allografts. Consequently, degradable polymer coating strategies that deliver small molecule growth factors such as FTY720 represent a novel therapeutic strategy for harnessing endogenous bone marrow-derived progenitors and enhancing healing in load-bearing bone defects. PMID:26125501

Quantification of ligand density and stoichiometry on the surface of liposomes using single-molecule fluorescence imaging.

PubMed

Belfiore, Lisa; Spenkelink, Lisanne M; Ranson, Marie; van Oijen, Antoine M; Vine, Kara L

2018-05-28

Despite the longstanding existence of liposome technology in drug delivery applications, there have been no ligand-directed liposome formulations approved for clinical use to date. This lack of translation is due to several factors, one of which is the absence of molecular tools for the robust quantification of ligand density on the surface of liposomes. We report here for the first time the quantification of proteins attached to the surface of small unilamellar liposomes using single-molecule fluorescence imaging. Liposomes were surface-functionalized with fluorescently labeled human proteins previously validated to target the cancer cell surface biomarkers plasminogen activator inhibitor-2 (PAI-2) and trastuzumab (TZ, Herceptin®). These protein-conjugated liposomes were visualized using a custom-built wide-field fluorescence microscope with single-molecule sensitivity. By counting the photobleaching steps of the fluorescently labeled proteins, we calculated the number of attached proteins per liposome, which was 11 ± 4 proteins for single-ligand liposomes. Imaging of dual-ligand liposomes revealed stoichiometries of the two attached proteins in accordance with the molar ratios of protein added during preparation. Preparation of PAI-2/TZ dual-ligand liposomes via two different methods revealed that the post-insertion method generated liposomes with a more equal representation of the two differently sized proteins, demonstrating the ability of this preparation method to enable better control of liposome protein densities. We conclude that the single-molecule imaging method presented here is an accurate and reliable quantification tool for determining ligand density and stoichiometry on the surface of liposomes. This method has the potential to allow for comprehensive characterization of novel ligand-directed liposomes that should facilitate the translation of these nanotherapies through to the clinic. Copyright © 2018 Elsevier B.V. All rights reserved.

Tunable ultrasmall visible-to-extended near-infrared emitting silver sulfide quantum dots for integrin-targeted cancer imaging.

PubMed

Tang, Rui; Xue, Jianpeng; Xu, Baogang; Shen, Duanwen; Sudlow, Gail P; Achilefu, Samuel

2015-01-27

The large size of many near-infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. In this study, we developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from 500 to 1200 nm and a QD core diameter between 1.5 and 9 nm. Conjugation of a tumor-avid cyclic pentapeptide (Arg-Gly-Asp-DPhe-Lys) resulted in monodisperse, water-soluble QDs (hydrodynamic diameter < 10 nm) without loss of the peptide's high binding affinity to tumor-associated integrins (KI = 1.8 nM/peptide). Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and noncytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.

177 Lu-Labeled Phosphoramidate-Based PSMA Inhibitors: The Effect of an Albumin Binder on Biodistribution and Therapeutic Efficacy in Prostate Tumor-Bearing Mice

DOE PAGES

Choy, Cindy J.; Ling, Xiaoxi; Geruntho, Jonathan J.; ...

2017-04-27

Prostate-specific membrane antigen (PSMA) continues to be an active biomarker for small-molecule PSMA-targeted imaging and therapeutic agents for prostate cancer and various non-prostatic tumors that are characterized by PSMA expression on their neovasculature. One of the challenges for small-molecule PSMA inhibitors with respect to delivering therapeutic payloads is their rapid renal clearance. In order to overcome this pharmacokinetic challenge, we outfitted a 177Lu-labeled phosphoramidate-based PSMA inhibitor (CTT1298) with an albumin-binding motif (CTT1403) and compared its in vivo performance with that of an analogous compound lacking the albumin-binding motif (CTT1401). The radiolabeling of CTT1401 and CTT1403 was achieved using click chemistrymore » to connect 177Lu-DOTA-N3 to the dibenzocyclooctyne (DBCO)-bearing CTT1298 inhibitor cores. A direct comparison in vitro and in vivo performance was made for CTT1401 and CTT1403; the specificity and efficacy by means of cellular uptake and internalization, biodistribution, and therapeutic efficacy were determined for both compounds. And while both compounds displayed excellent uptake and rapid internalization in PSMA+ PC3-PIP cells, the albumin binding moiety in CTT1403 conferred clear advantages to the PSMA-inhibitor scaffold including increased circulating half-life and prostate tumor uptake that continued to increase up to 168 h post-injection. This then increased tumor uptake translated into superior therapeutic efficacy of CTT1403 in PSMA+ PC3-PIP human xenograft tumors.« less

177 Lu-Labeled Phosphoramidate-Based PSMA Inhibitors: The Effect of an Albumin Binder on Biodistribution and Therapeutic Efficacy in Prostate Tumor-Bearing Mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choy, Cindy J.; Ling, Xiaoxi; Geruntho, Jonathan J.

Prostate-specific membrane antigen (PSMA) continues to be an active biomarker for small-molecule PSMA-targeted imaging and therapeutic agents for prostate cancer and various non-prostatic tumors that are characterized by PSMA expression on their neovasculature. One of the challenges for small-molecule PSMA inhibitors with respect to delivering therapeutic payloads is their rapid renal clearance. In order to overcome this pharmacokinetic challenge, we outfitted a 177Lu-labeled phosphoramidate-based PSMA inhibitor (CTT1298) with an albumin-binding motif (CTT1403) and compared its in vivo performance with that of an analogous compound lacking the albumin-binding motif (CTT1401). The radiolabeling of CTT1401 and CTT1403 was achieved using click chemistrymore » to connect 177Lu-DOTA-N3 to the dibenzocyclooctyne (DBCO)-bearing CTT1298 inhibitor cores. A direct comparison in vitro and in vivo performance was made for CTT1401 and CTT1403; the specificity and efficacy by means of cellular uptake and internalization, biodistribution, and therapeutic efficacy were determined for both compounds. And while both compounds displayed excellent uptake and rapid internalization in PSMA+ PC3-PIP cells, the albumin binding moiety in CTT1403 conferred clear advantages to the PSMA-inhibitor scaffold including increased circulating half-life and prostate tumor uptake that continued to increase up to 168 h post-injection. This then increased tumor uptake translated into superior therapeutic efficacy of CTT1403 in PSMA+ PC3-PIP human xenograft tumors.« less

Label-free detection of protein molecules secreted from an organ-on-a-chip model for drug toxicity assays

NASA Astrophysics Data System (ADS)

Morales, Andres W.; Zhang, Yu S.; Aleman, Julio; Alerasool, Parissa; Dokmeci, Mehmet R.; Khademhosseini, Ali; Ye, Jing Yong

2016-03-01

Clinical attrition is about 30% from failure of drug candidates due to toxic side effects, increasing the drug development costs significantly and slowing down the drug discovery process. This partly originates from the fact that the animal models do not accurately represent human physiology. Hence there is a clear unmet need for developing drug toxicity assays using human-based models that are complementary to traditional animal models before starting expensive clinical trials. Organ-on-a-chip techniques developed in recent years have generated a variety of human organ models mimicking different human physiological conditions. However, it is extremely challenging to monitor the transient and long-term response of the organ models to drug treatments during drug toxicity tests. First, when an organ-on-a-chip model interacts with drugs, a certain amount of protein molecules may be released into the medium due to certain drug effects, but the amount of the protein molecules is limited, since the organ tissue grown inside microfluidic bioreactors have minimum volume. Second, traditional fluorescence techniques cannot be utilized for real-time monitoring of the concentration of the protein molecules, because the protein molecules are continuously secreted from the tissue and it is practically impossible to achieve fluorescence labeling in the dynamically changing environment. Therefore, direct measurements of the secreted protein molecules with a label-free approach is strongly desired for organs-on-a-chip applications. In this paper, we report the development of a photonic crystal-based biosensor for label-free assays of secreted protein molecules from a liver-on-a-chip model. Ultrahigh detection sensitivity and specificity have been demonstrated.

Label-Free Determination of Protein Binding in Aqueous Solution using Overlayer Enhanced Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (OE-ATR-FTIR)

NASA Astrophysics Data System (ADS)

Ruthenburg, Travis; Aweda, Tolulope; Park, Simon; Meares, Claude; Land, Donald

2009-03-01

Protein binding/affinity studies are often performed using Surface Plasmon Resonance techniques that don't produce much spectral information. Measurement of protein binding affinity using FTIR is traditionally performed using high protein concentration or deuterated solvent. By immobilizing a protein near the surface of a gold-coated germanium internal reflection element interactions can be measured between an immobilized protein and free proteins or small molecules in aqueous solution. By monitoring the on and off rates of these interactions, the dissociation constant for the system can be determined. The dissociation constant for the molecule Yttrium-DOTA binding to the antibody 2D12.5 system was determined to be 100nM. Results will also be presented from our measurements of Bovine Serum Albumin (BSA) binding to anti-BSA.

Synthesis, Radiolabelling and In Vitro Characterization of the Gallium-68-, Yttrium-90- and Lutetium-177-Labelled PSMA Ligand, CHX-A''-DTPA-DUPA-Pep.

PubMed

Baur, Benjamin; Solbach, Christoph; Andreolli, Elena; Winter, Gordon; Machulla, Hans-Jürgen; Reske, Sven N

2014-04-29

Since prostate-specific membrane antigen (PSMA) has been identified as a diagnostic target for prostate cancer, many urea-based small PSMA-targeting molecules were developed. First, the clinical application of these Ga-68 labelled compounds in positron emission tomography (PET) showed their diagnostic potential. Besides, the therapy of prostate cancer is a demanding field, and the use of radiometals with PSMA bearing ligands is a valid approach. In this work, we describe the synthesis of a new PSMA ligand, CHX-A''-DTPA-DUPA-Pep, the subsequent labelling with Ga-68, Lu-177 and Y-90 and the first in vitro characterization. In cell investigations with PSMA-positive LNCaP C4-2 cells, KD values of ≤14.67 ± 1.95 nM were determined, indicating high biological activities towards PSMA. Radiosyntheses with Ga-68, Lu-177 and Y-90 were developed under mild reaction conditions (room temperature, moderate pH of 5.5 and 7.4, respectively) and resulted in nearly quantitative radiochemical yields within 5 min.

Synthesis, Radiolabelling and In Vitro Characterization of the Gallium-68-, Yttrium-90- and Lutetium-177-Labelled PSMA Ligand, CHX-A''-DTPA-DUPA-Pep

PubMed Central

Baur, Benjamin; Solbach, Christoph; Andreolli, Elena; Winter, Gordon; Machulla, Hans-Jürgen; Reske, Sven N.

2014-01-01

Since prostate-specific membrane antigen (PSMA) has been identified as a diagnostic target for prostate cancer, many urea-based small PSMA-targeting molecules were developed. First, the clinical application of these Ga-68 labelled compounds in positron emission tomography (PET) showed their diagnostic potential. Besides, the therapy of prostate cancer is a demanding field, and the use of radiometals with PSMA bearing ligands is a valid approach. In this work, we describe the synthesis of a new PSMA ligand, CHX-A''-DTPA-DUPA-Pep, the subsequent labelling with Ga-68, Lu-177 and Y-90 and the first in vitro characterization. In cell investigations with PSMA-positive LNCaP C4-2 cells, KD values of ≤14.67 ± 1.95 nM were determined, indicating high biological activities towards PSMA. Radiosyntheses with Ga-68, Lu-177 and Y-90 were developed under mild reaction conditions (room temperature, moderate pH of 5.5 and 7.4, respectively) and resulted in nearly quantitative radiochemical yields within 5 min. PMID:24787458

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study

PubMed Central

Shotton, D.; Thompson, K.; Wofsy, L.; Branton, D.

1978-01-01

We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others. PMID:10605454

Molecular self-assembly for biological investigations and nanoscale lithography

NASA Astrophysics Data System (ADS)

Cheunkar, Sarawut

Small, diffusible molecules when recognized by their binding partners, such as proteins and antibodies, trigger enzymatic activity, cell communication, and immune response. Progress in analytical methods enabling detection, characterization, and visualization of biological dynamics at the molecular level will advance our exploration of complex biological systems. In this dissertation, analytical platforms were fabricated to capture membrane-associated receptors, which are essential proteins in cell signaling pathways. The neurotransmitter serotonin and its biological precursor were immobilized on gold substrates coated with self-assembled monolayers (SAMs) of oligo(ethylene glycol)alkanethiols and their reactive derivatives. The SAM-coated substrates present the biologically selective affinity of immobilized molecules to target native membrane-associated receptors. These substrates were also tested for biospecificity using antibodies. In addition, small-molecule-functionalized platforms, expressing neurotransmitter pharmacophores, were employed to examine kinetic interactions between G-protein-coupled receptors and their associated neurotransmitters. The binding interactions were monitored using a quartz crystal microbalance equipped with liquid-flow injection. The interaction kinetics of G-protein-coupled serotonin 1A receptor and 5-hydroxytyptophan-functionalized surfaces were studied in a real-time, label-free environment. Key binding parameters, such as equilibrium dissociation constants, binding rate constants, and dissociative half-life, were extracted. These parameters are critical for understanding and comparing biomolecular interactions in modern biomedical research. By integrating self-assembly, surface functionalization, and nanofabrication, small-molecule microarrays were created for high-throughput screening. A hybrid soft-lithography, called microcontact insertion printing, was used to pattern small molecules at the dilute scales necessary for highly selective biorecognition. By carefully tuning the polar surface energy of polymeric stamps, problems associated with patterning hydrophilic tether molecules inserted into hydrophilic preformed SAMs are surmounted. The patterned substrates presenting neurotransmitter precursors selectively capture membrane-associated receptors. These advances provide new avenues for fabricating small-molecule arrays. Furthermore, a novel strategy based on a conventional microcontact printing, called chemical lift-off lithography, was invented to overcome the micrometer-scale resolution limits of molecular ink diffusion in soft lithography. Self-assembled monolayers of hydroxyl-terminated alkanethiols, preformed on gold substrates, were selectively removed by oxygen-plasma-treated polymeric stamps in a subtractive stamping process with high pattern fidelity. The covalent interactions formed at the stamp-substrate interface are believed to be responsible for removing not only alkanethiol molecules but also a monolayer of gold atoms from the substrates. A variety of high-resolution patterned features were fabricated, and stamps were cleaned and reused many times without feature deterioration. The remaining SAMs acted as resists for etching exposed gold features. Monolayer backfilling into lifted-off areas enabled patterned protein capture, and 40-nanometer chemical patterns were achieved.

Label free biosensor incorporating a replica-molded, vertically emitting distributed feedback laser

NASA Astrophysics Data System (ADS)

Lu, M.; Choi, S. S.; Wagner, C. J.; Eden, J. G.; Cunningham, B. T.

2008-06-01

A label free biosensor based upon a vertically emitting distributed feedback (DFB) laser has been demonstrated. The DFB laser comprises a replica-molded, one-dimensional dielectric grating coated with laser dye-doped polymer as the gain medium. Adsorption of biomolecules onto the laser surface alters the DFB laser emission wavelength, thereby permitting the kinetic adsorption of a protein polymer monolayer or the specific binding of small molecules to be quantified. A bulk sensitivity of 16.6nm per refractive index unit and the detection of a monolayer of the protein polymer poly(Lys, Phe) have been observed with this biosensor. The sensor represents a departure from conventional passive resonant optical sensors from the standpoint that the device actively generates its own narrowband high intensity output without stringent requirements on the coupling alignments, resulting in a simple, robust illumination and detection configuration.

Metabolic labelling of the carbohydrate core in bacterial peptidoglycan and its applications

PubMed Central

Liang, Hai; DeMeester, Kristen E.; Hou, Ching-Wen; Parent, Michelle A.; Caplan, Jeffrey L.; Grimes, Catherine L.

2017-01-01

Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults. A component of this material, N-acetyl-muramic acid (NAM), serves as a core structural element for innate immune recognition of PG fragments. We report the synthesis of modifiable NAM carbohydrate derivatives and the installation of these building blocks into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. Whole cells are labelled via click chemistry and visualized using super-resolution microscopy, revealing higher resolution PG structural details and allowing the cell wall biosynthesis, as well as its destruction in immune cells, to be tracked. This study will assist in the future identification of mechanisms that the immune system uses to recognize bacteria, glean information about fundamental cell wall architecture and aid in the design of novel antibiotics. PMID:28425464

Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.

1987-06-25

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher massmore » (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.« less

Synthesis and biodistribution of novel magnetic-poly(HEMA-APH) nanopolymer radiolabeled with iodine-131 and investigation its fate in vivo for cancer therapy

NASA Astrophysics Data System (ADS)

Avcıbaşı, Uğur; Avcıbaşı, Nesibe; Akalın, Hilmi Arkut; Ediz, Melis; Demiroğlu, Hasan; Gümüşer, Fikriye Gül; Özçalışkan, Emir; Türkcan, Ceren; Uygun, Deniz Aktaş; Akgöl, Sinan

2013-10-01

Herein, we investigated the biological uptake, distribution, and radiopharmaceutical potential of a novel molecule based on 2-hydroxyethyl methacrylate (HEMA) and anilinephtalein (APH) in the metabolism of Albino Wistar rats. In order to achieve this, we synthesized APH using organic synthesis methods and copolymerized APH with HEMA using a common polymerization method, surfactant-free emulsion polymerization. In the presence of Fe3O4 particles, we obtained a new generation magnetic-nano-scale polymer, magnetic-poly(HEMA-APH). This new molecule was chemically identified and approved by several characterization methods using Fourier transform infrared spectroscopy, scanning electron microscope, energy dispersive X-ray spectroscopy, electron spin resonance, atomic force microscope, and Zeta particle-size analysis. To evaluate the biological activity in live metabolism and anti-cancer potential of mag-poly(HEMA-APH), molecule was radioiodinated by a widely used labeling technique, iodogen method, with a gamma diffuser radionuclide, 131I. Thin-layer radiochromatography experiments demonstrated that 131I binded to nanopolymer with the labeling yield of 90 %. Lipophilicity and stability experiments were conducted to determine the condition of cold and labeled mag-poly(HEMA-APH) in rat blood and lipid medium. Results demonstrated that radioiodinated molecule stayed as an intact complex in rat metabolism for 24 h and experimental lipophilicity was determined as 0.12 ± 0.02. In vivo results obtained by imaging and biological distribution experiments indicated that mag-poly(HEMA-APH) labeled with 131I [131I-mag-poly(HEMA-APH)] highly incorporated into tissues of the uterus, the ovarian, the prostate, and the lungs in rat metabolism. Based on these results, it may be evaluated that novel mag-poly(HEMA-APH) molecule labeled with 131I is a compound which has a significant potential for being used as an anti-cancer agent. Certain results can only be obtained whether this molecule is applied to adenocarcinoma cell models and tumor-bearing animals.

Consistent assignment of the vibrations of symmetric and asymmetric ortho-disubstituted benzenes

NASA Astrophysics Data System (ADS)

Tuttle, William D.; Gardner, Adrian M.; Andrejeva, Anna; Kemp, David J.; Wakefield, Jonathan C. A.; Wright, Timothy G.

2018-02-01

The form of molecular vibrations, and changes in these, give valuable insights into geometric and electronic structure upon electronic excitation or ionization, and within families of molecules. Here, we give a description of the phenyl-ring-localized vibrational modes of the ground (S0) electronic states of a wide range of ortho-disubstituted benzene molecules including both symmetrically- and asymmetrically-substituted cases. We conclude that the use of the commonly-used Wilson or Varsányi mode labels, which are based on the vibrational motions of benzene itself, is misleading and ambiguous. In addition, we also find the use of the Mi labels for monosubstituted benzenes [A.M. Gardner, T.G. Wright. J. Chem. Phys. 135 (2011) 114305], or the recently-suggested labels for para-disubstituted benzenes [A. Andrejeva, A.M. Gardner, W.D. Tuttle, T.G. Wright, J. Molec. Spectrosc. 321, 28 (2016)] are not appropriate. Instead, we label the modes consistently based upon the Mulliken (Herzberg) method for the modes of ortho-difluorobenzene (pDFB) under Cs symmetry, since we wish the labelling scheme to cover both symmetrically- and asymmetrically-substituted molecules. By studying the vibrational wavenumbers from the same force field while varying the mass of the substituent, we are able to identify the corresponding modes across a wide range of molecules and hence provide consistent assignments. We assign the vibrations of the following sets of molecules: the symmetric o-dihalobenzenes, o-xylene and catechol (o-dihydroxybenzene); and the asymmetric o-dihalobenzenes, o-halotoluenes, o-halophenols and o-cresol. In the symmetrically-substituted species, we find a pair of in-phase and out-of-phase carbon-substituent stretches, and this motion persists in asymmetrically-substituted molecules for heavier substituents. When at least one of the substituents is light, then we find that these evolve into localized carbon-substituent stretches.

Biosensing via light scattering from plasmonic core-shell nanospheres coated with DNA molecules

NASA Astrophysics Data System (ADS)

Xie, Huai-Yi; Chen, Minfeng; Chang, Yia-Chung; Moirangthem, Rakesh Singh

2017-05-01

We present both experimental and theoretical studies for investigating DNA molecules attached on metallic nanospheres. We have developed an efficient and accurate numerical method to investigate light scattering from plasmonic nanospheres on a substrate covered by a shell, based on the Green's function approach with suitable spherical harmonic basis. Next, we use this method to study optical scattering from DNA molecules attached to metallic nanoparticles placed on a substrate and compare with experimental results. We obtain fairly good agreement between theoretical predictions and the measured ellipsometric spectra. The metallic nanoparticles were used to detect the binding with DNA molecules in a microfluidic setup via spectroscopic ellipsometry (SE), and a detectable change in ellipsometric spectra was found when DNA molecules are captured on Au nanoparticles. Our theoretical simulation indicates that the coverage of Au nanosphere by a submonolayer of DNA molecules, which is modeled by a thin layer of dielectric material (which may absorb light), can lead to a small but detectable spectroscopic shift in both the Ψ and Δ spectra with more significant change in Δ spectra in agreement with experimental results. Our studies demonstrated the ultrasensitive capability of SE for sensing submonolayer coverage of DNA molecules on Au nanospheres. Hence the spectroscopic ellipsometric measurements coupled with theoretical analysis via an efficient computation method can be an effective tool for detecting DNA molecules attached on Au nanoparticles, thus achieving label-free, non-destructive, and high-sensitivity biosensing with nanoscale resolution.

Ultra-small dye-doped silica nanoparticles via modified sol-gel technique.

PubMed

Riccò, R; Nizzero, S; Penna, E; Meneghello, A; Cretaio, E; Enrichi, F

2018-01-01

In modern biosensing and imaging, fluorescence-based methods constitute the most diffused approach to achieve optimal detection of analytes, both in solution and on the single-particle level. Despite the huge progresses made in recent decades in the development of plasmonic biosensors and label-free sensing techniques, fluorescent molecules remain the most commonly used contrast agents to date for commercial imaging and detection methods. However, they exhibit low stability, can be difficult to functionalise, and often result in a low signal-to-noise ratio. Thus, embedding fluorescent probes into robust and bio-compatible materials, such as silica nanoparticles, can substantially enhance the detection limit and dramatically increase the sensitivity. In this work, ultra-small fluorescent silica nanoparticles (NPs) for optical biosensing applications were doped with a fluorescent dye, using simple water-based sol-gel approaches based on the classical Stöber procedure. By systematically modulating reaction parameters, controllable size tuning of particle diameters as low as 10 nm was achieved. Particles morphology and optical response were evaluated showing a possible single-molecule behaviour, without employing microemulsion methods to achieve similar results. Graphical abstractWe report a simple, cheap, reliable protocol for the synthesis and systematic tuning of ultra-small (< 10 nm) dye-doped luminescent silica nanoparticles.

A small-molecule-linked DNA-graphene oxide-based fluorescence-sensing system for detection of biotin.

PubMed

Zhang, Hao; Li, Yan; Su, Xingguang

2013-11-15

In this paper, we establish a novel fluorescence-sensing system for the detection of biotin based on the interaction between DNA and graphene oxide and on protection of the terminal of the biotinylated single-stranded DNA fluorescent probe by streptavidin. In this system, streptavidin binds to the biotinylated DNA, which protects the DNA from hydrolysis by exonuclease I. The streptavidin-DNA conjugate is then adsorbed to the graphene oxide resulting in the fluorescence being quenched. Upon the addition of free biotin, it competes with the labeled biotin for the binding sites of streptavidin and then the exonuclease I digests the unbound DNA probe from the 3' to the 5' terminal, releasing the fluorophore from the DNA. Because of the weak affinity between the fluorophore and graphene oxide, the fluorescence is recovered. Under optimal conditions, the fluorescence intensity is proportional to the concentration of biotin in the concentration range of 0.5-20nmol/L. The detection limit for biotin is 0.44nmol/L. The proposed fluorescence-sensing system was applied to the determination of biotin in some real samples with satisfactory reproducibility and accuracy. This work could provide a common platform for detecting small biomolecules based on protein-small molecule ligand binding. Copyright © 2013 Elsevier Inc. All rights reserved.

Distribution of Particles, Small Molecules and Polymeric Formulation Excipients in the Suprachoroidal Space after Microneedle Injection

PubMed Central

Chiang, Bryce; Venugopal, Nitin; Edelhauser, Henry F.; Prausnitz, Mark R.

2016-01-01

The purpose of this work was to determine the effect of injection volume, formulation composition, and time on circumferential spread of particles, small molecules and polymeric formulation excipients in the suprachoroidal space (SCS) after microneedle injection into New Zealand White rabbit eyes ex vivo and in vivo. Microneedle injections of 25–150 μL Hank’s Balanced Salt Solution (HBSS) containing 0.2 μm red-fluorescent particles and a model small molecule (fluorescein) were performed in rabbit eyes ex vivo, and visualized via flat mount. Particles with diameters of 0.02 – 2 μm were co-injected into SCS in vivo with fluorescein or a polymeric formulation excipient: fluorescein isothiocyanate (FITC)-labeled Discovisc or FITC-labeled carboxymethyl cellulose (CMC). Fluorescent fundus images were acquired over time to determine area of particle, fluorescein and polymeric formulation excipient spread, as well as their co-localization. We found that fluorescein covered a significantly larger area than co-injected particles when suspended in HBSS, and that this difference was present from 3 min post-injection onwards. We further showed that there was no difference in initial area covered by FITC-Discovisc and particles; the transport time (i.e., the time until the FITC-Discovisc and particle area began dissociating) was 2 d. There was also no difference in initial area covered by FITC-CMC and particles; the transport time in FITC-CMC was 4 d. We also found that particle size (20 nm – 2 μm) had no effect on spreading area when delivered in HBSS or Discovisc. We conclude that (i) the area of particle spread in SCS during injection generally increased with increasing injection volume, was unaffected by particle size and was significantly less than the area of fluorescein spread, (ii) particles suspended in low-viscosity HBSS formulation were entrapped in the SCS after injection, whereas fluorescein was not and (iii) particles co-injected with viscous polymeric formulation excipients co-localized near the site of injection in the SCS, continued to co-localize while spreading over larger areas for 2 – 4 days, and then no longer co-localized as the polymeric formulation excipients were cleared within 1 – 3 weeks and the particles remained largely in place. These data suggest that particles encounter greater barriers to flow in SCS compared to molecules and that co-localization of particles and polymeric formulation excipients allow spreading over larger areas of the SCS until the particles and excipients dissociate. PMID:27742547

Distribution of particles, small molecules and polymeric formulation excipients in the suprachoroidal space after microneedle injection.

PubMed

Chiang, Bryce; Venugopal, Nitin; Edelhauser, Henry F; Prausnitz, Mark R

2016-12-01

The purpose of this work was to determine the effect of injection volume, formulation composition, and time on circumferential spread of particles, small molecules, and polymeric formulation excipients in the suprachoroidal space (SCS) after microneedle injection into New Zealand White rabbit eyes ex vivo and in vivo. Microneedle injections of 25-150 μL Hank's Balanced Salt Solution (HBSS) containing 0.2 μm red-fluorescent particles and a model small molecule (fluorescein) were performed in rabbit eyes ex vivo, and visualized via flat mount. Particles with diameters of 0.02-2 μm were co-injected into SCS in vivo with fluorescein or a polymeric formulation excipient: fluorescein isothiocyanate (FITC)-labeled Discovisc or FITC-labeled carboxymethyl cellulose (CMC). Fluorescent fundus images were acquired over time to determine area of particle, fluorescein, and polymeric formulation excipient spread, as well as their co-localization. We found that fluorescein covered a significantly larger area than co-injected particles when suspended in HBSS, and that this difference was present from 3 min post-injection onwards. We further showed that there was no difference in initial area covered by FITC-Discovisc and particles; the transport time (i.e., the time until the FITC-Discovisc and particle area began dissociating) was 2 d. There was also no difference in initial area covered by FITC-CMC and particles; the transport time in FITC-CMC was 4 d. We also found that particle size (20 nm-2 μm) had no effect on spreading area when delivered in HBSS or Discovisc. We conclude that (i) the area of particle spread in SCS during injection generally increased with increasing injection volume, was unaffected by particle size, and was significantly less than the area of fluorescein spread, (ii) particles suspended in low-viscosity HBSS formulation were entrapped in the SCS after injection, whereas fluorescein was not and (iii) particles co-injected with viscous polymeric formulation excipients co-localized near the site of injection in the SCS, continued to co-localize while spreading over larger areas for 2-4 days, and then no longer co-localized as the polymeric formulation excipients were cleared within 1-3 weeks and the particles remained largely in place. These data suggest that particles encounter greater barriers to flow in SCS compared to molecules and that co-localization of particles and polymeric formulation excipients allows spreading over larger areas of the SCS until the particles and excipients dissociate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recent Advances in Biosensing With Photonic Crystal Surfaces: A Review

PubMed Central

Cunningham, B.T.; Zhang, M.; Zhuo, Y.; Kwon, L.; Race, C.

2016-01-01

Photonic crystal surfaces that are designed to function as wavelength-selective optical resonators have become a widely adopted platform for label-free biosensing, and for enhancement of the output of photon-emitting tags used throughout life science research and in vitro diagnostics. While some applications, such as analysis of drug-protein interactions, require extremely high resolution and the ability to accurately correct for measurement artifacts, others require sensitivity that is high enough for detection of disease biomarkers in serum with concentrations less than 1 pg/ml. As the analysis of cells becomes increasingly important for studying the behavior of stem cells, cancer cells, and biofilms under a variety of conditions, approaches that enable high resolution imaging of live cells without cytotoxic stains or photobleachable fluorescent dyes are providing new tools to biologists who seek to observe individual cells over extended time periods. This paper will review several recent advances in photonic crystal biosensor detection instrumentation and device structures that are being applied towards direct detection of small molecules in the context of high throughput drug screening, photonic crystal fluorescence enhancement as utilized for high sensitivity multiplexed cancer biomarker detection, and label-free high resolution imaging of cells and individual nanoparticles as a new tool for life science research and single-molecule diagnostics. PMID:27642265

Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.

PubMed

DeRocco, Vanessa; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A; Weninger, Keith

2010-11-01

To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.

Four-color single molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes

PubMed Central

DeRocco, Vanessa C.; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A.; Weninger, Keith

2010-01-01

To allow studies of conformational changes within multi-molecular complexes, we present a simultaneous, 4-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera based, wide-field detection. We further demonstrate labeling histidine-tagged proteins non-covalently with tris-Nitrilotriacetic acid (tris-NTA) conjugated dyes to achieve single molecule detection. We combine these methods to co-localize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes. PMID:21091445

Light-Activated Staudinger–Bertozzi Ligation within Living Animals

PubMed Central

Shah, Lisa; Laughlin, Scott T.; Carrico, Isaac S.

2016-01-01

The ability to regulate small molecule chemistry in vivo will enable new avenues of exploration in imaging and pharmacology. However, realization of these goals will require reactions with high specificity and precise control. Here we demonstrate photocontrol over the highly specific Staudinger–Bertozzi ligation in vitro and in vivo. Our simple approach, photocaging the key phosphine atom, allows for the facile production of reagents with photochemistry that can be engineered for specific applications. The resulting compounds, which are both stable and efficiently activated, enable the spatial labeling of metabolically introduced azides in vitro and on live zebrafish. PMID:27010217

NMR signal enhancement by effective SABRE labeling of oligopeptides.

PubMed

Ratajczyk, Tomasz; Gutmann, Torsten; Bernatowicz, Piotr; Buntkowsky, Gerd; Frydel, Jaroslaw; Fedorczyk, Bartlomiej

2015-09-01

Signal amplification by reversible exchange (SABRE) can enhance nuclear magnetic resonance signals by several orders of magnitude. However, until now this was limited to a small number of model target molecules. Here, a new convenient method for SABRE activation applicable to a variety of synthetic model oligopeptides is demonstrated. For the first time, a highly SABRE-active pyridine-based biocompatible molecular framework is incorporated into synthetic oligopeptides. The SABRE activity is preserved, demonstrating the importance of such earmarking. Finally, a crucial exchange process responsible for SABRE activity is identified and discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proximity labeling of cis-ligands of CD22/Siglec-2 reveals stepwise α2,6 sialic acid-dependent and -independent interactions.

PubMed

Alborzian Deh Sheikh, Amin; Akatsu, Chizuru; Imamura, Akihiro; Abdu-Allah, Hajjaj H M; Takematsu, Hiromu; Ando, Hiromune; Ishida, Hideharu; Tsubata, Takeshi

2018-01-01

Lectins expressed on the cell surface are often bound and regulated by the membrane molecules containing the glycan ligands on the same cell (cis-ligands). However, molecular nature and function of cis-ligands are generally poorly understood partly because of weak interaction between lectins and glycan ligands. Cis-ligands are most extensively studied in CD22 (also known as Siglec-2), an inhibitory B lymphocyte receptor specifically recognizing α2,6 sialic acids. CD22, CD45 and IgM are suggested to be ligands of CD22. Here we labeled molecules in the proximity of CD22 in situ on B cell surface using biotin-tyramide. Molecules including CD22, CD45 and IgM were labeled in wild-type but not ST6GalI -/- B cells that lack α2,6 sialic acids, indicating that these molecules associate with CD22 by lectin-glycan interaction, and are therefore cis-ligands. In ST6GalI -/- B cells, these cis-ligands are located in a slightly more distance from CD22. Thus, the lectin-glycan interaction recruits cis-ligands already located in the relative proximity of CD22 through non-lectin-glycan interaction to the close proximity. Moreover, cis-ligands are labeled in Cmah -/- B cells that lack Neu5Gc preferred by mouse CD22 as efficiently as in wild-type B cells, indicating that very low affinity lectin-glycan interaction is sufficient for recruiting cis-ligands, and can be detected by proximity labeling. Thus, proximity labeling with tyramide appears to be a useful method to identify cis-ligands and to analyze their interaction with the lectins. Copyright © 2017 Elsevier Inc. All rights reserved.

Overview of Single-Molecule Speckle (SiMS) Microscopy and Its Electroporation-Based Version with Efficient Labeling and Improved Spatiotemporal Resolution.

PubMed

Yamashiro, Sawako; Watanabe, Naoki

2017-07-06

Live-cell single-molecule imaging was introduced more than a decade ago, and has provided critical information on remodeling of the actin cytoskeleton, the motion of plasma membrane proteins, and dynamics of molecular motor proteins. Actin remodeling has been the best target for this approach because actin and its associated proteins stop diffusing when assembled, allowing visualization of single-molecules of fluorescently-labeled proteins in a state specific manner. The approach based on this simple principle is called Single-Molecule Speckle (SiMS) microscopy. For instance, spatiotemporal regulation of actin polymerization and lifetime distribution of actin filaments can be monitored directly by tracking actin SiMS. In combination with fluorescently labeled probes of various actin regulators, SiMS microscopy has contributed to clarifying the processes underlying recycling, motion and remodeling of the live-cell actin network. Recently, we introduced an electroporation-based method called eSiMS microscopy, with high efficiency, easiness and improved spatiotemporal precision. In this review, we describe the application of live-cell single-molecule imaging to cellular actin dynamics and discuss the advantages of eSiMS microscopy over previous SiMS microscopy.

Nonlinear nonlocal infrared plasmonic arrays for pump-probe studies on protein monolayers

NASA Astrophysics Data System (ADS)

Erramilli, Shyamsunder; Adato, Ronen; Gabel, Alan; Yanik, Ahmet Ali; Altug, Hatice; Hong, Mi K.

2010-03-01

Infrared spectroscopy is an exquisite bond-specific tool for studying biomolecules with characteristic vibrational normal modes that serve as a molecular ``fingerprint''. Intrinsic absorption cross-sections for proteins are significant (˜10-19 -10-21 cm^2), although small compared to label-based fluorescence methods. We have shown that carefully designed plasmonic nanoantenna arrays can enhance the vibrational signatures by ˜ 10^5 (Adato et al, Proc Natl Acad Sci USA, 2009). Theoretical modeling combined with polarized FTIR-microscopy show that enhancement is due both to localized effects and nonlocal collective effects, governed by the dielectric properties of silicon and gold nanoantennae, coupled to protein molecules. The resonance properties can be modulated by photoinduced excitation of charge carriers and excitons, causing both a shift in the resonance frequency and a change in the enhancement factor. An ultrafast visible pump laser can then be used to extend visible pump-infrared probe studies to protein molecules even when the molecules lack a chromophore. This provides a toolkit for biophysical studies in which the nonlinear, nonlocal interaction between a 35-fs visible or near-infrared laser and the designed plasmonic nanoantenna arrays are used to study dynamics of protein molecules.

Influence of DOTA chelator position on biodistribution and targeting properties of (111)In-labeled synthetic anti-HER2 affibody molecules.

PubMed

Perols, Anna; Honarvar, Hadis; Strand, Joanna; Selvaraju, Ramkumar; Orlova, Anna; Karlström, Amelie Eriksson; Tolmachev, Vladimir

2012-08-15

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with (99m)Tc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K(D) values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1)-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-Z(HER2:S1) (64 °C) and DOTA-K58-Z(HER2:S1) (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with (111)In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1)and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.

Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.

PubMed

Lemcke, Heiko; Peukert, Janine; Voronina, Natalia; Skorska, Anna; Steinhoff, Gustav; David, Robert

2016-09-01

Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens.

PubMed

Li, Jie; Lin, Shixian; Wang, Jie; Jia, Shang; Yang, Maiyun; Hao, Ziyang; Zhang, Xiaoyu; Chen, Peng R

2013-05-15

Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

Frequency-domain photoacoustic and fluorescence microscopy: application on labeled and unlabeled cells

NASA Astrophysics Data System (ADS)

Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Pfeffer, Karoline; Wohlfarth, Sven; Hannesschläger, Günther; Klar, Thomas A.; Berer, Thomas

2018-02-01

In this paper, multimodal optical-resolution frequency-domain photoacoustic and fluorescence scanning microscopy is presented on labeled and unlabeled cells. In many molecules, excited electrons relax radiatively and non-radiatively, leading to fluorescence and photoacoustic signals, respectively. Both signals can then be detected simultaneously. There also exist molecules, e.g. hemoglobin, which do not exhibit fluorescence, but provide photoacoustic signals solely. Other molecules, especially fluorescent dyes, preferentially exhibit fluorescence. The fluorescence quantum yield of a molecule and with it the strength of photoacoustic and fluorescence signals depends on the local environment, e.g. on the pH. Therefore, the local distribution of the simultaneously recorded photoacoustic and fluorescence signals may be used in order to obtain information about the local chemistry.

Detecting Intramolecular Conformational Dynamics of Single Molecules in Short Distance Range with Sub-Nanometer Sensitivity

PubMed Central

Zhou, Ruobo; Kunzelmann, Simone; Webb, Martin R.; Ha, Taekjip

2011-01-01

Single molecule detection is useful for characterizing nanoscale objects such as biological macromolecules, nano-particles and nano-devices with nano-meter spatial resolution. Fluorescence resonance energy transfer (FRET) is widely used as a single-molecule assay to monitor intramolecular dynamics in the distance range of 3–8 nm. Here we demonstrate that self-quenching of two rhodamine derivatives can be used to detect small conformational dynamics corresponding to sub-nanometer distance changes in a FRET-insensitive short range at the single molecule level. A ParM protein mutant labeled with two rhodamines works as a single molecule ADP sensor which has 20 times brighter fluorescence signal in the ADP bound state than the unbound state. Single molecule time trajectories show discrete transitions between fluorescence on and off states that can be directly ascribed to ADP binding and dissociation events. The conformational changes observed with 20:1 contrast are only 0.5 nm in magnitude and are between crystallographic distances of 1.6 nm and 2.1 nm, demonstrating exquisite sensitivity to short distance scale changes. The systems also allowed us to gain information on the photophysics of self-quenching induced by rhodamine stacking: (1) photobleaching of either of the two rhodamines eliminates quenching of the other rhodamine fluorophore and (2) photobleaching from the highly quenched, stacked state is only two-fold slower than from the unstacked state. PMID:22023515

Electron Paramagnetic Resonance of a Single NV Nanodiamond Attached to an Individual Biomolecule

NASA Astrophysics Data System (ADS)

Teeling-Smith, Richelle M.; Jung, Young Woo; Scozzaro, Nicolas; Cardellino, Jeremy; Rampersaud, Isaac; North, Justin A.; Šimon, Marek; Bhallamudi, Vidya P.; Rampersaud, Arfaan; Johnston-Halperin, Ezekiel; Poirier, Michael G.; Hammel, P. Chris

2016-05-01

A key limitation of electron paramagnetic resonance (EPR), an established and powerful tool for studying atomic-scale biomolecular structure and dynamics is its poor sensitivity, samples containing in excess of 10^12 labeled biomolecules are required in typical experiments. In contrast, single molecule measurements provide improved insights into heterogeneous behaviors that can be masked by ensemble measurements and are often essential for illuminating the molecular mechanisms behind the function of a biomolecule. We report EPR measurements of a single labeled biomolecule that merge these two powerful techniques. We selectively label an individual double-stranded DNA molecule with a single nanodiamond containing nitrogen-vacancy (NV) centers, and optically detect the paramagnetic resonance of NV spins in the nanodiamond probe. Analysis of the spectrum reveals that the nanodiamond probe has complete rotational freedom and that the characteristic time scale for reorientation of the nanodiamond probe is slow compared to the transverse spin relaxation time. This demonstration of EPR spectroscopy of a single nanodiamond labeled DNA provides the foundation for the development of single molecule magnetic resonance studies of complex biomolecular systems.

Sensing small molecule interactions with lipid membranes by local pH modulation.

PubMed

Huang, Da; Zhao, Tao; Xu, Wei; Yang, Tinglu; Cremer, Paul S

2013-11-05

Herein, we utilized a label-free sensing platform based on pH modulation to detect the interactions between tetracaine, a positively charged small molecule used as a local anesthetic, and planar supported lipid bilayers (SLBs). The SLBs were patterned inside a flow cell, allowing for various concentrations of tetracaine to be introduced over the surface in a buffer solution. Studies with membranes containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) yielded an equilibrium dissociation constant value of Kd = 180 ± 47 μm for this small molecule-membrane interaction. Adding cholesterol to the SLBs decreased the affinity between tetracaine and the bilayers, while this interaction tightened when POPE (1-hexadecanoyl-2-(9-Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine) was added. Studies were also conducted with three negatively charged membrane lipids, POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)), POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (sodium salt)), and ganglioside GM1. All three measurements gave rise to a similar tightening of the apparent Kd value compared with pure POPC membranes. The lack of chemical specificity with the identity of the negatively charged lipid indicated that the tightening was largely electrostatic. Through a direct comparison with ITC measurements, it was found that the pH modulation sensor platform offers a facile, inexpensive, highly sensitive, and rapid method for the detection of interactions between putative drug candidates and lipid bilayers. As such, this technique may potentially be exploited as a screen for drug development and analysis.

Phase sensitive spectral domain interferometry for label free biomolecular interaction analysis and biosensing applications

NASA Astrophysics Data System (ADS)

Chirvi, Sajal

Biomolecular interaction analysis (BIA) plays vital role in wide variety of fields, which include biomedical research, pharmaceutical industry, medical diagnostics, and biotechnology industry. Study and quantification of interactions between natural biomolecules (proteins, enzymes, DNA) and artificially synthesized molecules (drugs) is routinely done using various labeled and label-free BIA techniques. Labeled BIA (Chemiluminescence, Fluorescence, Radioactive) techniques suffer from steric hindrance of labels on interaction site, difficulty of attaching labels to molecules, higher cost and time of assay development. Label free techniques with real time detection capabilities have demonstrated advantages over traditional labeled techniques. The gold standard for label free BIA is surface Plasmon resonance (SPR) that detects and quantifies the changes in refractive index of the ligand-analyte complex molecule with high sensitivity. Although SPR is a highly sensitive BIA technique, it requires custom-made sensor chips and is not well suited for highly multiplexed BIA required in high throughput applications. Moreover implementation of SPR on various biosensing platforms is limited. In this research work spectral domain phase sensitive interferometry (SD-PSI) has been developed for label-free BIA and biosensing applications to address limitations of SPR and other label free techniques. One distinct advantage of SD-PSI compared to other label-free techniques is that it does not require use of custom fabricated biosensor substrates. Laboratory grade, off-the-shelf glass or plastic substrates of suitable thickness with proper surface functionalization are used as biosensor chips. SD-PSI is tested on four separate BIA and biosensing platforms, which include multi-well plate, flow cell, fiber probe with integrated optics and fiber tip biosensor. Sensitivity of 33 ng/ml for anti-IgG is achieved using multi-well platform. Principle of coherence multiplexing for multi-channel label-free biosensing applications is introduced. Simultaneous interrogation of multiple biosensors is achievable with a single spectral domain phase sensitive interferometer by coding the individual sensograms in coherence-multiplexed channels. Experimental results demonstrating multiplexed quantitative biomolecular interaction analysis of antibodies binding to antigen coated functionalized biosensor chip surfaces on different platforms are presented.

Using Fluorescent Viruses for Detecting Bacteria in Water

NASA Technical Reports Server (NTRS)

Tabacco, Mary Beth; Qian, Xiaohua; Russo, Jaimie A.

2009-01-01

A method of detecting water-borne pathogenic bacteria is based partly on established molecular-recognition and fluorescent-labeling concepts, according to which bacteria of a species of interest are labeled with fluorescent reporter molecules and the bacteria can then be detected by fluorescence spectroscopy. The novelty of the present method lies in the use of bacteriophages (viruses that infect bacteria) to deliver the fluorescent reporter molecules to the bacteria of the species of interest.

Anionized and cationized hemeundecapeptides as probes for cell surface charge and permeability studies: differentiated labeling of endothelial plasmalemmal vesicles

PubMed Central

1985-01-01

To obtain small membrane markers easily accessible to the charged groups of the cell surface, we prepared, from hemeundecapeptide (HUP), three derivatives that maintain the peroxidatic activity: the anionized hemeundecapeptide, Mr 1,963, estimated diameter 1.68 nm, pl 3.5, for the detection of basic groups; and both a cationized hemeundecapeptide containing predominantly tertiary amino groups, Mr 2,215, estimated diameter 1.75 nm, pl 9.0, and a cationized hemeundecapeptide containing only primary amino groups, Mr 2,271, estimated diameter 1.75 nm, pl 10.6, for labeling acidic residues. The markers were perfused in situ in mice to label the luminal surface of fenestrated endothelium of pancreatic capillaries. Specimens were processed through the cytochemical reaction for peroxidatic activity and examined by electron microscopy. The anionized HUP and HUP (pl 4.85) marked the plasmalemma proper, the coated pits, and the membrane and diaphragms of plasmalemmal vesicles and transendothelial channels. The cationized HUP containing predominantly tertiary amino groups (pl 9.0) decorated all cell surface components with the exception of plasmalemmal vesicles and channels; the latter were, however, labeled by the cationized HUP containing only primary groups (pl 10.6), which suggests that these structures contain on their luminal surface very weak acidic residues of high pKa values. The fact that the membrane of plasmalemmal vesicles can discriminate against permeant cationic macromolecules only up to a pl of approximately 9.0 indicates that in the electrostatic restriction there is a charge limit. In the case of fenestrated capillary endothelium, the upper charge limit seems to be a pl of approximately 9.0. In these vessels, the charge discrimination is effective for molecules as small as 2 nm. PMID:3968182

Anionized and cationized hemeundecapeptides as probes for cell surface charge and permeability studies: differentiated labeling of endothelial plasmalemmal vesicles.

PubMed

Ghinea, N; Simionescu, N

1985-02-01

To obtain small membrane markers easily accessible to the charged groups of the cell surface, we prepared, from hemeundecapeptide (HUP), three derivatives that maintain the peroxidatic activity: the anionized hemeundecapeptide, Mr 1,963, estimated diameter 1.68 nm, pl 3.5, for the detection of basic groups; and both a cationized hemeundecapeptide containing predominantly tertiary amino groups, Mr 2,215, estimated diameter 1.75 nm, pl 9.0, and a cationized hemeundecapeptide containing only primary amino groups, Mr 2,271, estimated diameter 1.75 nm, pl 10.6, for labeling acidic residues. The markers were perfused in situ in mice to label the luminal surface of fenestrated endothelium of pancreatic capillaries. Specimens were processed through the cytochemical reaction for peroxidatic activity and examined by electron microscopy. The anionized HUP and HUP (pl 4.85) marked the plasmalemma proper, the coated pits, and the membrane and diaphragms of plasmalemmal vesicles and transendothelial channels. The cationized HUP containing predominantly tertiary amino groups (pl 9.0) decorated all cell surface components with the exception of plasmalemmal vesicles and channels; the latter were, however, labeled by the cationized HUP containing only primary groups (pl 10.6), which suggests that these structures contain on their luminal surface very weak acidic residues of high pKa values. The fact that the membrane of plasmalemmal vesicles can discriminate against permeant cationic macromolecules only up to a pl of approximately 9.0 indicates that in the electrostatic restriction there is a charge limit. In the case of fenestrated capillary endothelium, the upper charge limit seems to be a pl of approximately 9.0. In these vessels, the charge discrimination is effective for molecules as small as 2 nm.

Optical characterization of porous silicon microcavities for glucose oxidase biosensing

NASA Astrophysics Data System (ADS)

Palestino, G.; Agarwal, V.; Garcia, D. B.; Legros, R.; Pérez, E.; Gergely, C.

2008-04-01

PSi microcavity (PSiMc) is characterized by a narrow resonance peak in the optical spectrum that is very sensitive to small changes in the refractive index. We report that the resonant optical cavities of PSi structures can be used to enhance the detection of labeled fluorescent biomolecules. Various PSi configurations were tested in order to compare the optical response of the PSi devices to the capture of organic molecules. Morphological and topographical analyses were performed on PSiMc using Atomic Force (AFM) and Scanning Electron (SEM) microscopies. The heterogeneity in pores lengths resulting from etching process assures a better penetration of larger molecules into the pores and sensor sensitivity depends on the pore size. Molecular detection is monitored by the successive red shifts in the reflectance spectra after the stabilization of PSiMc with 3-aminopropyltriethoxysilane (APTES). The glucose oxidase was cross linked into the PSiMc structures following a silane-glutaraldehyde (GTA) chemistry.

Nanobiodevices for Biomolecule Analysis and Imaging

NASA Astrophysics Data System (ADS)

Yasui, Takao; Kaji, Noritada; Baba, Yoshinobu

2013-06-01

Nanobiodevices have been developed to analyze biomolecules and cells for biomedical applications. In this review, we discuss several nanobiodevices used for disease-diagnostic devices, molecular imaging devices, regenerative medicine, and drug-delivery systems and describe the numerous advantages of nanobiodevices, especially in biological, medical, and clinical applications. This review also outlines the fabrication technologies for nanostructures and nanomaterials, including top-down nanofabrication and bottom-up molecular self-assembly approaches. We describe nanopillar arrays and nanowall arrays for the ultrafast separation of DNA or protein molecules and nanoball materials for the fast separation of a wide range of DNA molecules, and we present examples of applications of functionalized carbon nanotubes to obtain information about subcellular localization on the basis of mobility differences between free fluorophores and fluorophore-labeled carbon nanotubes. Finally, we discuss applications of newly synthesized quantum dots to the screening of small interfering RNA, highly sensitive detection of disease-related proteins, and development of cancer therapeutics and diagnostics.

Detecting single DNA molecule interactions with optical microcavities (Presentation Recording)

NASA Astrophysics Data System (ADS)

Vollmer, Frank

2015-09-01

Detecting molecules and their interactions lies at the heart of all biosensor devices, which have important applications in health, environmental monitoring and biomedicine. Achieving biosensing capability at the single molecule level is, moreover, a particularly important goal since single molecule biosensors would not only operate at the ultimate detection limit by resolving individual molecular interactions, but they could also monitor biomolecular properties which are otherwise obscured in ensemble measurements. For example, a single molecule biosensor could resolve the fleeting interaction kinetics between a molecule and its receptor, with immediate applications in clinical diagnostics. We have now developed a label-free biosensing platform that is capable of monitoring single DNA molecules and their interaction kinetics[1], hence achieving an unprecedented sensitivity in the optical domain, Figure 1. We resolve the specific contacts between complementary oligonucleotides, thereby detecting DNA strands with less than 2.4 kDa molecular weight. Furthermore we can discern strands with single nucleotide mismatches by monitoring their interaction kinetics. Our device utilizes small glass microspheres as optical transducers[1,2, 3], which are capable of increasing the number of interactions between a light beam and analyte molecules. A prism is used to couple the light beam into the microsphere. Ourr biosensing approach resolves the specific interaction kinetics between single DNA fragments. The optical transducer is assembled in a simple three-step protocol, and consists of a gold nanorod attached to a glass microsphere, where the surface of the nanorod is further modified with oligonucleotide receptors. The interaction kinetics of an oligonucleotide receptor with DNA fragments in the surrounding aqueous solution is monitored at the single molecule level[1]. The light remains confined inside the sphere where it is guided by total internal reflections along a circular optical path, similar to an acoustic wave guided along the wall of St. Paul's Cathedral. These so called whispering gallery modes (WGM) propagate with little loss, so that even a whisper can be heard on the other side of the gallery. In the optical case, the light beam can travel many thousand times around the inside of the microsphere before being scattered or absorbed, thereby making numerous interactions with an analyte molecule, bound to microsphere from surrounding sample solution. The most part of the light intensity, however, remains inside the microsphere, just below the reflecting glass surface, resulting in a relatively weak interaction between the light and the bound molecule. To enhance this interaction further, we attach tiny 42 nm x 12 nm gold nanorods to the glass surface. When passing a nanorod, the lightwave induces oscillations of conduction electrons, resulting in so called plasmon resonance. These nanorod plasmons greatly enhance the light intensity on the nanorod, so that the interaction of the light with a molecule attached to the nanorod is also enhanced[4-6]. This enhanced interaction results in an increase in sensitivity by more than a factor of one thousand, putting our experiments of single DNA molecule detection within reach. For the specific detection of nucleic acids, we attach single-stranded DNA to the nanorod and immerse our device in a liquid solution. When a matching, i.e. complementary DNA fragment binds from solution to the "bait" on the nanorod, the enhanced interaction with the light results in an observable shift of the WGM wavelength. Since light propagates in a WGM only for a very precise resonance wavelength or frequency, this shift can be detected with great accuracy[3]. On our current biosensor platform, we detect wavelength shifts with an accuracy of less than one femtometer, resulting in an extremely high sensitivity for biosensing, which we leverage for the specific detection of single 8 mer oligonucleotides as well as the detection of less than 1 kDa intercalating small molecules[1]. [1] M. D. Baaske, M. R. Foreman, and F. Vollmer, "Single molecule nucleic acid interactions monitored on a label-free microcavity biosensing platform," Nature Nanotechnology, vol. 9, pp. 933-939, 2014. [2] Y. Wu, D. Y. Zhang, P. Yin, and F. Vollmer, "Ultraspecific and Highly Sensitive Nucleic Acid Detection by Integrating a DNA Catalytic Network with a Label-Free Microcavity," Small, vol. 10, pp. 2067-2076, 2014. [3] M. R. Foreman, W.-L. Jin, and F. Vollmer, "Optimizing Detection Limits in Whispering Gallery Mode Biosensing," Optics Express, vol. 22, pp. 5491-5511, 2014. [4] M. A. Santiago-Cordoba, S. V. Boriskina, F. Vollmer, and M. C. Demirel, "Nanoparticle-based protein detection by optical shift of a resonant microcavity," Applied Physics Letters, vol. 99, Aug 2011. [5] M. R. Foreman and F. Vollmer, "Theory of resonance shifts of whispering gallery modes by arbitrary plasmonic nanoparticles," New Journal of Physics, vol. 15, p. 083006, Aug 2013. [6] M. R. Foreman and F. Vollmer "Level repulsion in hybrid photonic-plasmonic microresonators for enhanced biodetection" Phys. Rev. A 88, 023831 (2013).

Current Status of Prostate-Specific Membrane Antigen Targeting in Nuclear Medicine: Clinical Translation of Chelator Containing Prostate-Specific Membrane Antigen Ligands Into Diagnostics and Therapy for Prostate Cancer.

PubMed

Kratochwil, Clemens; Afshar-Oromieh, Ali; Kopka, Klaus; Haberkorn, Uwe; Giesel, Frederik L

2016-09-01

The prostate-specific membrane antigen (PSMA) is expressed by approximately 90% of prostate carcinomas. The expression correlates with unfavorable prognostic factors, such as a high Gleason score, infiltrative growth, metastasis, and hormone-independence. The high specificity, especially in the undifferentiated stage, makes it an excellent target for diagnosis and therapy. Therefore, antibodies and small molecule inhibitors have been developed for imaging and therapy. In 2011 PSMA-11, a ligand that consists of the Glu-urea-motif and the chelator HBED-CC, which can be exclusively radiolabeled with (68)Ga for PET imaging, presented the clinical breakthrough for prostate cancer diagnostics. In two large diagnostic studies (n = 319 and n = 248) PET/CT with PSMA-11 successfully localized the recurrent tumor in approximately 90% of patients with biochemical relapse. Integrating PSMA-PET/CT into the planning phase of radiotherapy, the treatment concept is changed in 30%-50% of the patients. The combination of the Glu-urea-motif with DOTA, which can be labeled with several diagnostic and therapeutic radionuclides, opened new avenues for therapeutic usage of the small-molecule PSMA ligands. In the beginning of 2016, there are four confirmative reports (n = 19, n = 24, n = 30, and n = 56) from four different centers reporting a PSA response in approximately 70% of patients treated with (177)Lu-labeled PSMA ligands. In conclusion, the data available up to now indicate a widespread use of PSMA ligands for diagnostic applications with respect to staging, detection of recurrence, or metastases in patients with rising tumor markers and for therapy in case of failure of guideline-compliant treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

PubMed

Gao, Zhuangqiang; Qiu, Zhenli; Lu, Minghua; Shu, Jian; Tang, Dianping

2017-03-15

This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer. Copyright © 2016 Elsevier B.V. All rights reserved.

The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα.

PubMed

Wang, Yan-Yang; Zhou, Shun; Zhao, Ren; Hai, Ping; Zhe, Hong

2016-01-01

CDDO-Me has exhibited a potent anticancer effect in human esophageal squamous cell carcinoma (ESCC) cells in our previous study, but the molecular interactome remains elusive. We applied the approach of stable-isotope labeling by amino acids in cell culture (SILAC) to assess the proteomic responses of CDDO-Me treatment in human ESCC Ec109 cells. The data were subsequently validated using Western blot assay. The results of our study revealed that CDDO-Me increased the expression level of 543 protein molecules, but decreased the expression level of 709 protein molecules in Ec109 cells. Among these modulated protein molecules, calcium/calmodulin-dependent protein kinase type II subunit α (CaMKIIα) was highly expressed in all tested ESCC cell lines, whereas its expression levels were substantially lower in normal control cell line. Its silencing by small interfering RNA inhibited CDDO-Me induced apoptosis and autophagy in ESCC cells. Collectively, these data demonstrate that the therapeutic response of CDDO-Me in the human ESCC cells is mediated by CaMKIIα.

Proximity-dependent isothermal cycle amplification for small-molecule detection based on surface enhanced Raman scattering.

PubMed

Li, Ying; Zeng, Yan; Mao, Yaning; Lei, Chengcun; Zhang, Shusheng

2014-01-15

A novel proximity-dependent isothermal cycle amplification (PDICA) strategy has been proposed and successfully used for the determination of cocaine coupled with surface enhanced Raman scattering (SERS). For enhancing the SERS signal, Raman dye molecules modified bio-barcode DNA and gold nanoparticles (AuNPs) are used to prepare the Raman probes. Magnetic beads (MBs) are used as the carrier of amplification template and signal output products for circumventing the problem of high background induced by excess bio-barcode DNA. In the presence of target molecules, two label-free proximity probes can hybridize with each other and subsequently opens the hairpin connector-probe to perform the PDICA reaction including the target recycling amplification and strand-displacement amplification. As a result, abundant AuNPs Raman probes can be anchored on the surface of MBs and a low detection limit of 0.1 nM for cocaine is obtained. This assay also exhibits an excellent selectivity and has been successfully performed in human serum, which confirms the reliability and practicality of this protocol. © 2013 Elsevier B.V. All rights reserved.

Single molecule optical measurements of orientation and rotations of biological macromolecules.

PubMed

Shroder, Deborah Y; Lippert, Lisa G; Goldman, Yale E

2016-11-22

Subdomains of macromolecules often undergo large orientation changes during their catalytic cycles that are essential for their activity. Tracking these rearrangements in real time opens a powerful window into the link between protein structure and functional output. Site-specific labeling of individual molecules with polarized optical probes and measurement of their spatial orientation can give insight into the crucial conformational changes, dynamics, and fluctuations of macromolecules. Here we describe the range of single molecule optical technologies that can extract orientation information from these probes, review the relevant types of probes and labeling techniques, and highlight the advantages and disadvantages of these technologies for addressing specific inquiries.

Expedited Synthesis of Fluorine-18 Labeled Phenols. A Missing Link in PET Radiochemistry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katzenellenbogen, John A.; Zhou, Dong

Fluorine-18 (F-18) is arguably the most valuable radionuclide for positron emission tomographic (PET) imaging. However, while there are many methods for labeling small molecules with F-18 at aliphatic positions and on electron-deficient aromatic rings, there are essentially no reliable and practical methods to label electron-rich aromatic rings such as phenols, with F-18 at high specific activity. This is disappointing because fluorine-labeled phenols are found in many drugs; there are also many interesting plant metabolites and hormones that contain either phenols or other electron-rich aromatic systems such as indoles whose metabolism, transport, and distribution would be interesting to study if theymore » could readily be labeled with F-18. Most approaches to label phenols with F-18 involve the labeling of electron-poor precursor arenes by nucleophilic aromatic substitution, followed by subsequent conversion to phenols by oxidation or other multi-step sequences that are often inefficient and time consuming. Thus, the lack of good methods for labeling phenols and other electron-rich aromatics with F-18 at high specific activity represents a significant methodological gap in F-18 radiochemistry that can be considered a “Missing Link in PET Radiochemistry”. The objective of this research project was to develop and optimize a series of unusual synthetic transformations that will enable phenols (and other electron-rich aromatic systems) to be labeled with F-18 at high specific activity, rapidly, reliably, and conveniently, thereby bridging this gap. Through the studies conducted with support of this project, we have substantially advanced synthetic methodology for the preparation of fluorophenols. Our progress is presented in detail in the sections below, and much has been published or presented publication; other components are being prepared for publication. In essence, we have developed a completely new method to prepare o-fluorophenols from non-aromatic precursors (diazocyclohexenones) by a novel reaction sequence that uses fluoride ion as a precursor and various activating electrophiles, and we have improved methods for the preparation of heterodiaryl iodonium salts. Both methods have been used to prepare interesting potential radiotracers. Other advances have been made in labeling dendrimeric nanoparticle structures of increasing interest for multimodal imaging and in advancing labeling through fluorosilane bonds. Thus, the progress we have made substantially fills the significant gap in PET radiochemistry that we originally identified, and it provides for the field new methodology that can be applied to a number of current challenges, including the preparation of several molecules of interest as radiotracers, such as 2-[18F]Fluoroestradiol (2-FES) and m-fluorotyrosine, which we have illustrated. These methods can be used by any skilled radiochemist interesting in preparing these agents or similar fluorine-18 labeled electron-rich arene systems of interested for PET biological imaging in the most general sense.« less

Quantitative gene expression analysis in Caenorhabditis elegans using single molecule RNA FISH.

PubMed

Bolková, Jitka; Lanctôt, Christian

2016-04-01

Advances in fluorescent probe design and synthesis have allowed the uniform in situ labeling of individual RNA molecules. In a technique referred to as single molecule RNA FISH (smRNA FISH), the labeled RNA molecules can be imaged as diffraction-limited spots and counted using image analysis algorithms. Single RNA counting has provided valuable insights into the process of gene regulation. This microscopy-based method has often revealed a high cell-to-cell variability in expression levels, which has in turn led to a growing interest in investigating the biological significance of gene expression noise. Here we describe the application of the smRNA FISH technique to samples of Caenorhabditis elegans, a well-characterized model organism. Copyright © 2015 Elsevier Inc. All rights reserved.

Proteomic response to 5,6-dimethylxanthenone 4-acetic acid (DMXAA, vadimezan) in human non-small cell lung cancer A549 cells determined by the stable-isotope labeling by amino acids in cell culture (SILAC) approach

PubMed Central

Pan, Shu-Ting; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Yang, Yin-Xue; Wang, Dong; Qiu, Jia-Xuan; Zhou, Shu-Feng

2015-01-01

5,6-Dimethylxanthenone 4-acetic acid (DMXAA), also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC) and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC) approach. The proteomic data showed that treatment with DMXAA modulated the expression of 588 protein molecules in A549 cells, with 281 protein molecules being up regulated and 306 protein molecules being downregulated. Ingenuity pathway analysis (IPA) identified 256 signaling pathways and 184 cellular functional proteins that were regulated by DMXAA in A549 cells. These targeted molecules and signaling pathways were mostly involved in cell proliferation and survival, redox homeostasis, sugar, amino acid and nucleic acid metabolism, cell migration, and invasion and programed cell death. Subsequently, the effects of DMXAA on cell cycle distribution, apoptosis, autophagy, and reactive oxygen species (ROS) generation were experimentally verified. Flow cytometric analysis showed that DMXAA significantly induced G1 phase arrest in A549 cells. Western blotting assays demonstrated that DMXAA induced apoptosis via a mitochondria-dependent pathway and promoted autophagy, as indicated by the increased level of cytosolic cytochrome c, activation of caspase 3, and enhanced expression of beclin 1 and microtubule-associated protein 1A/1B-light chain 3 (LC3-II) in A549 cells. Moreover, DMXAA significantly promoted intracellular ROS generation in A549 cells. Collectively, this SILAC study quantitatively evaluates the proteomic response to treatment with DMXAA that helps to globally identify the potential molecular targets and elucidate the underlying mechanism of DMXAA in the treatment of NSCLC. PMID:25733813

Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

PubMed

Koh, Hye Ran; Wang, Xinlei; Myong, Sua

2016-08-01

TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. Copyright © 2016 Elsevier Inc. All rights reserved.

Selective detection of carbon-13, nitrogen-15, and deuterium labeled metabolites by capillary gas chromatography-chemical reaction interface/mass spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chace, D.H.; Abramson, F.P.

1989-12-15

We have applied a new chemical reaction interface/mass spectrometer technique (CRIMS) to the selective detection of 13C-, 15N-, and 2H-labeled phenytoin and its metabolites in urine following separation by capillary gas chromatography. The microwave-powered chemical reaction interface converts materials from their original forms into small molecules whose mass spectra serve to identify and quantify the nuclides that make up each analyte. The presence of each element is followed by monitoring the isotopic variants of CO2, NO, or H2 that are produced by the chemical reaction interface. Chromatograms showing only enriched 13C and 15N were produced by subtracting the abundance ofmore » naturally occurring isotopes from the observed M + 1 signal. A selective chromatogram of 2H (D) was obtained by measuring HD at m/z 3.0219 with a resolution of 2000. Metabolites representing less than 1.5% of the total labeled compounds could be identified in the chromatogram. Detection limits from urine of 380 pg/mL of a 15N-labeled metabolite, 7 ng/mL of a 13C-labeled metabolite, and 16 ng/mL of a deuterium labeled metabolite were determined at a signal to noise ratio of 2. Depending on the isotope examined, a linear dynamic range of 250-1000 was observed using CRIMS. To identify many of these labeled peaks (metabolites), the chromatographic analysis was repeated with the chemical reaction interface turned off and mass spectra obtained at the retention times found in the CRIMS experiment. CRIMS is a new analytical method that appears to be particularly useful for metabolism studies.« less

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

Bioactive lipid coating of bone allografts directs engraftment and fate determination of bone marrow-derived cells in rat GFP chimeras.

PubMed

Das, Anusuya; Segar, Claire E; Chu, Yihsuan; Wang, Tiffany W; Lin, Yong; Yang, Chunxi; Du, Xeujun; Ogle, Roy C; Cui, Quanjun; Botchwey, Edward A

2015-09-01

Bone grafting procedures are performed to treat wounds incurred during wartime trauma, accidents, and tumor resections. Endogenous mechanisms of repair are often insufficient to ensure integration between host and donor bone and subsequent restoration of function. We investigated the role that bone marrow-derived cells play in bone regeneration and sought to increase their contributions by functionalizing bone allografts with bioactive lipid coatings. Polymer-coated allografts were used to locally deliver the immunomodulatory small molecule FTY720 in tibial defects created in rat bone marrow chimeras containing genetically-labeled bone marrow for monitoring cell origin and fate. Donor bone marrow contributed significantly to both myeloid and osteogenic cells in remodeling tissue surrounding allografts. FTY720 coatings altered the phenotype of immune cells two weeks post-injury, which was associated with increased vascularization and bone formation surrounding allografts. Consequently, degradable polymer coating strategies that deliver small molecule growth factors such as FTY720 represent a novel therapeutic strategy for harnessing endogenous bone marrow-derived progenitors and enhancing healing in load-bearing bone defects. Copyright © 2015 Elsevier Ltd. All rights reserved.

Direct assessment of the metabolic origin of carbon atoms in glutamate from illuminated leaves using 13 C-NMR.

PubMed

Abadie, Cyril; Lothier, Jérémy; Boex-Fontvieille, Edouard; Carroll, Adam; Tcherkez, Guillaume

2017-12-01

Glutamate (Glu) is the cornerstone of nitrogen assimilation and photorespiration in illuminated leaves. Despite this crucial role, our knowledge of the flux to Glu de novo synthesis is rather limited. Here, we used isotopic labelling with 13 CO 2 and 13 C-NMR analyses to examine the labelling pattern and the appearance of multi-labelled species of Glu molecules to trace the origin of C-atoms found in Glu. We also compared this with 13 C-labelling patterns in Ala and Asp, which reflect citrate (and thus Glu) precursors, that is, pyruvate and oxaloacetate. Glu appeared to be less 13 C-labelled than Asp and Ala, showing that the Glu pool was mostly formed by 'old' carbon atoms. There were modest differences in intramolecular 13 C- 13 C couplings between Glu C-2 and Asp C-3, showing that oxaloacetate metabolism to Glu biosynthesis did not involve C-atom redistribution by the Krebs cycle. The apparent carbon allocation increased with carbon net photosynthesis. However, when expressed relative to CO 2 fixation, it was clearly higher at low CO 2 while it did not change in 2% O 2 , as compared to standard conditions. We conclude that Glu production from current photosynthetic carbon represents a small flux that is controlled by the gaseous environment, typically upregulated at low CO 2 . © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Bioorthogonal Metabolic DNA Labelling using Vinyl Thioether-Modified Thymidine and o-Quinolinone Quinone Methide.

PubMed

Gubu, Amu; Li, Long; Ning, Yan; Zhang, Xiaoyun; Lee, Seonghyun; Feng, Mengke; Li, Qiang; Lei, Xiaoguang; Jo, Kyubong; Tang, Xinjing

2018-04-17

Bioorthogonal metabolic DNA labeling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Here, we report the development of a new DNA metabolic labeling strategy enabled by the catalyst-free bioorthogonal ligation using vinyl thioether modified thymidine and o-quinolinone quinone methide. With the newly designed vinyl thioether-modified thymidine (VTdT), we added labeling tags on cellular DNA, which could further be linked to fluorochromes in cells. Therefore, we successfully visualized the DNA localization within cells as well as single DNA molecules without other staining reagents. In addition, we further characterized this bioorthogonal DNA metabolic labeling using DNase I digestion, MS characterization of VTdT as well as VTdT-oQQF conjugate in cell nuclei or mitochondria. This technique provides a powerful strategy to study DNA in cells, which paves the way to achieve future spatiotemporal deciphering of DNA synthesis and functions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

PubMed Central

Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P.; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; Piehler, Jacob; Hensel, Michael

2016-01-01

The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells. PMID:27534893

Optical Microfibre Based Photonic Components and Their Applications in Label-Free Biosensing

PubMed Central

Wang, Pengfei; Bo, Lin; Semenova, Yuliya; Farrell, Gerald; Brambilla, Gilberto

2015-01-01

Optical microfibre photonic components offer a variety of enabling properties, including large evanescent fields, flexibility, configurability, high confinement, robustness and compactness. These unique features have been exploited in a range of applications such as telecommunication, sensing, optical manipulation and high Q resonators. Optical microfibre biosensors, as a class of fibre optic biosensors which rely on small geometries to expose the evanescent field to interact with samples, have been widely investigated. Due to their unique properties, such as fast response, functionalization, strong confinement, configurability, flexibility, compact size, low cost, robustness, ease of miniaturization, large evanescent field and label-free operation, optical microfibres based biosensors seem a promising alternative to traditional immunological methods for biomolecule measurements. Unlabeled DNA and protein targets can be detected by monitoring the changes of various optical transduction mechanisms, such as refractive index, absorption and surface plasmon resonance, since a target molecule is capable of binding to an immobilized optical microfibre. In this review, we critically summarize accomplishments of past optical microfibre label-free biosensors, identify areas for future research and provide a detailed account of the studies conducted to date for biomolecules detection using optical microfibres. PMID:26287252

Advances in identification and validation of protein targets of natural products without chemical modification.

PubMed

Chang, J; Kim, Y; Kwon, H J

2016-05-04

Covering: up to February 2016Identification of the target proteins of natural products is pivotal to understanding the mechanisms of action to develop natural products for use as molecular probes and potential therapeutic drugs. Affinity chromatography of immobilized natural products has been conventionally used to identify target proteins, and has yielded good results. However, this method has limitations, in that labeling or tagging for immobilization and affinity purification often result in reduced or altered activity of the natural product. New strategies have recently been developed and applied to identify the target proteins of natural products and synthetic small molecules without chemical modification of the natural product. These direct and indirect methods for target identification of label-free natural products include drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), and bioinformatics-based analysis of connectivity. This review focuses on and reports case studies of the latest advances in target protein identification methods for label-free natural products. The integration of newly developed technologies will provide new insights and highlight the value of natural products for use as biological probes and new drug candidates.

Optical Microfibre Based Photonic Components and Their Applications in Label-Free Biosensing.

PubMed

Wang, Pengfei; Bo, Lin; Semenova, Yuliya; Farrell, Gerald; Brambilla, Gilberto

2015-07-22

Optical microfibre photonic components offer a variety of enabling properties, including large evanescent fields, flexibility, configurability, high confinement, robustness and compactness. These unique features have been exploited in a range of applications such as telecommunication, sensing, optical manipulation and high Q resonators. Optical microfibre biosensors, as a class of fibre optic biosensors which rely on small geometries to expose the evanescent field to interact with samples, have been widely investigated. Due to their unique properties, such as fast response, functionalization, strong confinement, configurability, flexibility, compact size, low cost, robustness, ease of miniaturization, large evanescent field and label-free operation, optical microfibres based biosensors seem a promising alternative to traditional immunological methods for biomolecule measurements. Unlabeled DNA and protein targets can be detected by monitoring the changes of various optical transduction mechanisms, such as refractive index, absorption and surface plasmon resonance, since a target molecule is capable of binding to an immobilized optical microfibre. In this review, we critically summarize accomplishments of past optical microfibre label-free biosensors, identify areas for future research and provide a detailed account of the studies conducted to date for biomolecules detection using optical microfibres.

Using mass spectrometry to study the photo-affinity labeling of protein tyrosine phosphatase 1B

NASA Astrophysics Data System (ADS)

Leriche, Tammy; Skorey, Kathryn; Roy, Patrick; McKay, Dan; Bateman, Kevin P.

2004-11-01

Protein tyrosine phosphatase 1B (PTP1B) is a potential target for the treatment of Type II diabetes and several companies are developing small molecule inhibitors of this enzyme. Part of the characterization of these compounds as PTP1B inhibitors is the understanding of how they bind in the enzyme active site. The use of photo-activated inhibitors that target the active site can provide such insight. This paper describes the characterization of a photoprobe directed at the active site of PTP1B. Mass spectrometry revealed the specific binding of the probe to the intact protein. Digestion of the labeled protein followed by LC-MS and LC-MS/MS was used to show that the photoprobe binds to a specific active site amino acid. This was confirmed by comparison with the X-ray structure of PTP1B with a PTP1B inhibitor. The probe labels a conserved acidic residue (Asp) that is required for catalytic activity. This photoprobe may prove to be a useful tool for the development of a PTP1B inhibitor or for the study of PTPs in general.

A spin labelling study of immunomodulating peptidoglycan monomer and adamantyltripeptides entrapped into liposomes.

PubMed

Frkanec, Ruza; Noethig-Laslo, Vesna; Vranesić, Branka; Mirosavljević, Krunoslav; Tomasić, Jelka

2003-04-01

The interaction of immunostimulating compounds, the peptidoglycan monomer (PGM) and structurally related adamantyltripeptides (AdTP1 and AdTP2), respectively, with phospholipids in liposomal bilayers were investigated by electron paramagnetic resonance spectroscopy. (1). The fatty acids bearing the nitroxide spin label at different positions along the acyl chain were used to investigate the interaction of tested compounds with negatively charged multilamellar liposomes. Electron spin resonance (ESR) spectra were studied at 290 and 310 K. The entrapment of the adamantyltripeptides affected the motional properties of all spin labelled lipids, while the entrapment of PGM had no effect. (2). Spin labelled PGM was prepared and the novel compound bearing the spin label attached via the amino group of diaminopimelic acid was chromatographically purified and chemically characterized. The rotational correlation time of the spin labelled molecule dissolved in buffer at pH 7.4 was studied as a function of temperature. The conformational change was observed above 300 K. The same effect was observed with the spin labelled PGM incorporated into liposomes. Such effect was not observed when the spin labelled PGM was studied at alkaline pH, probably due to the hydrolysis of PGM molecule. The study of possible interaction with liposomal membrane is relevant to the use of tested compounds incorporated into liposomes, as adjuvants in vivo.

A matrix-assisted laser desorption/ionization mass spectroscopy method for the analysis of small molecules by integrating chemical labeling with the supramolecular chemistry of cucurbituril.

PubMed

Ding, Jun; Xiao, Hua-Ming; Liu, Simin; Wang, Chang; Liu, Xin; Feng, Yu-Qi

2018-10-05

Although several methods have realized the analysis of low molecular weight (LMW) compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) by overcoming the problem of interference with MS signals in the low mass region derived from conventional organic matrices, this emerging field still requires strategies to address the issue of analyzing complex samples containing LMW components in addition to the LMW compounds of interest, and solve the problem of lack of universality. The present study proposes an integrated strategy that combines chemical labeling with the supramolecular chemistry of cucurbit [n]uril (CB [n]) for the MALDI MS analysis of LMW compounds in complex samples. In this strategy, the target LMW compounds are first labeled by introducing a series of bifunctional reagents that selectively react with the target analytes and also form stable inclusion complexes with CB [n]. Then, the labeled products act as guest molecules that readily and selectively form stable inclusion complexes with CB [n]. This strategy relocates the MS signals of the LMW compounds of interest from the low mass region suffering high interference to the high mass region where interference with low mass components is absent. Experimental results demonstrate that a wide range of LMW compounds, including carboxylic acids, aldehydes, amines, thiol, and cis-diols, can be successfully detected using the proposed strategy, and the limits of detection were in the range of 0.01-1.76 nmol/mL. In addition, the high selectivity of the labeling reagents for the target analytes in conjunction with the high selectivity of the binding between the labeled products and CB [n] ensures an absence of signal interference with the non-targeted LMW components of complex samples. Finally, the feasibility of the proposed strategy for complex sample analysis is demonstrated by the accurate and rapid quantitative analysis of aldehydes in saliva and herbal medicines. As such, this work not only provides an alternative method for the detection of various LMW compounds using MALDI MS, but also can be applied to the selective and high-throughput analysis of LMW analytes in complex samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Portable SERS sensor for malachite green and other small dye molecules

NASA Astrophysics Data System (ADS)

Qiu, Suyan; Zhao, Fusheng; Li, Jingting; Shih, Wei-Chuan

2017-02-01

Sensitive detection of specific chemicals on site can be extremely powerful in many fields. Owing to its molecular fingerprinting capability, surface-enhanced Raman scattering has been one of the technological contenders. In this paper, we describe the novel use of DNA topological nanostructure on nanoporous gold nanoparticle (NPG-NP) array chip for chemical sensing. NPG-NP features large surface area and high-density plasmonic field enhancement known as "hotspots". Hence, NPG-NP array chip has found many applications in nanoplasmonic sensor development. This technique can provide novel label-free molecular sensing capability and enables high sensitivity and specificity detection using a portable Raman spectrometer.

A highly efficient dual-diazonium reagent for protein crosslinking and construction of a virus-based gel.

PubMed

Ma, Dejun; Zhang, Jie; Zhang, Changyu; Men, Yuwen; Sun, Hongyan; Li, Lu-Yuan; Yi, Long; Xi, Zhen

2018-05-09

A new bench-stable reagent with double diazonium sites was designed and synthesized for protein crosslinking. Based on the highly efficient diazonium-Tyr coupling reaction, a direct mixture of the reagent and tobacco mosaic virus led to the formation of a new hydrogel, which could be degraded by chemicals and could be used to encapsulate small molecules for sustained release. Because plant viruses exhibit many chemical characteristics like protein labelling and nucleic acid packaging, the virus-based hydrogel will have large chemical space for further functionalization. Besides, this dual-diazonium reagent should be a generally useful crosslinker for chemical biology and biomaterials.

Hemoglobin Cleavage Site-Specificity of the Plasmodium falciparum Cysteine Proteases Falcipain-2 and Falcipain-3

PubMed Central

Subramanian, Shoba; Hardt, Markus; Choe, Youngchool; Niles, Richard K.; Johansen, Eric B.; Legac, Jennifer; Gut, Jiri; Kerr, Iain D.; Craik, Charles S.; Rosenthal, Philip J.

2009-01-01

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P1 – P4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P2 position. Second, with overlapping peptides spanning α and β globin and proteolysis-dependent 18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents. PMID:19357776

Single molecule optical measurements of orientation and rotations of biological macromolecules

PubMed Central

Shroder, Deborah Y; Lippert, Lisa G; Goldman, Yale E

2016-01-01

The subdomains of macromolecules often undergo large orientation changes during their catalytic cycles that are essential for their activity. Tracking these rearrangements in real time opens a powerful window into the link between protein structure and functional output. Site-specific labeling of individual molecules with polarized optical probes and measuring their spatial orientation can give insight into the crucial conformational changes, dynamics, and fluctuations of macromolecules. Here we describe the range of single molecule optical technologies that can extract orientation information from these probes, we review the relevant types of probes and labeling techniques, and we highlight the advantages and disadvantages of these technologies for addressing specific inquiries. PMID:28192292

Novel diagnostics of metabolic dysfunction detected in breath and plasma by selective isotope-assisted labeling.

PubMed

Haviland, Julia A; Tonelli, Marco; Haughey, Dermot T; Porter, Warren P; Assadi-Porter, Fariba M

2012-08-01

Metabolomics is the study of a unique fingerprint of small molecules present in biological systems under healthy and disease conditions. One of the major challenges in metabolomics is validation of fingerprint molecules to identify specifically perturbed pathways in metabolic aberrations. This step is crucial to the understanding of budding metabolic pathologies and the ability to identify early indicators of common diseases such as obesity, type 2 diabetes mellitus, metabolic syndrome, polycystic ovary syndrome, and cancer. We present a novel approach to diagnosing aberrations in glucose utilization including metabolic pathway switching in a disease state. We used a well-defined prenatally exposed glucocorticoid mouse model that results in adult females with metabolic dysfunction. We applied the complementary technologies of nuclear magnetic resonance spectroscopy and cavity ring-down spectroscopy to analyze serial plasma samples and real-time breath measurements following selective (13)C-isotope-assisted labeling. These platforms allowed us to trace metabolic markers in whole animals and identify key metabolic pathway switching in prenatally glucocorticoid-treated animals. Total glucose flux is significantly proportionally increased through the major oxidative pathways of glycolysis and the pentose phosphate pathway in the prenatally glucocorticoid-treated animals relative to the control animals. This novel diagnostics approach is fast, noninvasive, and sensitive for determining specific pathway utilization, and provides a direct translational application in the health care field. Copyright © 2012 Elsevier Inc. All rights reserved.

Label-free liquid crystal biosensor based on specific oligonucleotide probes for heavy metal ions.

PubMed

Yang, Shengyuan; Wu, Chao; Tan, Hui; Wu, Yan; Liao, Shuzhen; Wu, Zhaoyang; Shen, Guoli; Yu, Ruqin

2013-01-02

In this study, to enhance the capability of metal ions disturbing the orientation of liquid crystals (LCs), we designed a new label-free LC biosensor for the highly selective and sensitive detection of heavy metal ions. This strategy makes use of the target-induced DNA conformational change to enhance the disruption of target molecules for the orientation of LC leading to an amplified optical signal. The Hg(2+) ion, which possesses a unique property to bind specifically to two DNA thymine (T) bases, is used as a model heavy metal ion. In the presence of Hg(2+), the specific oligonucleotide probes form a conformational reorganization of the oligonucleotide probes from hairpin structure to duplex-like complexes. The duplex-like complexes are then bound on the triethoxysilylbutyraldehyde/N,N-dimethyl-N-octadecyl (3-aminopropyl) trimethoxysilyl chloride (TEA/DMOAP)-coated substrate modified with capture probes, which can greatly distort the orientational profile of LC, making the optical image of LC cell birefringent as a result. The optical signal of LC sensor has a visible change at the Hg(2+) concentration of low to 0.1 nM, showing good detection sensitivity. The cost-effective LC sensing method can translate the concentration signal of heavy metal ions in solution into the presence of DNA duplexes and is expected to be a sensitive detection platform for heavy metal ions and other small molecule monitors.

Prostate-specific membrane antigen for prostate cancer theranostics: from imaging to targeted therapy.

PubMed

Arsenault, Frédéric; Beauregard, Jean-Mathieu; Pouliot, Frédéric

2018-06-22

In recent years, major advances in molecular imaging of prostate cancers (PCa) were made with the development and clinical validation of highly accurate PET tracers to stage and restage the disease. Prostate-specific membrane antigen (PSMA) is a transmembrane protein highly expressed in PCa, and its expression has led to the development of PSMA-binding radiopharmaceuticals for molecular imaging or radioligand therapy (RLT). We herein review the recent literature published on diagnostic and therapeutic (i.e. theranostic) PSMA tracers. Development in small PSMA-targeted molecules labeled with gallium-68 and fluorine-18 show promising results for primary staging and detection of disease at biochemical recurrence using PET/computed tomography (PET/CT). Studies show a higher sensitivity and specificity, along with an improved detection rate over conventional imaging (CT scan and bone scan) or choline PET tracers, especially for restaging after prostate-specific antigen failure following loco-regional therapy. In addition, some PSMA tracers can be labeled with beta-minus and alpha particle emitters, yielding encouraging response rates and low toxicity, and potentially offering a new line of targeted therapy for metastatic castration-resistant PCa. PSMA-targeted tracers have shown unprecedented accuracy to stage and restage PCa using PET/CT. Given their specific biodistribution toward PCa tissue, PSMA RLT now offers new therapeutic possibilities to target metastatic PCa. Prospective multicenter randomized studies investigating the clinical impact management impacts of PSMA-targeted molecules are urgently needed.

The Morphology of Emulsion Polymerized Latex Particles

DOE R&D Accomplishments Database

Wignall, G. D.; Ramakrishnan, V. R.; Linne, M. A.; Klein, A.; Sperling, L. H.; Wai, M. P.; Gelman, R. A.; Fatica, M. G.; Hoerl, R. H.; Fisher, L. W.

1987-11-01

Under monomer starved feed conditions, emulsion polymerization of perdeuterated methyl methacrylate and styrene in the presence of preformed polymethylmethacrylate latexes resulted in particles with a core-shell morphology, as determined by small-angle neutron scattering (SANS) analysis for a hollow sphere. The locus of polymerization of the added deuterated monomer is therefore at the particle surface. In similar measurements a statistical copolymer of styrene and methyl methacrylate was used as seed particles for further polymerization of trideuteromethyl methacrylate. The resulting polymer latex was again shown to have a core-shell morphological structure as determined by SANS. SANS experiments were also undertaken on polystyrene latexes polymerized by equilibrium swelling methods, with deuterated polymer forming the first or second step. The experiments covered a molecular weight range of 6 x 10{sup 4} 10{sup 6} the molecular weights are consistent with the experimental errors, indicating that the deuterium labeled molecules are randomly distributed in the latex. These results led to the finding that the polymer chains were constrained in the latex particles by factors of 2 to 4 from the relaxed coil dimensions. For M < 10{sup 6} g/mol SANS gave zero angle scattering intensities much higher than expected on the basis of a random distribution of labeled molecules. Several models were examined, including the possible development of core-shell structures at lower molecular weights.

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

PubMed Central

Lindhoud, Simon; Westphal, Adrie H.; van Mierlo, Carlo P. M.; Visser, Antonie J. W. G.; Borst, Jan Willem

2014-01-01

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. PMID:25535076

Chemical reactivation of quenched fluorescent protein molecules enables resin-embedded fluorescence microimaging

PubMed Central

Xiong, Hanqing; Zhou, Zhenqiao; Zhu, Mingqiang; Lv, Xiaohua; Li, Anan; Li, Shiwei; Li, Longhui; Yang, Tao; Wang, Siming; Yang, Zhongqin; Xu, Tonghui; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

2014-01-01

Resin embedding is a well-established technique to prepare biological specimens for microscopic imaging. However, it is not compatible with modern green-fluorescent protein (GFP) fluorescent-labelling technique because it significantly quenches the fluorescence of GFP and its variants. Previous empirical optimization efforts are good for thin tissue but not successful on macroscopic tissue blocks as the quenching mechanism remains uncertain. Here we show most of the quenched GFP molecules are structurally preserved and not denatured after routine embedding in resin, and can be chemically reactivated to a fluorescent state by alkaline buffer during imaging. We observe up to 98% preservation in yellow-fluorescent protein case, and improve the fluorescence intensity 11.8-fold compared with unprocessed samples. We demonstrate fluorescence microimaging of resin-embedded EGFP/EYFP-labelled tissue block without noticeable loss of labelled structures. This work provides a turning point for the imaging of fluorescent protein-labelled specimens after resin embedding. PMID:24886825

Luminescent probes for optical in vivo imaging

NASA Astrophysics Data System (ADS)

Texier, Isabelle; Josserand, Veronique; Garanger, Elisabeth; Razkin, Jesus; Jin, Zhaohui; Dumy, Pascal; Favrot, Marie; Boturyn, Didier; Coll, Jean-Luc

2005-04-01

Going along with instrumental development for small animal fluorescence in vivo imaging, we are developing molecular fluorescent probes, especially for tumor targeting. Several criteria have to be taken into account for the optimization of the luminescent label. It should be adapted to the in vivo imaging optical conditions : red-shifted absorption and emission, limited overlap between absorption and emission for a good signal filtering, optimized luminescence quantum yield, limited photo-bleaching. Moreover, the whole probe should fulfill the biological requirements for in vivo labeling : adapted blood-time circulation, biological conditions compatibility, low toxicity. We here demonstrate the ability of the imaging fluorescence set-up developed in LETI to image the bio-distribution of molecular probes on short times after injection. Targeting with Cy5 labeled holo-transferrin of subcutaneous TS/Apc (angiogenic murine breast carcinoma model) or IGROV1 (human ovarian cancer) tumors was achieved. Differences in the kinetics of the protein uptake by the tumors were evidenced. IGROV1 internal metastatic nodes implanted in the peritoneal cavity could be detected in nude mice. However, targeted metastatic nodes in lung cancer could only be imaged after dissection of the mouse. These results validate our fluorescence imaging set-up and the use of Cy5 as a luminescent label. New fluorescent probes based on this dye and a molecular delivery template (the RAFT molecule) can thus be envisioned.

Comparative Evaluation of Anti-HER2 Affibody Molecules Labeled with 64Cu Using NOTA and NODAGA

PubMed Central

Yim, Cheng-Bin; Rajander, Johan; Perols, Anna; Karlström, Amelie Eriksson; Haaparanta-Solin, Merja; Grönroos, Tove J.; Solin, Olof; Orlova, Anna

2017-01-01

Imaging using affibody molecules enables discrimination between breast cancer metastases with high and low expression of HER2, making appropriate therapy selection possible. This study aimed to evaluate if the longer half-life of 64Cu (T1/2 = 12.7 h) would make 64Cu a superior nuclide compared to 68Ga for PET imaging of HER2 expression using affibody molecules. The synthetic ZHER2:S1 affibody molecule was conjugated with the chelators NOTA or NODAGA and labeled with 64Cu. The tumor-targeting properties of 64Cu-NOTA-ZHER2:S1 and 64Cu-NODAGA-ZHER2:S1 were evaluated and compared with the targeting properties of 68Ga-NODAGA-ZHER2:S1 in mice. Both 64Cu-NOTA-ZHER2:S1 and 64Cu-NODAGA-ZHER2:S1 demonstrated specific targeting of HER2-expressing xenografts. At 2 h after injection of 64Cu-NOTA-ZHER2:S1, 64Cu-NODAGA-ZHER2:S1, and 68Ga-NODAGA-ZHER2:S1, tumor uptakes did not differ significantly. Renal uptake of 64Cu-labeled conjugates was dramatically reduced at 6 and 24 h after injection. Notably, radioactivity uptake concomitantly increased in blood, lung, liver, spleen, and intestines, which resulted in decreased tumor-to-organ ratios compared to 2 h postinjection. Organ uptake was lower for 64Cu-NODAGA-ZHER2:S1. The most probable explanation for this biodistribution pattern was the release and redistribution of renal radiometabolites. In conclusion, monoamide derivatives of NOTA and NODAGA may be suboptimal chelators for radiocopper labeling of anti-HER2 affibody molecules and, possibly, other scaffold proteins with high renal uptake. PMID:29097939

NIR-to-visible upconversion nanoparticles for fluorescent labeling and targeted delivery of siRNA

NASA Astrophysics Data System (ADS)

Jiang, Shan; Zhang, Yong; Lim, Kian Meng; Sim, Eugene K. W.; Ye, Lei

2009-04-01

Near-infrared (NIR)-to-visible upconversion fluorescent nanoparticles were synthesized and used for imaging and targeted delivery of small interfering RNA (siRNA) to cancer cells. Silica-coated NaYF4 upconversion nanoparticles (UCNs) co-doped with lanthanide ions (Yb/Er) were synthesized. Folic acid and anti-Her2 antibody conjugated UCNs were used to fluorescently label the folate receptors of HT-29 cells and Her2 receptors of SK-BR-3 cells, respectively. The intracellular uptake of the folic acid and antibody conjugated UCNs was visualized using a confocal fluorescence microscope equipped with an NIR laser. siRNA was attached to anti-Her2 antibody conjugated UCNs and the delivery of these nanoparticles to SK-BR-3 cells was studied. Meanwhile, a luciferase assay was established to confirm the gene silencing effect of siRNA. Upconversion nanoparticles can serve as a fluorescent probe and delivery system for simultaneous imaging and delivery of biological molecules.

Label-Free QCM Immunosensor for the Detection of Ochratoxin A

PubMed Central

Ertekin, Özlem; Laguna, Duygu Ercan; Özen, Fehime Şeyma; Öztürk, Zafer Ziya; Öztürk, Selma

2018-01-01

Ochratoxin A (OTA) is a potent mycotoxin that poses a risk in food and feed moieties and subject to worldwide regulation. Laboratory-based analytical methods are traditionally employed for reliable OTA quantification, but these methods cannot provide rapid and on-site analysis, where biosensors fill this gap. In this study a label-free quartz crystal microbalance (QCM)-based immunosensor for the detection of OTA, which is one of the most important small molecule contaminants, was developed by direct immobilization of OTA to amine-bearing sensor surfaces using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) chemistry. The protein-free sensor surface enabled regeneration of sensor surface with 50 mM NaOH and 1% SDS up to 13 times without loss of performance, which would disrupt a protein-containing sensor surface. We developed a QCM immunosensor using the developed sensor surface with a 17.2–200 ng/mL detection range which can be used for on-site detection of feedstuffs. PMID:29641432

Label-Free QCM Immunosensor for the Detection of Ochratoxin A.

PubMed

Pirinçci, Şerife Şeyda; Ertekin, Özlem; Laguna, Duygu Ercan; Özen, Fehime Şeyma; Öztürk, Zafer Ziya; Öztürk, Selma

2018-04-11

Ochratoxin A (OTA) is a potent mycotoxin that poses a risk in food and feed moieties and subject to worldwide regulation. Laboratory-based analytical methods are traditionally employed for reliable OTA quantification, but these methods cannot provide rapid and on-site analysis, where biosensors fill this gap. In this study a label-free quartz crystal microbalance (QCM)-based immunosensor for the detection of OTA, which is one of the most important small molecule contaminants, was developed by direct immobilization of OTA to amine-bearing sensor surfaces using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) chemistry. The protein-free sensor surface enabled regeneration of sensor surface with 50 mM NaOH and 1% SDS up to 13 times without loss of performance, which would disrupt a protein-containing sensor surface. We developed a QCM immunosensor using the developed sensor surface with a 17.2-200 ng/mL detection range which can be used for on-site detection of feedstuffs.

Spot Surface Labeling of Magnetic Microbeads and Application in Biological Force Measurements

NASA Astrophysics Data System (ADS)

Estes, Ashley; O'Brien, E. Tim; Hill, David; Superfine, Richard

2006-11-01

Biological force measurements on single molecules and macromolecular structures often use microbeads for the application of force. These techniques are often complicated by multiple attachments and nonspecific binding. In one set of experiments, we are applying a magnetic force microscope that allows us to pull on magnetic beads attached to ciliated human bronchial epithelial cells. These experiments provide a means to measure the stall force of cilia and understand how cilia propel fluids. However, because we are using beads with diameters of one and 2.8 microns, and the diameter of human airway cilia is approximately 200 nm, we cannot be assured that the bead is bound to a single cilium. To address this, we have developed a sputter coating technique to block the biotin binding capability of the streptavidin labeled bead over its entire surface except for a small spot. These beads may also have applications in other biological experiments such as DNA force experiments in which binding of a single target to an individual bead is critical.

Single-Molecule Optical Spectroscopy and Imaging: From Early Steps to Recent Advances

NASA Astrophysics Data System (ADS)

Moerner, William E.

The initial steps toward optical detection and spectroscopy of single molecules arose out of the study of spectral hole-burning in inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989. In the early 1990s, many fascinating physical effects were observed for individual molecules such as spectral diffusion, optical switching, vibrational spectra, and magnetic resonance of a single molecular spin. Since the mid-1990s when experiments moved to room temperature, a wide variety of biophysical effects may be explored, and a number of physical phenomena from the low temperature studies have analogs at high temperature. Recent advances worldwide cover a huge range, from in vitro studies of enzymes, proteins, and oligonucleotides, to observations in real time of a single protein performing a specific function inside a living cell. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation of individual fluorophores leads naturally to localization beyond the optical diffraction limit. Combining this with active optical control of the number of emitting molecules leads to superresolution imaging, a new frontier for optical microscopy beyond the optical diffraction limit and for chemical design of photoswitchable fluorescent labels. Finally, to study one molecule in aqueous solution without surface perturbations, a new electrokinetic trap is described (the ABEL trap) which can trap single small biomolecules without the need for large dielectric beads.

DNA Immobilization and Hybridization Detection by the Intrinsic Molecular Charge Using Capacitive Field-Effect Sensors Modified with a Charged Weak Polyelectrolyte Layer.

PubMed

Bronder, Thomas S; Poghossian, Arshak; Scheja, Sabrina; Wu, Chunsheng; Keusgen, Michael; Mewes, Dieter; Schöning, Michael J

2015-09-16

Miniaturized setup, compatibility with advanced micro- and nanotechnologies, and ability to detect biomolecules by their intrinsic molecular charge favor the semiconductor field-effect platform as one of the most attractive approaches for the development of label-free DNA chips. In this work, a capacitive field-effect EIS (electrolyte-insulator-semiconductor) sensor covered with a layer-by-layer prepared, positively charged weak polyelectrolyte layer of PAH (poly(allylamine hydrochloride)) was used for the label-free electrical detection of DNA (deoxyribonucleic acid) immobilization and hybridization. The negatively charged probe single-stranded DNA (ssDNA) molecules were electrostatically adsorbed onto the positively charged PAH layer, resulting in a preferentially flat orientation of the ssDNA molecules within the Debye length, thus yielding a reduced charge-screening effect and a higher sensor signal. Each sensor-surface modification step (PAH adsorption, probe ssDNA immobilization, hybridization with complementary target DNA (cDNA), reducing an unspecific adsorption by a blocking agent, incubation with noncomplementary DNA (ncDNA) solution) was monitored by means of capacitance-voltage and constant-capacitance measurements. In addition, the surface morphology of the PAH layer was studied by atomic force microscopy and contact-angle measurements. High hybridization signals of 34 and 43 mV were recorded in low-ionic strength solutions of 10 and 1 mM, respectively. In contrast, a small signal of 4 mV was recorded in the case of unspecific adsorption of fully mismatched ncDNA. The density of probe ssDNA and dsDNA molecules as well as the hybridization efficiency was estimated using the experimentally measured DNA immobilization and hybridization signals and a simplified double-layer capacitor model. The results of field-effect experiments were supported by fluorescence measurements, verifying the DNA-immobilization and hybridization event.

In vitro cell studies of technetium-99m labeled RGD-HYNIC peptide, a comparison of tricine and EDDA as co-ligands.

PubMed

Su, Zi-Fen; He, Jiang; Rusckowski, Mary; Hnatowich, Donald J

2003-02-01

The level of alpha(V)beta(3) integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to alpha(V)beta(3) integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with (99m)Tc. The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N'-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture. The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated. The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the (99m)Tc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of alpha(V)beta(3) integrin proteins are expressed on the cells. Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD/EDDA may not be improved compared to RGD/tricine since quantitation of the cell binding results suggests that the number of alpha(V)beta(3) integrin proteins per cell might be limited.

Comparing the Properties of Electrochemical-Based DNA Sensors Employing Different Redox Tags

PubMed Central

Kang, Di; Zuo, Xiaolei; Yang, Renqiang; Xia, Fan; Plaxco, Kevin W.; White, Ryan J.

2009-01-01

Many electrochemical biosensor approaches developed in recent years utilize redox labeled (most commonly methylene blue or ferrocene) oligonucleotide probes site-specifically attached to an interrogating electrode. Sensors in this class have been reported employing a range of probe architectures, including single- and double-stranded DNA, more complex DNA structures, DNA and RNA aptamers and, most recently, DNA-small molecule chimeras. Signaling in this class of sensors is generally predicated on binding-induced changes in the efficiency with which the covalently attached redox label transfers electrons with the interrogating electrode. Here we have investigated how the properties of the redox tag affect the performance of such sensors. Specifically, we compare the differences in signaling and stability of electrochemical DNA sensors (E-DNA sensors) fabricated using either ferrocene or methylene blue as the signaling redox moiety. We find that while both tags support efficient E-DNA signaling, ferrocene produces slightly improved signal gain and target affinity. These small advantages, however, come at a potentially significant price: the ferrocene-based sensors are far less stable than their methylene blue counterparts, particularly with regards to stability to long-term storage, repeated electrochemical interrogations, repeated sensing/regeneration iterations, and employment in complex sample matrices such as blood serum. PMID:19810694

High-Frequency Electron Paramagnetic Resonance Spectroscopy of Nitroxide-Functionalized Nanodiamonds in Aqueous Solution.

PubMed

Akiel, R D; Stepanov, V; Takahashi, S

2017-06-01

Nanodiamond (ND) is an attractive class of nanomaterial for fluorescent labeling, magnetic sensing of biological molecules, and targeted drug delivery. Many of those applications require tethering of target biological molecules on the ND surface. Even though many approaches have been developed to attach macromolecules to the ND surface, it remains challenging to characterize dynamics of tethered molecule. Here, we show high-frequency electron paramagnetic resonance (HF EPR) spectroscopy of nitroxide-functionalized NDs. Nitroxide radical is a commonly used spin label to investigate dynamics of biological molecules. In the investigation, we developed a sample holder to overcome water absorption of HF microwave. Then, we demonstrated HF EPR spectroscopy of nitroxide-functionalized NDs in aqueous solution and showed clear spectral distinction of ND and nitroxide EPR signals. Moreover, through EPR spectral analysis, we investigate dynamics of nitroxide radicals on the ND surface. The demonstration sheds light on the use of HF EPR spectroscopy to investigate biological molecule-functionalized nanoparticles.

A cost-effective approach to produce 15N-labelled amino acids employing Chlamydomonas reinhardtii CC503.

PubMed

Nicolás Carcelén, Jesús; Marchante-Gayón, Juan Manuel; González, Pablo Rodríguez; Valledor, Luis; Cañal, María Jesús; Alonso, José Ignacio García

2017-08-18

The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15 N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15 NH 4 Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15 N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13 C-labelled fatty acids have been isolated from labelled microalga biomass as valuable industrial products. In this study, we propose Chlamydomonas reinhardtii CC503 as a feasible microorganism and strain to produce labelled biomass from which a standard containing sixteen 15 N-labelled amino acids could be obtained.

Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor.

PubMed

Adams, André A; Okagbare, Paul I; Feng, Juan; Hupert, Matuesz L; Patterson, Don; Göttert, Jost; McCarley, Robin L; Nikitopoulos, Dimitris; Murphy, Michael C; Soper, Steven A

2008-07-09

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.

Highly Efficient Circulating Tumor Cell Isolation from Whole Blood and Label-Free Enumeration Using Polymer-Based Microfluidics with an Integrated Conductivity Sensor

PubMed Central

Adams, André A.; Okagbare, Paul I.; Feng, Juan; Hupert, Matuesz L.; Patterson, Don; Göttert, Jost; McCarley, Robin L.; Nikitopoulos, Dimitris; Murphy, Michael C.; Soper, Steven A.

2008-01-01

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (≥1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 μm width × 150 μm depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation. PMID:18557614

Method for performing site-specific affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

1999-01-01

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

Miniaturized reaction vessel system, method for performing site-specific biochemical reactions and affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

2000-01-01

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

Method for performing site-specific affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, A.D.; Lysov, Y.P.; Dubley, S.A.

1999-05-18

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between the cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting the extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to the extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from the array. 14 figs.

Covalent dye attachment influences the dynamics and conformational properties of flexible peptides

PubMed Central

Crevenna, Alvaro H.; Bomblies, Rainer; Lamb, Don C.

2017-01-01

Fluorescence spectroscopy techniques like Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have become important tools for the in vitro and in vivo investigation of conformational dynamics in biomolecules. These methods rely on the distance-dependent quenching of the fluorescence signal of a donor fluorophore either by a fluorescent acceptor fluorophore (FRET) or a non-fluorescent quencher, as used in FCS with photoinduced electron transfer (PET). The attachment of fluorophores to the molecule of interest can potentially alter the molecular properties and may affect the relevant conformational states and dynamics especially of flexible biomolecules like intrinsically disordered proteins (IDP). Using the intrinsically disordered S-peptide as a model system, we investigate the impact of terminal fluorescence labeling on the molecular properties. We perform extensive molecular dynamics simulations on the labeled and unlabeled peptide and compare the results with in vitro PET-FCS measurements. Experimental and simulated timescales of end-to-end fluctuations were found in excellent agreement. Comparison between simulations with and without labels reveal that the π-stacking interaction between the fluorophore labels traps the conformation of S-peptide in a single dominant state, while the unlabeled peptide undergoes continuous conformational rearrangements. Furthermore, we find that the open to closed transition rate of S-peptide is decreased by at least one order of magnitude by the fluorophore attachment. Our approach combining experimental and in silico methods provides a benchmark for the simulations and reveals the significant effect that fluorescence labeling can have on the conformational dynamics of small biomolecules, at least for inherently flexible short peptides. The presented protocol is not only useful for comparing PET-FCS experiments with simulation results but provides a strategy to minimize the influence on molecular properties when chosing labeling positions for fluorescence experiments. PMID:28542243

Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays

PubMed Central

2011-01-01

Background With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles. Results RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts. Conclusions An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment. PMID:21235785

Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening

PubMed Central

Cheong, Wing-Lam; Tsang, Ming-San; So, Pui-Kin; Chung, Wai-Hong; Leung, Yun-Chung; Chan, Pak-Ho

2014-01-01

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening. PMID:25074398

Transfer of arachidonate from phosphatidylcholine to phosphatidylethanolamine and triacylglycerol in guinea pig alveolar macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nijssen, J.G.; Oosting, R.S.; Nkamp, F.Pv.

1986-10-01

Guinea pig alveolar macrophages were labeled by incubation with either arachidonate or linoleate. Arachidonate labeled phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triglycerides (TG) equally well, with each lipid containing about 30% of total cellular radioactivity. In comparison to arachidonate, linoleate was recovered significantly less in PE (7%) and more in TG (47%). To investigate whether redistributions of acyl chains among lipid classes took place, the macrophages were incubated with 1-acyl-2-(1-/sup 14/C)arachidonoyl PC or 1-acyl-2-(1-/sup 14/C)linoleoyl PC. After harvesting, the cells incubated with 1-acyl-2-(1-/sup 14/C)linoleoyl PC contained 86% of the recovered cellular radioactivity in PC, with only small amounts of label beingmore » transferred to PE and TG (3 and 6%, respectively). More extensive redistributions were observed with arachidonate-labeled PC. In this case, only 60% of cellular radioactivity was still associated with PC, while 22 and 12%, respectively, had been transferred to PE and TG. Arachidonate transfer from PC to PE was unaffected by an excess of free arachidonate which inhibited this transfer to TG for over 90%, indicating that different mechanisms or arachidonoyl CoA pools were involved in the transfer of arachidonate from PC to PE and TG. Cells prelabeled with 1-acyl-2-(1-/sup 14/C)arachidonoyl PC released /sup 14/C-label into the medium upon further incubation. This release was slightly stimulated by zymosan and threefold higher in the presence of the Ca2+-ionophore A23187. Labeling of macrophages with intact phospholipid molecules appears to be a suitable method for studying acyl chain redistribution and release reactions.« less

Fluorescence Immunofiltration Assay of Brucella Melitensis.

DTIC Science & Technology

1995-01-01

second urease -labelled antibody directed against fluorescein. The assay system is useful for measuring protein, virus and bacteria in aqueous...binding site for the signal-generating urease -labelled antibody, it is a highly fluorescent molecule and has signal-generating capacity of its own

Light-Driven Nano-oscillators for Label-Free Single-Molecule Monitoring of MicroRNA.

PubMed

Chen, Zixuan; Peng, Yujiao; Cao, Yue; Wang, Hui; Zhang, Jian-Rong; Chen, Hong-Yuan; Zhu, Jun-Jie

2018-06-13

Here, we present a mapping tool based on individual light-driven nano-oscillators for label-free single-molecule monitoring of microRNA. This design uses microRNA as a single-molecule damper for nano-oscillators by forming a rigid dual-strand structure in the gap between nano-oscillators and the immobilized surface. The ultrasensitive detection is attributed to comparable dimensions of the gap and microRNA. A developed surface plasmon-coupled scattering imaging technology enables us to directly measure the real-time gap distance vibration of multiple nano-oscillators with high accuracy and fast dynamics. High-level and low-level states of the oscillation amplitude indicate melting and hybridization statuses of microRNA. Lifetimes of two states reveal that the hybridization rate of microRNA is determined by the three-dimensional diffusion. This imaging technique contributes application potentials in a single-molecule detection and nanomechanics study.

Site-Specific Three-Color Labeling of α-Synuclein via Conjugation to Uniquely Reactive Cysteines during Assembly by Native Chemical Ligation.

PubMed

Lee, Taehyung C; Moran, Crystal R; Cistrone, Philip A; Dawson, Philip E; Deniz, Ashok A

2018-04-12

Single-molecule fluorescence is widely used to study conformational complexity in proteins, and has proven especially valuable with intrinsically disordered proteins (IDPs). Protein studies using dual-color single-molecule Förster resonance energy transfer (smFRET) are now quite common, but many could benefit from simultaneous measurement of multiple distances through multi-color labeling. Such studies, however, have suffered from limitations in site-specific incorporation of more than two dyes per polypeptide. Here we present a fully site-specific three-color labeling scheme for α-synuclein, an IDP with important putative functions and links to Parkinson disease. The convergent synthesis combines native chemical ligation with regiospecific cysteine protection of expressed protein fragments to permit highly controlled labeling via standard cysteine-maleimide chemistry, enabling more global smFRET studies. Furthermore, this modular approach is generally compatible with recombinant proteins and expandable to accommodate even more complex experiments, such as by labeling with additional colors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Label-free, real-time interaction and adsorption analysis 1: surface plasmon resonance.

PubMed

Fee, Conan J

2013-01-01

A key requirement for the development of proteins for use in nanotechnology is an understanding of how individual proteins bind to other molecules as they assemble into larger structures. The introduction of labels to enable the detection of biomolecules brings the inherent risk that the labels themselves will influence the nature of biomolecular interactions. Thus, there is a need for label-free interaction and adsorption analysis. In this and the following chapter, two biosensor techniques are reviewed: surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM). Both allow real-time analysis of biomolecular interactions and both are label-free. The first of these, SPR, is an optical technique that is highly sensitive to the changes in refractive index that occur with protein (or other molecule) accumulation near an illuminated gold surface. Unlike QCM ( Chapter 18 ) SPR is not affected by the water that may be associated with the adsorbed layer nor by conformational changes in the adsorbed species. SPR thus provides unique information about the interaction of a protein with its binding partners.

In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies.

PubMed

Kanje, Sara; Hober, Sophia

2015-04-01

Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Resistive-pulse measurements with nanopipettes: detection of Au nanoparticles and nanoparticle-bound anti-peanut IgY.

PubMed

Wang, Yixian; Kececi, Kaan; Mirkin, Michael V; Mani, Vigneshwaran; Sardesai, Naimish; Rusling, James F

2013-02-01

Solid-state nanopores have been widely employed in sensing applications from Coulter counters to DNA sequencing devices. The analytical signal in such experiments is the change in ionic current flowing through the orifice caused by the large molecule or nanoparticle translocation through the pore. Conceptually similar nanopipette-based sensors can offer several advantages including the ease of fabrication and small physical size essential for local measurements and experiments in small spaces. This paper describes the first evaluation of nanopipettes with well characterized geometry for resistive-pulse sensing of Au nanoparticles (AuNP), nanoparticles coated with an allergen epitope peptide layer, and AuNP-peptide particles with bound antipeanut antibodies (IgY) on the peptide layer. The label-free signal produced by IgY-conjugated particles was strikingly different from those obtained with other analytes, thus suggesting the possibility of selective and sensitive resistive-pulse sensing of antibodies.

Resistive-pulse measurements with nanopipettes: detection of Au nanoparticles and nanoparticle-bound anti-peanut IgY†

PubMed Central

Wang, Yixian; Kececi, Kaan; Mani, Vigneshwaran; Sardesai, Naimish

2013-01-01

Solid-state nanopores have been widely employed in sensing applications from Coulter counters to DNA sequencing devices. The analytical signal in such experiments is the change in ionic current flowing through the orifice caused by the large molecule or nanoparticle translocation through the pore. Conceptually similar nanopipette-based sensors can offer several advantages including the ease of fabrication and small physical size essential for local measurements and experiments in small spaces. This paper describes the first evaluation of nanopipettes with well characterized geometry for resistive-pulse sensing of Au nanoparticles (AuNP), nanoparticles coated with an allergen epitope peptide layer, and AuNP–peptide particles with bound antipeanut antibodies (IgY) on the peptide layer. The label-free signal produced by IgY-conjugated particles was strikingly different from those obtained with other analytes, thus suggesting the possibility of selective and sensitive resistive-pulse sensing of antibodies. PMID:23991282

Microfluidic radiolabeling of biomolecules with PET radiometals

PubMed Central

Zeng, Dexing; Desai, Amit V.; Ranganathan, David; Wheeler, Tobias D.; Kenis, Paul J. A.; Reichert, David E.

2012-01-01

Introduction A robust, versatile and compact microreactor has been designed, fabricated and tested for the labeling of bifunctional chelate conjugated biomolecules (BFC-BM) with PET radiometals. Methods The developed microreactor was used to radiolabel a chelate, either 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) that had been conjugated to cyclo(Arg-Gly-Asp-DPhe-Lys) peptide, with both 64Cu and 68Ga respectively. The microreactor radiolabeling conditions were optimized by varying temperature, concentration and residence time. Results Direct comparisons between the microreactor approach and conventional methods showed improved labeling yields and increased reproducibility with the microreactor under identical labeling conditions, due to enhanced mass and heat transfer at the microscale. More importantly, over 90% radiolabeling yields (incorporation of radiometal) were achieved with a 1:1 stoichiometry of bifunctional chelate biomolecule conjugate (BFC-BM) to radiometal in the microreactor, which potentially obviates extensive chromatographic purification that is typically required to remove the large excess of unlabeled biomolecule in radioligands prepared using conventional methods. Moreover, higher yields for radiolabeling of DOTA-functionalized BSA protein (Bovine Serum Albumin) were observed with 64Cu/68Ga using the microreactor, which demonstrates the ability to label both small and large molecules. Conclusions A robust, reliable, compact microreactor capable of chelating radiometals with common chelates has been developed and validated. Based on our radiolabeling results, the reported microfluidic approach overall outperforms conventional radiosynthetic methods, and is a promising technology for the radiometal labeling of commonly utilized BFC-BM in aqueous solutions. PMID:23078875

Microfluidic radiolabeling of biomolecules with PET radiometals.

PubMed

Zeng, Dexing; Desai, Amit V; Ranganathan, David; Wheeler, Tobias D; Kenis, Paul J A; Reichert, David E

2013-01-01

A robust, versatile and compact microreactor has been designed, fabricated and tested for the labeling of bifunctional chelate conjugated biomolecules (BFC-BM) with PET radiometals. The developed microreactor was used to radiolabel a chelate, either 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) that had been conjugated to cyclo(Arg-Gly-Asp-DPhe-Lys) peptide, with both ⁶⁴Cu and ⁶⁸Ga respectively. The microreactor radiolabeling conditions were optimized by varying temperature, concentration and residence time. Direct comparisons between the microreactor approach and conventional methods showed improved labeling yields and increased reproducibility with the microreactor under identical labeling conditions, due to enhanced mass and heat transfer at the microscale. More importantly, over 90% radiolabeling yields (incorporation of radiometal) were achieved with a 1:1 stoichiometry of bifunctional chelate biomolecule conjugate (BFC-BM) to radiometal in the microreactor, which potentially obviates extensive chromatographic purification that is typically required to remove the large excess of unlabeled biomolecule in radioligands prepared using conventional methods. Moreover, higher yields for radiolabeling of DOTA-functionalized BSA protein (Bovine Serum Albumin) were observed with ⁶⁴Cu/⁶⁸Ga using the microreactor, which demonstrates the ability to label both small and large molecules. A robust, reliable, compact microreactor capable of chelating radiometals with common chelates has been developed and validated. Based on our radiolabeling results, the reported microfluidic approach overall outperforms conventional radiosynthetic methods, and is a promising technology for the radiometal labeling of commonly utilized BFC-BM in aqueous solutions. Copyright © 2013 Elsevier Inc. All rights reserved.

Nanopore arrays in a silicon membrane for parallel single-molecule detection: DNA translocation

NASA Astrophysics Data System (ADS)

Zhang, Miao; Schmidt, Torsten; Jemt, Anders; Sahlén, Pelin; Sychugov, Ilya; Lundeberg, Joakim; Linnros, Jan

2015-08-01

Optical nanopore sensing offers great potential in single-molecule detection, genotyping, or DNA sequencing for high-throughput applications. However, one of the bottle-necks for fluorophore-based biomolecule sensing is the lack of an optically optimized membrane with a large array of nanopores, which has large pore-to-pore distance, small variation in pore size and low background photoluminescence (PL). Here, we demonstrate parallel detection of single-fluorophore-labeled DNA strands (450 bps) translocating through an array of silicon nanopores that fulfills the above-mentioned requirements for optical sensing. The nanopore array was fabricated using electron beam lithography and anisotropic etching followed by electrochemical etching resulting in pore diameters down to ∼7 nm. The DNA translocation measurements were performed in a conventional wide-field microscope tailored for effective background PL control. The individual nanopore diameter was found to have a substantial effect on the translocation velocity, where smaller openings slow the translocation enough for the event to be clearly detectable in the fluorescence. Our results demonstrate that a uniform silicon nanopore array combined with wide-field optical detection is a promising alternative with which to realize massively-parallel single-molecule detection.

Continuous high throughput molecular adhesion based cell sorting using ridged microchannels

NASA Astrophysics Data System (ADS)

Tasadduq, Bushra; Wang, Gonghao; Alexeev, Alexander; Sarioglu, Ali Fatih; Sulchek, Todd

2016-11-01

Cell molecular interactions govern important physiological processes such as stem cell homing, inflammation and cancer metastasis. But due to a lack of effective separation technologies selective to these interactions it is challenging to specifically sort cells. Other label free separation techniques based on size, stiffness and shape do not provide enough specificity to cell type, and correlation to clinical condition. We propose a novel microfluidic device capable of high throughput molecule dependent separation of cells by flowing them through a microchannel decorated with molecule specific coated ridges. The unique aspect of this sorting design is the use of optimized gap size which is small enough to lightly squeeze the cells while flowing under the ridged part of the channel to increase the surface area for interaction between the ligand on cell surface and coated receptor molecule but large enough so that biomechanical markers, stiffness and viscoelasticity, do not dominate the cell separation mechanism. We are able to separate Jurkat cells based on its expression of PSGL-1ligand using ridged channel coated with P selectin at a flow rate of 0.045ml/min and achieve 2-fold and 5-fold enrichment of PSGL-1 positive and negative Jurkat cells respectively.

Optical measurement of transverse molecular diffusion in a microchannel.

PubMed Central

Kamholz, A E; Schilling, E A; Yager, P

2001-01-01

Quantitative analysis of molecular diffusion is a necessity for the efficient design of most microfluidic devices as well as an important biophysical method in its own right. This study demonstrates the rapid measurement of diffusion coefficients of large and small molecules in a microfluidic device, the T-sensor, by means of conventional epifluorescence microscopy. Data were collected by monitoring the transverse flux of analyte from a sample stream into a second stream flowing alongside it. As indicated by the low Reynolds numbers of the system (< 1), flow is laminar, and molecular transport between streams occurs only by diffusion. Quantitative determinations were made by fitting data with predictions of a one-dimensional model. Analysis was made of the flow development and its effect on the distribution of diffusing analyte using a three-dimensional modeling software package. Diffusion coefficients were measured for four fluorescently labeled molecules: fluorescein-biotin, insulin, ovalbumin, and streptavidin. The resulting values differed from accepted results by an average of 2.4%. Microfluidic system parameters can be selected to achieve accurate diffusion coefficient measurements and to optimize other microfluidic devices that rely on precise transverse transport of molecules. PMID:11259309

Non-radioactive detection of trinucleotide repeat size variability.

PubMed

Tomé, Stéphanie; Nicole, Annie; Gomes-Pereira, Mario; Gourdon, Genevieve

2014-03-06

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

Analyte-Size-Dependent Ionization and Quantification of Monosaccharides in Human Plasma Using Cation-Exchanged Smectite Layers.

PubMed

Ding, Yuqi; Kawakita, Kento; Xu, Jiawei; Akiyama, Kazuhiko; Fujino, Tatsuya

2015-08-04

Smectite, a synthetic inorganic polymer with a saponite structure, was subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Typical organic matrix molecules 2,4,6-trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid (DHBA) were intercalated into the layer spacing of cation-exchanged smectite, and the complex was used as a new matrix for laser desorption/ionization mass spectrometry. Because of layer spacing limitations, only a small analyte that could enter the layer and bind to THAP or DHBA could be ionized. This was confirmed by examining different analyte/matrix preparation methods and by measuring saccharides with different molecular sizes. Because of the homogeneous distribution of THAP molecules in the smectite layer spacing, high reproducibility of the analyte peak intensity was achieved. By using isotope-labeled (13)C6-d-glucose as the internal standard, quantitative analysis of monosaccharides in pretreated human plasma sample was performed, and the value of 8.6 ± 0.3 μg/mg was estimated.

3'-End labeling of nucleic acids by a polymerase ribozyme.

PubMed

Samanta, Biswajit; Horning, David P; Joyce, Gerald F

2018-06-13

A polymerase ribozyme can be used to label the 3' end of RNA or DNA molecules by incorporating a variety of functionalized nucleotide analogs. Guided by a complementary template, the ribozyme adds a single nucleotide that may contain a fluorophore, biotin, azide or alkyne moiety, thus enabling the detection and/or capture of selectively labeled materials. Employing a variety of commercially available nucleotide analogs, efficient labeling was demonstrated for model RNAs and DNAs, human microRNAs and natural tRNA.

DNA nanomechanics allows direct digital detection of complementary DNA and microRNA targets.

PubMed

Husale, Sudhir; Persson, Henrik H J; Sahin, Ozgur

2009-12-24

Techniques to detect and quantify DNA and RNA molecules in biological samples have had a central role in genomics research. Over the past decade, several techniques have been developed to improve detection performance and reduce the cost of genetic analysis. In particular, significant advances in label-free methods have been reported. Yet detection of DNA molecules at concentrations below the femtomolar level requires amplified detection schemes. Here we report a unique nanomechanical response of hybridized DNA and RNA molecules that serves as an intrinsic molecular label. Nanomechanical measurements on a microarray surface have sufficient background signal rejection to allow direct detection and counting of hybridized molecules. The digital response of the sensor provides a large dynamic range that is critical for gene expression profiling. We have measured differential expressions of microRNAs in tumour samples; such measurements have been shown to help discriminate between the tissue origins of metastatic tumours. Two hundred picograms of total RNA is found to be sufficient for this analysis. In addition, the limit of detection in pure samples is found to be one attomolar. These results suggest that nanomechanical read-out of microarrays promises attomolar-level sensitivity and large dynamic range for the analysis of gene expression, while eliminating biochemical manipulations, amplification and labelling.

An Infrared Actin Probe for Deep-Cell Electroporation-Based Single-Molecule Speckle (eSiMS) Microscopy

PubMed Central

Yamashiro, Sawako; Watanabe, Naoki

2017-01-01

Single-molecule speckle (SiMS) microscopy is a powerful method to directly elucidate biochemical reactions in live cells. However, since the signal from an individual fluorophore is extremely faint, the observation area by epi-fluorescence microscopy is restricted to the thin cell periphery to reduce autofluorescence, or only molecules near the plasma membrane are visualized by total internal reflection fluorescence (TIRF) microscopy. Here, we introduce a new actin probe labeled with near infrared (NIR) emissive CF680R dye for easy-to-use, electroporation-based SiMS microscopy (eSiMS) for deep-cell observation. CF680R-labeled actin (CF680R-actin) incorporated into actin structures and showed excellent brightness and photostability suitable for single-molecule imaging. Importantly, the intensity of autofluorescence with respect to SiMS brightness was reduced to approximately 13% compared to DyLight 550-labeled actin (DL550-actin). CF680R-actin enabled the monitoring of actin SiMS in actomyosin bundles associated with adherens junctions (AJs) located at 3.5–4 µm above the basal surfaces of epithelial monolayers. These favorable properties of CF680R-actin extend the application of eSiMS to actin turnover and flow analyses in deep cellular structures. PMID:28671584

Molecular targets for small-molecule modulators of circadian clocks

PubMed Central

He, Baokun; Chen, Zheng

2016-01-01

Background Circadian clocks are endogenous timing systems that regulate various aspects of mammalian metabolism, physiology and behavior. Traditional chronotherapy refers to the administration of drugs in a defined circadian time window to achieve optimal pharmacokinetic and therapeutic efficacies. In recent years, substantial efforts have been dedicated to developing novel small-molecule modulators of circadian clocks. Methods Here, we review the recent progress in the identification of molecular targets of small-molecule clock modulators and their efficacies in clock-related disorders. Specifically, we examine the clock components and regulatory factors as possible molecular targets of small molecules, and we review several key clock-related disorders as promising venues for testing the preventive/therapeutic efficacies of these small molecules. Finally, we also discuss circadian regulation of drug metabolism. Results Small molecules can modulate the period, phase and/or amplitude of the circadian cycle. Core clock proteins, nuclear hormone receptors, and clock-related kinases and other epigenetic regulators are promising molecular targets for small molecules. Through these targets small molecules exert protective effects against clock-related disorders including the metabolic syndrome, immune disorders, sleep disorders and cancer. Small molecules can also modulate circadian drug metabolism and response to existing therapeutics. Conclusion Small-molecule clock modulators target clock components or diverse cellular pathways that functionally impinge upon the clock. Target identification of new small-molecule modulators will deepen our understanding of key regulatory nodes in the circadian network. Studies of clock modulators will facilitate their therapeutic applications, alone or in combination, for clock-related diseases. PMID:26750111

Determination of helix orientations in a flexible DNA by multi-frequency EPR spectroscopy.

PubMed

Grytz, C M; Kazemi, S; Marko, A; Cekan, P; Güntert, P; Sigurdsson, S Th; Prisner, T F

2017-11-15

Distance measurements are performed between a pair of spin labels attached to nucleic acids using Pulsed Electron-Electron Double Resonance (PELDOR, also called DEER) spectroscopy which is a complementary tool to other structure determination methods in structural biology. The rigid spin label Ç, when incorporated pairwise into two helical parts of a nucleic acid molecule, allows the determination of both the mutual orientation and the distance between those labels, since Ç moves rigidly with the helix to which it is attached. We have developed a two-step protocol to investigate the conformational flexibility of flexible nucleic acid molecules by multi-frequency PELDOR. In the first step, a library with a broad collection of conformers, which are in agreement with topological constraints, NMR restraints and distances derived from PELDOR, was created. In the second step, a weighted structural ensemble of these conformers was chosen, such that it fits the multi-frequency PELDOR time traces of all doubly Ç-labelled samples simultaneously. This ensemble reflects the global structure and the conformational flexibility of the two-way DNA junction. We demonstrate this approach on a flexible bent DNA molecule, consisting of two short helical parts with a five adenine bulge at the center. The kink and twist motions between both helical parts were quantitatively determined and showed high flexibility, in agreement with a Förster Resonance Energy Transfer (FRET) study on a similar bent DNA motif. The approach presented here should be useful to describe the relative orientation of helical motifs and the conformational flexibility of nucleic acid structures, both alone and in complexes with proteins and other molecules.

Development of a Photo-Cross-Linkable Diaminoquinazoline Inhibitor for Target Identification in Plasmodium falciparum.

PubMed

Lubin, Alexandra S; Rueda-Zubiaurre, Ainoa; Matthews, Holly; Baumann, Hella; Fisher, Fabio R; Morales-Sanfrutos, Julia; Hadavizadeh, Kate S; Nardella, Flore; Tate, Edward W; Baum, Jake; Scherf, Artur; Fuchter, Matthew J

2018-04-13

Diaminoquinazolines represent a privileged scaffold for antimalarial discovery, including use as putative Plasmodium histone lysine methyltransferase inhibitors. Despite this, robust evidence for their molecular targets is lacking. Here we report the design and development of a small-molecule photo-cross-linkable probe to investigate the targets of our diaminoquinazoline series. We demonstrate the effectiveness of our designed probe for photoaffinity labeling of Plasmodium lysates and identify similarities between the target profiles of the probe and the representative diaminoquinazoline BIX-01294. Initial pull-down proteomics experiments identified 104 proteins from different classes, many of which are essential, highlighting the suitability of the developed probe as a valuable tool for target identification in Plasmodium falciparum.

Development of analytical method for ultrasensitive detection of salbutamol utilizing DNA labeled-immunoprobe.

PubMed

Lei, Yajing; Li, Xiaoyi; Akash, Muhammad Sajid Hamid; Zhou, Lifang; Tang, Xiaojing; Shi, Weixing; Liu, Zhiming; Chen, Shuqing

2015-03-25

Salbutamol (SAL) is known as quick-acting β-2 receptor agonist and its use in humans for pulmonary diseases and/or in animal feed is limited because of associated potential hazardous effects on health. Several techniques are available for the detection of SAL, but are expensive and time consuming. Here for the first time, a novel pre-assembled DNA immunoprobe-based immuno-PCR assay was developed to investigate the levels of SAL in human urine samples and compared the proposed immuno-PCR assay for the detection of SAL with that of direct competitive ELISA. Under the optimized conditions, the assay showed a linear range over seven orders of magnitude, whereas the limit of detection of SAL in buffer was found to be 21 fg/mL. Current immuno-PCR assay exhibited a 300-fold better detection limit for SAL as compared to one achieved with direct competitive ELISA using the same antibody. In conclusion, it has been found that the developed method is highly sensitive and simple method that can significantly enhance the detection sensitivity for small molecules. Moreover it can be modified and used for detecting other small molecules and/or chemicals that exist in the environment and are responsible for the environmental pollution. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparative Study of Three Methods for Affinity Measurements: Capillary Electrophoresis Coupled with UV Detection and Mass Spectrometry, and Direct Infusion Mass Spectrometry

NASA Astrophysics Data System (ADS)

Mironov, Gleb G.; Logie, Jennifer; Okhonin, Victor; Renaud, Justin B.; Mayer, Paul M.; Berezovski, Maxim V.

2012-07-01

We present affinity capillary electrophoresis and mass spectrometry (ACE-MS) as a comprehensive separation technique for label-free solution-based affinity analysis. The application of ACE-MS for measuring affinity constants between eight small molecule drugs [ibuprofen, s-flurbiprofen, diclofenac, phenylbutazone, naproxen, folic acid, resveratrol, and 4,4'-(propane-1,3-diyl) dibenzoic acid] and β-cyclodextrin is described. We couple on-line ACE with MS to combine the separation and kinetic capability of ACE together with the molecular weight and structural elucidation of MS in one system. To understand the full potential of ACE-MS, we compare it with two other methods: Direct infusion mass spectrometry (DIMS) and ACE with UV detection (ACE-UV). After the evaluation, DIMS provides less reliable equilibrium dissociation constants than separation-based ACE-UV and ACE-MS, and cannot be used solely for the study of noncovalent interactions. ACE-MS determines apparent dissociation constants for all reacting small molecules in a mixture, even in cases when drugs overlap with each other during separation. The ability of ACE-MS to interact, separate, and rapidly scan through m/z can facilitate the simultaneous affinity analysis of multiple interacting pairs, potentially leading to the high-throughput screening of drug candidates.

Study and development of label-free optical biosensors for biomedical applications

NASA Astrophysics Data System (ADS)

Choi, Charles J.

For the majority of assays currently performed, fluorescent or colorimetric chemical labels are commonly attached to the molecules under study so that they may be readily visualized. The methods of using labels to track biomolecular binding events are very sensitive and effective, and are employed as standardized assay protocol across research labs worldwide. However, using labels induces experimental uncertainties due to the effect of the label on molecular conformation, active binding sites, or inability to find an appropriate label that functions equivalently for all molecules in an experiment. Therefore, the ability to perform highly sensitive biochemical detection without the use of fluorescent labels would further simplify assay protocols and would provide quantitative kinetic data, while removing experimental artifacts from fluorescent quenching, shelf-life, and background fluorescence phenomena. In view of the advantages mentioned above, the study and development of optical label-free sensor technologies have been undertaken here. In general, label-free photonic crystal (PC) biosensors and metal nanodome array surface-enhanced Raman scattering (SERS) substrates, both of which are fabricated by nanoreplica molding process, have been used as the method to attack the problem. Chapter 1 shows the work on PC label-free biosensor incorporated microfluidic network for bioassay performance enhancement and kinetic reaction rate constant determination. Chapter 2 describes the work on theoretical and experimental comparison of label-free biosensing in microplate, microfluidic, and spot-based affinity capture assays. Chapter 3 shows the work on integration of PC biosensor with actuate-to-open valve microfluidic chip for pL-volume combinatorial mixing and screening application. In Chapter 4, the development and characterization of SERS nanodome array is shown. Lastly, Chapter 5 describes SERS nanodome sensor incorporated tubing for point-of-care monitoring of intravenous drugs and metabolites.

Recent advances in developing small molecules targeting RNA.

PubMed

Guan, Lirui; Disney, Matthew D

2012-01-20

RNAs are underexploited targets for small molecule drugs or chemical probes of function. This may be due, in part, to a fundamental lack of understanding of the types of small molecules that bind RNA specifically and the types of RNA motifs that specifically bind small molecules. In this review, we describe recent advances in the development and design of small molecules that bind to RNA and modulate function that aim to fill this void.

Microwave-assisted synthesis of water-soluble, fluorescent gold nanoclusters capped with small organic molecules and a revealing fluorescence and X-ray absorption study

NASA Astrophysics Data System (ADS)

Helmbrecht, C.; Lützenkirchen-Hecht, D.; Frank, W.

2015-03-01

Colourless solutions of blue light-emitting, water-soluble gold nanoclusters (AuNC) were synthesized from gold colloids under microwave irradiation using small organic molecules as ligands. Stabilized by 1,3,5-triaza-7-phosphaadamantane (TPA) or l-glutamine (GLU), fluorescence quantum yields up to 5% were obtained. AuNC are considered to be very promising for biological labelling, optoelectronic devices and light-emitting materials but the structure-property relationships have still not been fully clarified. To expand the knowledge about the AuNC apart from their fluorescent properties they were studied by X-ray absorption spectroscopy elucidating the oxidation state of the nanoclusters' gold atoms. Based on curve fitting of the XANES spectra in comparison to several gold references, optically transparent fluorescent AuNC are predicted to be ligand-stabilized Au5+ species. Additionally, their near edge structure compared with analogous results of polynuclear clusters known from the literature discloses an increasing intensity of the feature close to the absorption edge with decreasing cluster size. As a result, a linear relationship between the cluster size and the X-ray absorption coefficient can be established for the first time.Colourless solutions of blue light-emitting, water-soluble gold nanoclusters (AuNC) were synthesized from gold colloids under microwave irradiation using small organic molecules as ligands. Stabilized by 1,3,5-triaza-7-phosphaadamantane (TPA) or l-glutamine (GLU), fluorescence quantum yields up to 5% were obtained. AuNC are considered to be very promising for biological labelling, optoelectronic devices and light-emitting materials but the structure-property relationships have still not been fully clarified. To expand the knowledge about the AuNC apart from their fluorescent properties they were studied by X-ray absorption spectroscopy elucidating the oxidation state of the nanoclusters' gold atoms. Based on curve fitting of the XANES spectra in comparison to several gold references, optically transparent fluorescent AuNC are predicted to be ligand-stabilized Au5+ species. Additionally, their near edge structure compared with analogous results of polynuclear clusters known from the literature discloses an increasing intensity of the feature close to the absorption edge with decreasing cluster size. As a result, a linear relationship between the cluster size and the X-ray absorption coefficient can be established for the first time. Electronic supplementary information (ESI) available: The deconvoluted reference spectra are given in ESI Fig. 1-9. See DOI: 10.1039/c4nr07051h

Aluminum nanostructures with strong visible-range SERS activity for versatile micropatterning of molecular security labels.

PubMed

Lay, Chee Leng; Koh, Charlynn Sher Lin; Wang, Jing; Lee, Yih Hong; Jiang, Ruibin; Yang, Yijie; Yang, Zhe; Phang, In Yee; Ling, Xing Yi

2018-01-03

The application of aluminum (Al)-based nanostructures for visible-range plasmonics, especially for surface-enhanced Raman scattering (SERS), currently suffers from inconsistent local electromagnetic field distributions and/or inhomogeneous distribution of probe molecules. Herein, we lithographically fabricate structurally uniform Al nanostructures which enable homogeneous adsorption of various probe molecules. Individual Al nanostructures exhibit strong local electromagnetic field enhancements, in turn leading to intense SERS activity. The average SERS enhancement factor (EF) for individual nanostructures exceeds 10 4 for non-resonant probe molecules in the visible spectrum. These Al nanostructures also retain more than 70% of their original SERS intensities after one-month storage, displaying superb stability under ambient conditions. We further achieve tunable polarization-dependent SERS responses using anisotropic Al nanostructures, facilitating the design of sophisticated SERS-based security labels. Our micron-sized security label comprises two-tier security features, including a machine-readable hybrid quick-response (QR) code overlaid with a set of ciphertexts. Our work demonstrates the versatility of Al-based structures in low-cost modern chemical nano-analytics and forgery protection.

Modification degrees at specific sites on heparan sulphate: an approach to measure chemical modifications on biological molecules with stable isotope labelling

PubMed Central

Wu, Zhengliang L.; Lech, Miroslaw

2005-01-01

Chemical modification of biological molecules is a general mechanism for cellular regulation. A quantitative approach has been developed to measure the extent of modification on HS (heparan sulphates). Sulphation on HS by sulphotransferases leads to variable sulphation levels, which allows cells to tune their affinities to various extracellular proteins, including growth factors. With stable isotope labelling and HPLC-coupled MS, modification degrees at various O-sulphation sites could be determined. A bovine kidney HS sample was first saturated in vitro with 34S by an OST (O-sulphotransferase), then digested with nitrous acid and analysed with HPLC-coupled MS. The 34S-labelled oligosaccharides were identified based on their unique isotope clusters. The modification degrees at the sulphotransferase recognition sites were obtained by calculating the intensities of isotopic peaks in the isotope clusters. The modification degrees at 3-OST-1 and 6-OST-1 sites were examined in detail. This approach can also be used to study other types of chemical modifications on biological molecules. PMID:15743272

Developing Photo Activated Localization Microscopy

NASA Astrophysics Data System (ADS)

Hess, Harald

2015-03-01

Photo Activated Localization Microscopy, PALM, acquires super-resolution images by activating a subset of activatable fluorescent labels and estimating the center of the each molecular label to sub-diffractive accuracy. When this process is repeated thousands of times for different subsets of molecules, then an image can be rendered from all the center coordinates of the molecules. I will describe the circuitous story of its development that began with another super-resolution technique, NSOM, developed by my colleague Eric Betzig, who imaged single molecules at room temperature, and later we spectrally resolved individual luminescent centers of quantum wells. These two observations inspired a generalized path to localization microscopy, but that path was abandoned because no really useful fluorescent labels were available. After a decade of nonacademic industrial pursuits and the subsequent freedom of unemployment, we came across a class of genetically expressible fluorescent proteins that were switchable or convertible that enabled the concept to be implemented and be biologically promising. The past ten years have been very active with many groups exploring applications and enhancements of this concept. Demonstrating significant biological relevance will be the metric if its success.

Distribution of distances between DNA barcode labels in nanochannels close to the persistence length

NASA Astrophysics Data System (ADS)

Reinhart, Wesley F.; Reifenberger, Jeff G.; Gupta, Damini; Muralidhar, Abhiram; Sheats, Julian; Cao, Han; Dorfman, Kevin D.

2015-02-01

We obtained experimental extension data for barcoded E. coli genomic DNA molecules confined in nanochannels from 40 nm to 51 nm in width. The resulting data set consists of 1 627 779 measurements of the distance between fluorescent probes on 25 407 individual molecules. The probability density for the extension between labels is negatively skewed, and the magnitude of the skewness is relatively insensitive to the distance between labels. The two Odijk theories for DNA confinement bracket the mean extension and its variance, consistent with the scaling arguments underlying the theories. We also find that a harmonic approximation to the free energy, obtained directly from the probability density for the distance between barcode labels, leads to substantial quantitative error in the variance of the extension data. These results suggest that a theory for DNA confinement in such channels must account for the anharmonic nature of the free energy as a function of chain extension.

Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface

PubMed Central

Siegrist, M. Sloan; Swarts, Benjamin M.; Fox, Douglas M.; Lim, Shion An; Bertozzi, Carolyn R.

2015-01-01

The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology. PMID:25725012

High-Throughput Block Optical DNA Sequence Identification.

PubMed

Sagar, Dodderi Manjunatha; Korshoj, Lee Erik; Hanson, Katrina Bethany; Chowdhury, Partha Pratim; Otoupal, Peter Britton; Chatterjee, Anushree; Nagpal, Prashant

2018-01-01

Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label-free optical DNA sequencing technique will require nanoscale focusing of light, a high-throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the "fingerprinting region" from ≈400-1400 cm -1 . Here, surface-enhanced Raman spectroscopy is used to demonstrate label-free identification of DNA nucleobases with multiplexed 3D plasmonic nanofocusing. While nanometer-scale mode volumes prevent identification of single nucleobases within a DNA sequence, the block optical technique can identify A, T, G, and C content in DNA k-mers. The content of each nucleotide in a DNA block can be a unique and high-throughput method for identifying sequences, genes, and other biomarkers as an alternative to single-letter sequencing. Additionally, coupling two complementary vibrational spectroscopy techniques (infrared and Raman) can improve block characterization. These results pave the way for developing a novel, high-throughput block optical sequencing method with lossy genomic data compression using k-mer identification from multiplexed optical data acquisition. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Efficient Enzymatic Preparation of (13) N-Labelled Amino Acids: Towards Multipurpose Synthetic Systems.

PubMed

da Silva, Eunice S; Gómez-Vallejo, Vanessa; Baz, Zuriñe; Llop, Jordi; López-Gallego, Fernando

2016-09-12

Nitrogen-13 can be efficiently produced in biomedical cyclotrons in different chemical forms, and its stable isotopes are present in the majority of biologically active molecules. Hence, it may constitute a convenient alternative to Fluorine-18 and Carbon-11 for the preparation of positron-emitter-labelled radiotracers; however, its short half-life demands for the development of simple, fast, and efficient synthetic processes. Herein, we report the one-pot, enzymatic and non-carrier-added synthesis of the (13) N-labelled amino acids l-[(13) N]alanine, [(13) N]glycine, and l-[(13) N]serine by using l-alanine dehydrogenase from Bacillus subtilis, an enzyme that catalyses the reductive amination of α-keto acids by using nicotinamide adenine dinucleotide (NADH) as the redox cofactor and ammonia as the amine source. The integration of both l-alanine dehydrogenase and formate dehydrogenase from Candida boidinii in the same reaction vessel to facilitate the in situ regeneration of NADH during the radiochemical synthesis of the amino acids allowed a 50-fold decrease in the concentration of the cofactor without compromising reaction yields. After optimization of the experimental conditions, radiochemical yields were sufficient to carry out in vivo imaging studies in small rodents. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Triggered optical biosensor

DOEpatents

Song, Xuedong; Swanson, Basil I.

2001-10-02

An optical biosensor is provided for the detection of a multivalent target biomolecule, the biosensor including a substrate having a bilayer membrane thereon, a recognition molecule situated at the surface, the recognition molecule capable of binding with the multivalent target biomolecule, the recognition molecule further characterized as including a fluorescence label thereon and as being movable at the surface and a device for measuring a fluorescence change in response to binding between the recognition molecule and the multivalent target biomolecule.

A systematization of spectral data on the methanol molecule

NASA Astrophysics Data System (ADS)

Akhlyostin, A. Yu.; Voronina, S. S.; Lavrentiev, N. A.; Privezentsev, A. I.; Rodimova, O. B.; Fazliev, A. Z.

2015-11-01

Problems underlying a systematization of spectral data on the methanol molecule are formulated. Data on the energy levels and vacuum wavenumbers acquired from the published literature are presented in the form of information sources imported into the W@DIS information system. Sets of quantum numbers and labels used to describe the CH3OH molecular states are analyzed. The set of labels is different from universally accepted sets. A system of importing the data sources into W@DIS is outlined. The structure of databases characterizing transitions in an isolated CH3OH molecule is introduced and a digital library of the relevant published literature is discussed. A brief description is given of an imported data quality analysis and representation of the results obtained in the form of ontologies for subsequent computer processing.

Single DNA molecule detection using nanopipettes and nanoparticles.

PubMed

Karhanek, Miloslav; Kemp, Jennifer T; Pourmand, Nader; Davis, Ronald W; Webb, Chris D

2005-02-01

Single DNA molecules labeled with nanoparticles can be detected by blockades of ionic current as they are translocated through a nanopipette tip formed by a pulled glass capillary. The nanopipette detection technique can provide not only tools for detection and identification of single DNA and protein molecules but also deeper insight and understanding of stochastic interactions of various biomolecules with their environment.

Highly sensitive detection using microring resonator and nanopores

NASA Astrophysics Data System (ADS)

Bougot-Robin, K.; Hoste, J. W.; Le Thomas, N.; Bienstman, P.; Edel, J. B.

2016-04-01

One of the most significant challenges facing physical and biological scientists is the accurate detection and identification of single molecules in free-solution environments. The ability to perform such sensitive and selective measurements opens new avenues for a large number of applications in biological, medical and chemical analysis, where small sample volumes and low analyte concentrations are the norm. Access to information at the single or few molecules scale is rendered possible by a fine combination of recent advances in technologies. We propose a novel detection method that combines highly sensitive label-free resonant sensing obtained with high-Q microcavities and position control in nanoscale pores (nanopores). In addition to be label-free and highly sensitive, our technique is immobilization free and does not rely on surface biochemistry to bind probes on a chip. This is a significant advantage, both in term of biology uncertainties and fewer biological preparation steps. Through combination of high-Q photonic structures with translocation through nanopore at the end of a pipette, or through a solid-state membrane, we believe significant advances can be achieved in the field of biosensing. Silicon microrings are highly advantageous in term of sensitivity, multiplexing, and microfabrication and are chosen for this study. In term of nanopores, we both consider nanopore at the end of a nanopipette, with the pore being approach from the pipette with nanoprecise mechanical control. Alternatively, solid state nanopores can be fabricated through a membrane, supporting the ring. Both configuration are discussed in this paper, in term of implementation and sensitivity.

Development and Application of Chemical Probes for Vibrational Imaging by Stimulated Raman Scattering

NASA Astrophysics Data System (ADS)

Hu, Fanghao

During the last decade, Raman microscopy is experiencing rapid development and increasingly applied in biological and medical systems. Especially, stimulated Raman scattering (SRS) microscopy, which significantly improves the sensitivity of Raman scattering through stimulated emission, has allowed direct visualization of many species that are previously challenging with conventional fluorescence imaging. Compared to fluorescence, SRS imaging requires no label or small label on the target molecule, thus with minimal perturbation to the molecule of interest. Moreover, Raman scattering is free from complicated photophysical and photochemical processes such as photobleaching, and has intrinsically narrower linewidth than fluorescence emission. This allows multiplexed Raman imaging with minimal spectral crosstalk and excellent photo-stability. To achieve the full potential of Raman microscopy, vibrational probes have been developed for Raman imaging. Multiple Raman probes with a few atoms in size are applied in Raman imaging with high sensitivity and specificity. An overview of both fluorescence and Raman microscopy and their imaging probes is given in Chapter 1 with a brief discussion on the SRS theory. Built on the current progress of Raman microscopy and vibrational probes, I write on my research in the development of carbon-deuterium, alkyne and nitrile probes for visualizing choline metabolism (Chapter 2), glucose uptake activity (Chapter 3), complex brain metabolism (Chapter 4) and polymeric nanoparticles (Chapter 5) in live cells and tissues, as well as the development of polyyne-based vibrational probes for super-multiplexed imaging, barcoding and analysis (Chapter 6).

Label-free optical imaging of nonfluorescent molecules by stimulated radiation.

PubMed

Min, Wei

2011-12-01

Imaging contrasts other than fluorescence are highly desirable for label-free detection and interrogation of nonfluorescent molecular species inside live cells, tissues, and organisms. The recently developed stimulated Raman scattering (SRS) and stimulated emission microscopy techniques provide sensitive and specific contrast mechanisms for nonfluorescent species, by employing the light amplification aspect of stimulated radiation. Compared to their spontaneous counterparts, stimulated radiation can enhance the imaging performance significantly, making the previously 'dark' molecules observable. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques. Copyright © 2011 Elsevier Ltd. All rights reserved.

Size ratio performance in detecting cerebral aneurysm rupture status is insensitive to small vessel removal.

PubMed

Lauric, Alexandra; Baharoglu, Merih I; Malek, Adel M

2013-04-01

The variable definition of size ratio (SR) for sidewall (SW) vs bifurcation (BIF) aneurysms raises confusion for lesions harboring small branches, such as carotid ophthalmic or posterior communicating locations. These aneurysms are considered SW by many clinicians, but SR methodology classifies them as BIF. To evaluate the effect of ignoring small vessels and SW vs stringent BIF labeling on SR ruptured aneurysm detection performance in borderline aneurysms with small branches, and to reconcile SR-based labeling with clinical SW/BIF classification. Catheter rotational angiographic datasets of 134 consecutive aneurysms (60 ruptured) were automatically measured in 3-dimensional. Stringent BIF labeling was applied to clinically labeled aneurysms, with 21 aneurysms switching label from SW to BIF. Parent vessel size was evaluated both taking into account, and ignoring, small vessels. SR was defined accordingly as the ratio between aneurysm and parent vessel sizes. Univariate and multivariate statistics identified significant features. The square of the correlation coefficient (R(2)) was reported for bivariate analysis of alternative SR calculations. Regardless of SW/BIF labeling method, SR was equally significant in discriminating aneurysm ruptured status (P < .001). Bivariate analysis of alternative SR had a high correlation of R(2) = 0.94 on the whole dataset, and R = 0.98 on the 21 borderline aneurysms. Ignoring small branches from SR calculation maintains rupture status detection performance, while reducing postprocessing complexity and removing labeling ambiguity. Aneurysms adjacent to these vessels can be considered SW for morphometric analysis. It is reasonable to use the clinical SW/BIF labeling when using SR for rupture risk evaluation.

Selective Adsorption on Fluorinated Plastic Enables the Optical Detection of Molecular Pollutants in Water

NASA Astrophysics Data System (ADS)

Lanfranco, R.; Giavazzi, F.; Salina, M.; Tagliabue, G.; Di Nicolò, E.; Bellini, T.; Buscaglia, M.

2016-05-01

Amorphous fluorinated plastic can be produced with a refractive index similar to that of water, a condition that makes it essentially invisible when immersed in aqueous solutions. Because of this property, even a small amount of adsorbed molecules on the plastic-water interface provides a detectable optical signal. We investigate two distinct substrates made of this material, characterized by different interface areas: a prism and a microporous membrane. We demonstrate that both substrates enable the label-free detection of molecular compounds in water even without any surface functionalization. The adsorption of molecules on the planar surface of the prism provides an increase of optical reflectivity, whereas the adsorption on the internal surface of the microporous membrane yields an increase of scattered light. Despite the different mechanisms, we find a similar optical response upon adsorption. We confirm this result by a theoretical model accounting for both reflection and scattering. We investigate the spontaneous adsorption process for different kinds of molecules: surfactants with different charges, a protein (lysozyme), and a constituent of gasoline (hexane). The measured equilibrium and kinetic constants for adsorption differ by orders of magnitudes among the different classes of molecules. By suitable analytical models, accounting for the effects of mass limitation and transport, we find a simple and general scaling of the adsorption parameters with the molecular size.

Consistent assignment of the vibrations of symmetric and asymmetric meta-disubstituted benzenes

NASA Astrophysics Data System (ADS)

Kemp, David J.; Tuttle, William D.; Jones, Florence M. S.; Gardner, Adrian M.; Andrejeva, Anna; Wakefield, Jonathan C. A.; Wright, Timothy G.

2018-04-01

The assignment of vibrational structure in spectra gives valuable insights into geometric and electronic structure changes upon electronic excitation or ionization; particularly when such information is available for families of molecules. We give a description of the phenyl-ring-localized vibrational modes of the ground (S0) electronic states of sets of meta-disubstituted benzene molecules including both symmetrically- and asymmetrically-substituted cases. As in our earlier work on monosubstituted benzenes (Gardner and Wright, 2011), para-disubstituted benzenes (Andrejeva et al., 2016), and ortho-disubstituted benzenes (Tuttle et al., 2018), we conclude that the use of the commonly-used Wilson or Varsányi mode labels, which are based on the vibrational motions of benzene itself, is misleading and ambiguous. Instead, we label the phenyl-ring-localized modes consistently based upon the Mulliken (Herzberg) method for the modes of meta-difluorobenzene (mDFB) under Cs symmetry, since we wish the labelling scheme to cover both symmetrically- and asymmetrically-substituted molecules. By studying the vibrational wavenumbers obtained from the same force-field while varying the mass of the substituent, we are able to follow the evolving modes across a wide range of molecules and hence provide consistent assignments. We assign the vibrations of the following sets of molecules: the symmetric meta-dihalobenzenes, meta-xylene and resorcinol (meta-dihydroxybenzene); and the asymmetric meta-dihalobenzenes, meta-halotoluenes, meta-halophenols and meta-cresol. In the symmetrically-substituted species, we find two pairs of in-phase and out-of-phase carbon-substituent stretches, and this motion persists in asymmetrically-substituted molecules for heavier substituents; however, when at least one of the substituents is light, then we find that these evolve into localized carbon-substituent stretches.

Tutorial for the structure elucidation of small molecules by means of the LSD software.

PubMed

Nuzillard, Jean-Marc; Plainchont, Bertrand

2018-06-01

Automatic structure elucidation of small molecules by means of the "logic for structure elucidation" (LSD) software is introduced in the context of the automatic exploitation of chemical shift correlation data and with minimal input from chemical shift values. The first step in solving a structural problem by means of LSD is the extraction of pertinent data from the 1D and 2D spectra. This operation requires the labeling of the resonances and of their correlations; its reliability highly depends on the quality of the spectra. The combination of COSY, HSQC, and HMBC spectra results in proximity relationships between nonhydrogen atoms that are associated in order to build the possible solutions of a problem. A simple molecule, camphor, serves as an example for the writing of an LSD input file and to show how solution structures are obtained. An input file for LSD must contain a nonambiguous description of each atom, or atom status, which includes the chemical element symbol, the hybridization state, the number of bound hydrogen atoms and the formal electric charge. In case of atom status ambiguity, the pyLSD program performs clarification by systematically generating the status of the atoms. PyLSD also proposes the use of the nmrshiftdb algorithm in order to rank the solutions of a problem according to the quality of the fit between the experimental carbon-13 chemical shifts, and the ones predicted from the proposed structures. To conclude, some hints toward future uses and developments of computer-assisted structure elucidation by LSD are proposed. Copyright © 2017 John Wiley & Sons, Ltd.

Chemical biology-based approaches on fluorescent labeling of proteins in live cells.

PubMed

Jung, Deokho; Min, Kyoungmi; Jung, Juyeon; Jang, Wonhee; Kwon, Youngeun

2013-05-01

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (~4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.

Interspecies scaling and prediction of human clearance: comparison of small- and macro-molecule drugs

PubMed Central

Huh, Yeamin; Smith, David E.; Feng, Meihau Rose

2014-01-01

Human clearance prediction for small- and macro-molecule drugs was evaluated and compared using various scaling methods and statistical analysis.Human clearance is generally well predicted using single or multiple species simple allometry for macro- and small-molecule drugs excreted renally.The prediction error is higher for hepatically eliminated small-molecules using single or multiple species simple allometry scaling, and it appears that the prediction error is mainly associated with drugs with low hepatic extraction ratio (Eh). The error in human clearance prediction for hepatically eliminated small-molecules was reduced using scaling methods with a correction of maximum life span (MLP) or brain weight (BRW).Human clearance of both small- and macro-molecule drugs is well predicted using the monkey liver blood flow method. Predictions using liver blood flow from other species did not work as well, especially for the small-molecule drugs. PMID:21892879

The δ18O of Atmospheric Water Vapour is Recorded in the Oxygen Isotope Ratios of Leaf water and Organic Molecules at High Relative Humidity

NASA Astrophysics Data System (ADS)

Lehmann, M. M.; Goldsmith, G. R.; Schmid, L.; Siegwolf, R. T.; Gessler, A.; Saurer, M.

2016-12-01

The oxygen stable isotope ratios (δ18O) of water and organic molecules in plants hold information about plant physiology, ecohydrology, and environmental conditions. For instance, the δ18O ratio of leaf water reflects both the δ18O ratios of water in the soil and in the atmosphere. This water, which is incorporated into organic molecules at the time of synthesis, thus serves to record the environment in which the plant was growing. However, how δ18O of atmospheric water vapour affects the δ18O ratio of organic molecules remains poorly understood. In order to investigate the effects of fog and rain (e.g. high atmospheric water availability) on δ18O ratios of leaf water and organic molecules, we exposed oak tree saplings (Quercus robur) in wet and dry soil treatments to 18O-depleted water vapour at ca. 90% relative humidity for 5 h. We harvested plant material over 24 h to trace the movement of the isotopic label in water and organics throughout the plant from the leaves to the stem. The atmospheric water vapour caused a strong 18O-depletion in leaf and xylem water, as well as in leaf carbohydrates, with the most negative ratios observed at the end of the fogging. Moreover, the label was clearly observed in twig and stem phloem carbohydrates following a short delay. A detailed compound-specific isotope analysis of the leaf carbohydrates revealed that the label caused an 18O-depletion in fructose, glucose, and sucrose. Quercitol, an oak-specific alditol, did not show 18O-depletion. Clear soil moisture treatment effects were only observed for twig phloem carbohydrates, with a stronger 18O-depletion in wet plants than in dry plants, suggesting retarded leaf-to-phloem sugar export in trees under drought. We demonstrate that labelling with 18O-depleted water is a potential tool to trace the movement and incorporation of oxygen stable isotopes in plants. We clearly show that changes in δ18O of atmospheric water vapour are quickly imprinted on leaf water and ultimately incorporated into organic molecules.

Using the QCM Biosensor-Based T7 Phage Display Combined with Bioinformatics Analysis for Target Identification of Bioactive Small Molecule.

PubMed

Takakusagi, Yoichi; Takakusagi, Kaori; Sugawara, Fumio; Sakaguchi, Kengo

2018-01-01

Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 phage capsid. Here, we describe the T7 phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used.

Nanostructures based on quantum dots for application in promising methods of single- and multiphoton imaging and diagnostics

NASA Astrophysics Data System (ADS)

Nabiev, I. R.

2017-01-01

Molecules recognizing biomarkers of diseases (monoclonal antibodies (monoABs)) are often too large for biomedical applications, and the conditions that are used to bind them with nanolabels lead to disordered orientation of monoABs with respect to the nanoparticle surface. Extremely small nanoprobes, designed via oriented conjugation of quantum dots (QDs) with single-domain antibodies (sdABs) derived from the immunoglobulin of llama and produced in the E. coli culture, have a hydrodynamic diameter less than 12 nm and contain equally oriented sdAB molecules on the surface of each QD. These nanoprobes exhibit excellent specificity and sensitivity in quantitative determination of a small number of cells expressing biomarkers. In addition, the higher diffusion coefficient of sdABs makes it possible to perform immunohistochemical analysis in bulk tissue, inaccessible for conventional monoABs. The necessary conditions for implementing high-quality immunofluorescence diagnostics are a high specificity of labeling and clear differences between the fluorescence of nanoprobes and the autofluorescence of tissues. Multiphoton micros-copy with excitation in the near-IR spectral range, which is remote from the range of tissue autofluorescence excitation, makes it possible to solve this problem and image deep layers in biological tissues. The two-photon absorption cross sections of CdSe/ZnS QDs conjugated with sdABs exceed the corresponding values for organic fluorophores by several orders of magnitude. These nanoprobes provide clear discrimination between the regions of tumor and normal tissues with a ratio of the sdAB fluorescence to the tissue autofluorescence upon two-photon excitation exceeding that in the case of single-photon excitation by a factor of more than 40. The data obtained indicate that the sdAB-QD conjugates used as labels provide the same, or even better, quality as the "gold standard" of immunohistochemical diagnostics. The developed nanoprobes are expected to find wide application in high-efficiency imaging of tumor and multiparameter diagnostics.

Effects of 31 FDA approved small-molecule kinase inhibitors on isolated rat liver mitochondria.

PubMed

Zhang, Jun; Salminen, Alec; Yang, Xi; Luo, Yong; Wu, Qiangen; White, Matthew; Greenhaw, James; Ren, Lijun; Bryant, Matthew; Salminen, William; Papoian, Thomas; Mattes, William; Shi, Qiang

2017-08-01

The FDA has approved 31 small-molecule kinase inhibitors (KIs) for human use as of November 2016, with six having black box warnings for hepatotoxicity (BBW-H) in product labeling. The precise mechanisms and risk factors for KI-induced hepatotoxicity are poorly understood. Here, the 31 KIs were tested in isolated rat liver mitochondria, an in vitro system recently proposed to be a useful tool to predict drug-induced hepatotoxicity in humans. The KIs were incubated with mitochondria or submitochondrial particles at concentrations ranging from therapeutic maximal blood concentrations (Cmax) levels to 100-fold Cmax levels. Ten endpoints were measured, including oxygen consumption rate, inner membrane potential, cytochrome c release, swelling, reactive oxygen species, and individual respiratory chain complex (I-V) activities. Of the 31 KIs examined only three including sorafenib, regorafenib and pazopanib, all of which are hepatotoxic, caused significant mitochondrial toxicity at concentrations equal to the Cmax, indicating that mitochondrial toxicity likely contributes to the pathogenesis of hepatotoxicity associated with these KIs. At concentrations equal to 100-fold Cmax, 18 KIs were found to be toxic to mitochondria, and among six KIs with BBW-H, mitochondrial injury was induced by regorafenib, lapatinib, idelalisib, and pazopanib, but not ponatinib, or sunitinib. Mitochondrial liability at 100-fold Cmax had a positive predictive power (PPV) of 72% and negative predictive power (NPV) of 33% in predicting human KI hepatotoxicity as defined by product labeling, with the sensitivity and specificity being 62% and 44%, respectively. Similar predictive power was obtained using the criterion of Cmax ≥1.1 µM or daily dose ≥100 mg. Mitochondrial liability at 1-2.5-fold Cmax showed a 100% PPV and specificity, though the NPV and sensitivity were 32% and 14%, respectively. These data provide novel mechanistic insights into KI hepatotoxicity and indicate that mitochondrial toxicity at therapeutic levels can help identify hepatotoxic KIs.

Novel Raman Techniques for Imaging and Sensing

NASA Astrophysics Data System (ADS)

Edwards, Perry S.

Raman scattering spectroscopy is extensively demonstrated as a label-free, chemically selective sensing and imaging technique for a multitude of chemical and biological applications. The ability to detect "fingerprint" spectral signatures of individual molecules, without the need to introduce chemical labelers, makes Raman scattering a powerful sensing technique. However, spectroscopy based on spontaneous Raman scattering traditionally suffers from inherently weak signals due to small Raman scattering cross-sections. Thus, considerable efforts have been put forth to find pathways towards enhancing Raman signals to bolster sensitivity for detecting small concentrations of molecules or particles. The development of coherent Raman techniques that can offer orders of magnitude increase in signal have garnered significant interest in recent years for their application in imaging; such techniques include coherent anti-Stokes Raman scattering and stimulated Raman scattering. Additionally, methods to enhance the local field of either the pump or generated Raman signal, such as through surface enhanced Raman scattering, have been investigated for their orders of magnitude improvement in sensitivity and single molecule sensing capability. The work presented in this dissertation describes novel techniques for performing high speed and highly sensitive Raman imaging as well as sensing applications towards bioimaging and biosensing. Coherent anti-Stokes Raman scattering (CARS) is combined with holography to enable recording of high-speed (single laser shot), wide field CARS holograms which can be used to reconstruct the both the amplitude and the phase of the anti-Stokes field therefore allowing 3D imaging. This dissertation explores CARS holography as a viable label-free bio-imaging technique. A Raman scattering particle sensing system is also developed that utilizes wave guide properties of optical fibers and ring-resonators to perform enhanced particle sensing. Resonator-enhanced particle sensing is experimentally examined as a new method for enhancing Raman scattering from particles interacting with circulating optical fields within both a fiber ring-cavity and whispering gallery mode microtoroid microresonators. The achievements described in this dissertation include: (1) Demonstration of the bio-imaging capability of CARS holography by recording of CARS holograms of subcellular components in live cancer (HeLa) cells. (2) Label-free Raman microparticle sensing using a tapered optical fibers. A tapered fiber can guide light to particles adsorbed on the surface of the taper to generate scattered Raman signal which can be collected by a microRaman detection system. (3) Demonstration of the proof of concept of resonator-enhanced Raman spectroscopy in a fiber ring resonator consisting of a section of fiber taper. (4) A method for locking the pump laser to the resonate frequencies of a resonator. This is demonstrated using a fiber ring resonator and microtoroid microresonators. (5) Raman scattered signal from particles adhered to microtoroid microresonators is acquired using 5 seconds of signal integration time and with the pump laser locked to a cavity resonance. (6) Theoretical analysis is performed that indicates resonator-enhanced Raman scattering from microparticles adhered to microresonators can be achieved with the pump laser locked to the frequency of a high-Q cavity resonant mode.

Detecting RNA/DNA hybridization using double-labeled donor probes with enhanced fluorescence resonance energy transfer signals.

PubMed

Okamura, Yukio; Watanabe, Yuichiro

2006-01-01

Fluorescence resonance energy transfer (FRET) occurs when two fluorophores are in close proximity, and the emission energy of a donor fluorophore is transferred to excite an acceptor fluorophore. Using such fluorescently labeled oligonucleotides as FRET probes, makes possible specific detection of RNA molecules even if similar sequences are present in the environment. A higher ratio of signal to background fluorescence is required for more sensitive probe detection. We found that double-labeled donor probes labeled with BODIPY dye resulted in a remarkable increase in fluorescence intensity compared to single-labeled donor probes used in conventional FRET. Application of this double-labeled donor system can improve a variety of FRET techniques.

Iodine 125 Imaging in Mice Using NaI(Tl)/Flat Panel PMT Integral Assembly

NASA Astrophysics Data System (ADS)

Cinti, M. N.; Majewski, S.; Williams, M. B.; Bachmann, C.; Cominelli, F.; Kundu, B. K.; Stolin, A.; Popov, V.; Welch, B. L.; De Vincentis, G.; Bennati, P.; Betti, M.; Ridolfi, S.; Pani, R.

2007-06-01

Radiolabeled agents that bind to specific receptors have shown great promise in diagnosing and characterizing tumor cell biology. In vivo imaging of gene transcription and protein expression represents an other area of interest. The radioisotope I is commercially available as a label for molecular probes and utilized by researchers in small animal studies. We propose an advanced imaging detector based on planar NaI(T1) integral assembly with a Hamamatsu Flat Panel Photomultiplier (MA-PMT) representing one of the best trade-offs between spatial resolution and detection efficiency. We characterized the imaging performances of this planar detector, in comparison with a gamma camera based on a pixellated scintillator. We also tested the in-vivo image capability by acquiring images of mice as a part of a study of inflammatory bowel disease (IBD). In this study, four 25g mice with an IBD-like phenotype (SAMP1/YitFc) were injected with 375, 125, 60 and 30 muCi of I-labelled antibody against mucosal vascular addressin cell adhesion molecule (MAdCAM-1), which is up-regulated in the presence of inflammation. Two mice without bowel inflammation were injected with 150 and 60 muCi of the labeled anti-MAdCAM-1 antibody as controls. To better evaluate the performances of the integral assembly detector, we also acquired mice images with a dual modality (X and Gamma Ray) camera dedicated for small animal imaging. The results coming from this new detector are considerable: images of SAMP1/YitFc injected with 30 muCi activity show inflammation throughout the intestinal tract, with the disease very well defined at two hours post-injection.

Solvent effect on FRET spectroscopic ruler

NASA Astrophysics Data System (ADS)

Qu, Songyuan; Liu, Chuanbo; Liu, Qiong; Wu, Wei; Du, Baoji; Wang, Jin

2018-03-01

A discrepancy has emerged in recent years between single-molecule Förster resonance energy transfer (smFRET) measurements and small angle X-ray scattering (SAXS) or small angle neutron scattering experiments in the study of unfolded or intrinsically disordered proteins in denaturing solutions. Despite significant advances that have been made in identifying various factors which may have contributed to the manifestation of the so-called smFRET-SAXS discrepancy, no consensus has been reached so far on its original source or eventual resolution. In this study, we investigate this problem from the perspective of the solvent effect on FRET spectroscopic ruler (SEFSR), a generic term we use to describe various solvent-dependent factors affecting the accuracy of the FRET experimental method that is known as a "spectroscopic ruler." Some factors belonging to SEFSR, such as direct dye-solvent interaction and labeling configuration, seem to have not received due attention regarding their significance in contributing to the discrepancy. We identify SEFSR by measuring a rigid segment of a double-stranded DNA in various solutions using the smFRET method and evaluate its relative importance in smFRET experiments by measuring segments of a single-stranded DNA and polyethylene glycol (PEG) in solutions. We find that SEFSR can produce non-negligible FRET-inferred interdye distance changes in various solutions, with an intensity following the Hofmeister series in ionic solutions and dependent on labeling configurations. SEFSR is found to be significant in GuHCl and urea solutions, which can fully cover the apparent expansion signal of dye-labeled PEG. Our findings suggest that SEFSR may have played an important role in contributing to the smFRET-SAXS discrepancy.

Exploitation of phosphorescent labelling reagent of fullerol-fluorescein isothiocyanate and new method for the determination of trace alkaline phosphatase as well as forecast of human diseases.

PubMed

Liu, Jia-Ming; Huang, Xiao-Mei; Liu, Zhen-Bo; Lin, Shao-Qin; Li, Fei-Ming; Gao, Fei; Li, Zhi-Ming; Zeng, Li-Qing; Li, Lian-Ying; Ouyang, Ying

2009-08-26

A new phosphorescent labelling reagent consisting of fullerol, fluorescein isothiocyanate and N,N-dimethylaniline (F-ol-(FITC)(n)-DMA) was developed. The mode of action is based on the reactivity of the active -OH group in F-ol with the -COOH group of FITC to form an F-ol-(FITC)(n)-DMA complex containing several FITC molecules. F-ol-(FITC)(n)-DMA increased the number of luminescent molecules in the biological target of WGA-AP-WGA-F-ol-(FITC)(n)-DMA (WGA and AP are wheat germ agglutinin and alkaline phosphatase, respectively) which improved the sensitivity using solid substrate room temperature phosphorimetry (SSRTP) detection. The proposed method provided high sensitivity and strong specificity for WGA-AP. The limit of detection (LD) was 0.15 ag AP spot(-1) for F-ol and 0.097 ag AP spot(-1) for FITC in F-ol-(FITC)(n)-DMA, which was lower than the method using single luminescent molecules of F-ol-DMA and FITC-DMA to label WGA (0.20 ag AP spot(-1) for F-ol-DMA and 0.22 ag AP spot(-1) for FITC-DMA). Results for the determination of AP in human serum were in good agreement with those obtained by enzyme-linked immunosorbent assay. The mechanism of F-ol-(FITC)(n)-DMA labelling of WGA was discussed.

Facilities for small-molecule crystallography at synchrotron sources.

PubMed

Barnett, Sarah A; Nowell, Harriott; Warren, Mark R; Wilcox, Andrian; Allan, David R

2016-01-01

Although macromolecular crystallography is a widely supported technique at synchrotron radiation facilities throughout the world, there are, in comparison, only very few beamlines dedicated to small-molecule crystallography. This limited provision is despite the increasing demand for beamtime from the chemical crystallography community and the ever greater overlap between systems that can be classed as either small macromolecules or large small molecules. In this article, a very brief overview of beamlines that support small-molecule single-crystal diffraction techniques will be given along with a more detailed description of beamline I19, a dedicated facility for small-molecule crystallography at Diamond Light Source.

A post-labeling strategy based on dye-induced peeling of the aptamer off single-walled carbon nanotubes for electrochemical aptasensing.

PubMed

Fu, Yingchun; Wang, Ting; Bu, Lijuan; Xie, Qingji; Li, Penghao; Chen, Jinhua; Yao, Shouzhuo

2011-03-07

A simple and efficient post-labeling strategy based on dye-induced peeling of the aptamer molecules off single-walled carbon nanotubes was developed for electrochemical aptasensing of thrombin with a detection limit down to 3 pM.

New trends and applications in carboxylation for isotope chemistry.

PubMed

Bragg, Ryan A; Sardana, Malvika; Artelsmair, Markus; Elmore, Charles S

2018-05-08

Carboxylations are an important method for the incorporation of isotopically labeled 14 CO 2 into molecules. This manuscript will review labeled carboxylations since 2010 and will present a perspective on the potential of recent unlabeled methodology for labeled carboxylations. The perspective portion of the manuscript is broken into 3 major sections based on product type, arylcarboxylic acids, benzylcarboxylic acids, and alkyl carboxylic acids, and each of those sections is further subdivided by substrate. © 2018 AstraZeneca. Journal of Labelled Compounds and Radiopharmaceuticals Published by John Wiley & Sons, Ltd.

Systems Based Study of the Therapeutic Potential of Small Charged Molecules for the Inhibition of IL-1 Mediated Cartilage Degradation

PubMed Central

Kar, Saptarshi; Smith, David W.; Gardiner, Bruce S.; Grodzinsky, Alan J.

2016-01-01

Inflammatory cytokines are key drivers of cartilage degradation in post-traumatic osteoarthritis. Cartilage degradation mediated by these inflammatory cytokines has been extensively investigated using in vitro experimental systems. Based on one such study, we have developed a computational model to quantitatively assess the impact of charged small molecules intended to inhibit IL-1 mediated cartilage degradation. We primarily focus on the simplest possible computational model of small molecular interaction with the IL-1 system—direct binding of the small molecule to the active site on the IL-1 molecule itself. We first use the model to explore the uptake and release kinetics of the small molecule inhibitor by cartilage tissue. Our results show that negatively charged small molecules are excluded from the negatively charged cartilage tissue and have uptake kinetics in the order of hours. In contrast, the positively charged small molecules are drawn into the cartilage with uptake and release timescales ranging from hours to days. Using our calibrated computational model, we subsequently explore the effect of small molecule charge and binding constant on the rate of cartilage degradation. The results from this analysis indicate that the small molecules are most effective in inhibiting cartilage degradation if they are either positively charged and/or bind strongly to IL-1α, or both. Furthermore, our results showed that the cartilage structural homeostasis can be restored by the small molecule if administered within six days following initial tissue exposure to IL-1α. We finally extended the scope of the computational model by simulating the competitive inhibition of cartilage degradation by the small molecule. Results from this model show that small molecules are more efficient in inhibiting cartilage degradation by binding directly to IL-1α rather than binding to IL-1α receptors. The results from this study can be used as a template for the design and development of more pharmacologically effective osteoarthritis drugs, and to investigate possible therapeutic options. PMID:27977731

Label-free in-flow detection of single DNA molecules using glass nanopipettes.

PubMed

Gong, Xiuqing; Patil, Amol V; Ivanov, Aleksandar P; Kong, Qingyuan; Gibb, Thomas; Dogan, Fatma; deMello, Andrew J; Edel, Joshua B

2014-01-07

With the view of enhancing the functionality of label-free single molecule nanopore-based detection, we have designed and developed a highly robust, mechanically stable, integrated nanopipette-microfluidic device which combines the recognized advantages of microfluidic systems and the unique properties/advantages of nanopipettes. Unlike more typical planar solid-state nanopores, which have inherent geometrical constraints, nanopipettes can be easily positioned at any point within a microfluidic channel. This is highly advantageous, especially when taking into account fluid flow properties. We show that we are able to detect and discriminate between DNA molecules of varying lengths when motivated through a microfluidic channel, upon the application of appropriate voltage bias across the nanopipette. The effects of applied voltage and volumetric flow rates have been studied to ascertain translocation event frequency and capture rate. Additionally, by exploiting the advantages associated with microfluidic systems (such as flow control and concomitant control over analyte concentration/presence), we show that the technology offers a new opportunity for single molecule detection and recognition in microfluidic devices.

Water accelerated transformation of d-limonene induced by ultraviolet irradiation and air exposure.

PubMed

Li, Li Jun; Hong, Peng; Jiang, Ze Dong; Yang, Yuan Fan; Du, Xi Ping; Sun, Hao; Wu, Li Ming; Ni, Hui; Chen, Feng

2018-01-15

d-Limonene is a fragrant chemical that widely exists in aromatic products. Isotopic labelling of water molecules plus GC-MS and GC-PCI-Q-TOF analyses were used to investigate the influence of water molecules on chemical transformation of d-limonene induced by UV irradiation and air exposure. The results showed that the synergistic effect of UV irradiation, air exposure and water presence could facilitate d-limonene transformation into the limonene oxides: p-mentha-2,8-dienols, hydroperoxides, carveols, l-carvone and carvone oxide. UV irradiation, air exposure, or water alone, however, caused negligible d-limonene transformation. With the aid of isotopic labelling of water and oxygen molecules, it was found that water molecules were split into hydrogen radicals and hydroxyl radicals, and the hydrogen radicals, in particular, promoted the transformation reactions. This study has elucidated the mechanism and factors that influence the transformation of d-limonene, which will benefit industries involved in production and storage of d-limonene-containing products. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA origami-based shape IDs for single-molecule nanomechanical genotyping

NASA Astrophysics Data System (ADS)

Zhang, Honglu; Chao, Jie; Pan, Dun; Liu, Huajie; Qiang, Yu; Liu, Ke; Cui, Chengjun; Chen, Jianhua; Huang, Qing; Hu, Jun; Wang, Lianhui; Huang, Wei; Shi, Yongyong; Fan, Chunhai

2017-04-01

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ~10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level.

DNA origami-based shape IDs for single-molecule nanomechanical genotyping

PubMed Central

Zhang, Honglu; Chao, Jie; Pan, Dun; Liu, Huajie; Qiang, Yu; Liu, Ke; Cui, Chengjun; Chen, Jianhua; Huang, Qing; Hu, Jun; Wang, Lianhui; Huang, Wei; Shi, Yongyong; Fan, Chunhai

2017-01-01

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ∼10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level. PMID:28382928

A description of the reformatted spring small grains labeling procedure used in test 2, part 2 of the US/Canada wheat and barley exploratory experiment

NASA Technical Reports Server (NTRS)

Palmer, W. F.; Magness, E. R. (Principal Investigator)

1981-01-01

The reformatted spring small grains labeling procedure is designed to be used for assigning crop identification labels to a predetermined and selected number of dots. The development and description of this procedure is presented.

Click strategies for single-molecule protein fluorescence.

PubMed

Milles, Sigrid; Tyagi, Swati; Banterle, Niccolò; Koehler, Christine; VanDelinder, Virginia; Plass, Tilman; Neal, Adrian P; Lemke, Edward A

2012-03-21

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

Hybrid lymph node imaging using 64Cu-labeled mannose-conjugated human serum albumin with and without indocyanine green.

PubMed

Kang, Choong Mo; An, Gwang Il; Choe, Yearn Seong

2015-10-01

Human serum albumin (HSA), which has 58 Lys residues, one Cys residue, and indocyanine green (ICG) adsorption sites, can be used as a multifunctional platform for the development of hybrid imaging probes. In this study, we prepared 64Cu-labeled mannose-conjugated HSA with and without ICG ([64Cu]1-ICG and [64Cu]1, respectively) and compared hybrid PET/near-infrared fluorescence (NIRF) imaging with positron emission tomography (PET)/Cerenkov luminescence (CL) imaging of lymph nodes (LNs). 1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)/mannose-conjugated HSA (1) was synthesized by conjugating mannose molecules to Lys residues and a DOTA molecule to a Cys residue of HSA. Compound 1 was then labeled with Cu ([64Cu]1), and the resulting [64Cu]1 was adsorbed with ICG ([64Cu]1-ICG). PET/NIRF or PET/CL imaging and subsequent biodistribution studies were performed in ICR mice after injection of the probes into the foot pads. The numbers of mannose and DOTA molecules conjugated to HSA were 7.17 ± 0.49 and 0.95 ± 0.18, respectively. The site-specific conjugation of one DOTA molecule to HSA was sufficient for 64Cu-labeling with high efficiency (96.0 ± 1.1%). PET/NIRF and PET/CL imaging and subsequent biodistribution studies demonstrated that the probes were avidly taken up by the popliteal LNs (PO), with a slightly higher uptake ratio of the PO to the lumbar LNs by [64Cu]1. In-vivo studies suggest that [64Cu]1 has more specific and selective binding to mannose receptors in the PO than [64Cu]1-ICG.

Methods for immobilizing nucleic acids on a gel substrate

DOEpatents

Mirzabekov, Andrei Darievich; Proudnikov, Dimitri Y.; Timofeev, Edward N.; Kochetkova, Svetlana V.; Florentiev, Vladimir L.; Shick, Valentine V.

1999-01-01

A method for labeling oligonucleotide molecules, and for immobilizing oligonucleotide and DNA molecules is provided comprising modifying the molecules to create a chemically active group, and contacting activated fluorescent dyes to the region. A method for preparing an immobilization substrate is also provided comprising modifying a gel to contain desired functional groups which covalently interact with certain moieties of the oligonucleotide molecules. A method for immobilizing biomolecules and other molecules within a gel by copolymerization of allyl-substituted oligonucleotides, DNA and proteins with acrylamide is also provided.

Antibody-enabled small-molecule drug discovery.

PubMed

Lawson, Alastair D G

2012-06-29

Although antibody-based therapeutics have become firmly established as medicines for serious diseases, the value of antibodies as tools in the early stages of small-molecule drug discovery is only beginning to be realized. In particular, antibodies may provide information to reduce risk in small-molecule drug discovery by enabling the validation of targets and by providing insights into the design of small-molecule screening assays. Moreover, antibodies can act as guides in the quest for small molecules that have the ability to modulate protein-protein interactions, which have traditionally only been considered to be tractable targets for biological drugs. The development of small molecules that have similar therapeutic effects to current biologics has the potential to benefit a broader range of patients at earlier stages of disease.

Computer simulation of gene detection without PCR by single molecule detection

NASA Astrophysics Data System (ADS)

Davis, Lloyd M.; Williams, John G.; Lamb, Don T.

1999-01-01

Pioneer Hi-Bred is developing a low-cost method for rapid screening of DNA, for use in research on elite crop seed genetics. Unamplified genomic DNA with the requisite base sequence is simultaneously labeled by two different colored fluorescent probes, which hybridize near the selected gene. Dual-channel single molecule detection (SMD) within a flow cell, then provides a sensitive and specific assay for the gene. The technique has been demonstrated using frequency- doubled Nd:YAG laser excitation of two visible-wavelength dyes. A prototype instrument employing infrared fluorophores and laser diodes for excitation has been developed. Here, we report results from a Monte Carlo simulation of the new instrument, in which experimentally determined photophysical parameters for candidate infrared dyes are used for parametric studies of experimental operating conditions. Fluorophore photostability is found to be a key factor in determining the instrument sensitivity. Most infrared dyes have poor photostability, resulting in inefficient SMD. However, the normalized cross-correlation function of the photon signals from each of the two channels can still yield a discernable peak, provided that the concentration of dual- labeled molecules is sufficiently high. Further, for low concentrations, processing of the two photon streams with Gaussian -weighted sliding sum digital filters and selection of simultaneously occurring peaks can also provide a sensitive indicator of the presence of dual-labeled molecules, although accidental coincidences must be considered in the interpretation of results.

Electrochemiluminescent DNA sensor based on controlled Zn-mediated grafting of diazonium precursors.

PubMed

Torréns, Mabel; Ortiz, Mayreli; Bejarano-Nosas, Diego; O'Sullivan, Ciara K

2015-07-01

Controlled Zn-mediated grafting of a thin layer of a diazonium salt was used to functionalise a carbon electrode with ruthenium(II)-tris-bipyridine (Ru)-labelled DNA for use as a capture probe in an electrochemiluminescent genosensor. A secondary reporter probe was labelled with a ferrocene (Fc) molecule, and in the presence of the single-stranded DNA target a genocomplex formed, where the Fc-label effectively quenched the electrochemiluminescence of the signal emitted from the Ru-label. The spacing of the labels for maximum sensitivity and minimum detection limit was optimised, and the signal reproducibility and stability of the method was established.

Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching

NASA Astrophysics Data System (ADS)

Starke, Ilka; Johnson, Kathryn M.; Petersen, Jan; Gräber, Peter; Opipari, Anthony W.; Glick, Gary D.; Börsch, Michael

2016-03-01

Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme FoF1-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in FoF1-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C terminus of subunit γ. Imaging the threedimensional arrangement of mitochondria in living yeast cells was possible at superresolution using structured illumination microscopy, SIM. We measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy. Our data confirmed the binding of Cy5-labeled Bz-423 to the top of the F1 domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.

Individual breathing reactions measured in hemoglobin by hydrogen exchange methods.

PubMed Central

Englander, S W; Calhoun, D B; Englander, J J; Kallenbach, N R; Liem, R K; Malin, E L; Mandal, C; Rogero, J R

1980-01-01

Protein hydrogen exchange is generally believed to register some aspects of internal protein dynamics, but the kind of motion at work is not clear. Experiments are being done to identify the determinants of protein hydrogen exchange and to distinguish between local unfolding and accessibility-penetration mechanisms. Results with small molecules, polynucleotides, and proteins demonstrate that solvent accessibility is by no means sufficient for fast exchange. H-exchange slowing is quite generally connected with intramolecular H-bonding, and the exchange process depends pivotally on transient H-bond cleavage. At least in alpha-helical structures, the cooperative aspect of H-bond cleavage must be expressed in local unfolding reactions. Results obtained by use of a difference hydrogen exchange method appear to provide a direct measurement of transient, cooperative, local unfolding reactions in hemoglobin. The reality of these supposed coherent breathing units is being tested by using the difference H-exchange approach to tritium label the units one at a time and then attempting to locate the tritium by fragmenting the protein, separating the fragments, and testing them for label. Early results demonstrate the feasibility of this approach. PMID:7248462

Specifically and wash-free labeling of SNAP-tag fused proteins with a hybrid sensor to monitor local micro-viscosity.

PubMed

Wang, Chao; Song, Xinbo; Chen, Lingcheng; Xiao, Yi

2017-05-15

Viscosity, as one of the major factors of intracellular microenvironment, influences the function of proteins. To detect local micro-viscosity of a protein, it is a precondition to apply a viscosity sensor for specifically target to proteins. However, all the reported small-molecule probes are just suitable for sensing/imaging of macro-viscosity in biological fluids of entire cells or organelles. To this end, we developed a hybrid sensor BDP-V BG by connecting a viscosity-sensitive boron-dipyrromethene (BODIPY) molecular rotor (BDP-V) to O 6 -benzylguanine (BG) for specific detection of local micro-viscosity of SNAP-tag fused proteins. We measured and calculated the reaction efficiency between the sensor and SNAP-tag protein in vitro to confirm the high labeling specificity. We also found that the labeling reaction results in a 53-fold fluorescence enhancement for the rotor, which qualifies it as a wash-free sensor with ignorable background fluorescence. The high sensitivity of protein labeled sensor (BDP-V-SNAP) to the changes of local viscosity was evaluated by detecting the enhancement of fluorescence lifetimes. Further, with the sensor BDP-V BG, we achieved high specific labeling of cells expressing two SNAP-tag fused proteins (nuclear histone H2B and mitochondrial COX8A). Two-photon excited fluorescence lifetime imaging revealed that, the micro-viscosities nearby the SNAP-tag fused two proteins are distinct. The different changes of local micro-viscosity of SNAP-tag fused histone protein in apoptosis induced by three nucleus-targeted drugs were also characterized for the first time. Copyright © 2016 Elsevier B.V. All rights reserved.

Diazo Reagents with Small Steric Footprints for Simultaneous Arming/SAR Studies of Alcohol-Containing Natural Products via O–H Insertion

PubMed Central

Chamni, Supakarn; He, Qing-Li; Dang, Yongjun; Bhat, Shridhar; Liu, Jun O.; Romo, Daniel

2011-01-01

Natural products are essential tools for basic cellular studies leading to the identification of medically relevant protein targets and the discovery of potential therapeutic leads. The development of methods that enable mild and selective derivatization of natural products continues to be of significant interest for mining their information-rich content. Herein, we describe novel diazo reagents for simultaneous arming and structure-activity relationship (SAR) studies of alcohol-containing natural products with a small steric footprint, namely an α-trifluoroethyl (HTFB) substituted reagent. The Rh(II)-catalyzed O–H insertion reaction of several natural products, including the potent translation inhibitor lactimidomycin, was investigated and useful reactivity and both chemo- and site (chemosite) selectivities were observed. Differential binding to the known protein targets of both FK506 and fumagillol was demonstrated, validating the advantage of the smaller steric footprint of trifluoroethyl derivatives. A p-azidophenyl diazo reagent is also described that will prove useful for photoaffinity labeling of low affinity small molecule protein receptors. PMID:21894934

Small Molecule Chemical Probes of MicroRNA Function

PubMed Central

Velagapudi, Sai Pradeep; Vummidi, Balayeshwanth R.; Disney, Matthew D.

2015-01-01

MicroRNAs (miRNAs) are small, non-coding RNAs that control protein expression. Aberrant miRNA expression has been linked to various human diseases, and thus miRNAs have been explored as diagnostic markers and therapeutic targets. Although it is challenging to target RNA with small molecules in general, there have been successful campaigns that have identified small molecule modulators of miRNA function by targeting various pathways. For example, small molecules that modulate transcription and target nuclease processing sites in miRNA precursors have been identified. Herein, we describe challenges in developing chemical probes that target miRNAs and highlight aspects of miRNA cellular biology elucidated by using small molecule chemical probes. We expect that this area will expand dramatically in the near future as strides are made to understand small molecule recognition of RNA from a fundamental perspective. PMID:25500006

Labeled nucleotide phosphate (NP) probes

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2009-02-03

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Medium-Bandgap Small-Molecule Donors Compatible with Both Fullerene and Nonfullerene Acceptors.

PubMed

Huo, Yong; Yan, Cenqi; Kan, Bin; Liu, Xiao-Fei; Chen, Li-Chuan; Hu, Chen-Xia; Lau, Tsz-Ki; Lu, Xinhui; Sun, Chun-Lin; Shao, Xiangfeng; Chen, Yongsheng; Zhan, Xiaowei; Zhang, Hao-Li

2018-03-21

Much effort has been devoted to the development of new donor materials for small-molecule organic solar cells due to their inherent advantages of well-defined molecular weight, easy purification, and good reproducibility in photovoltaic performance. Herein, we report two small-molecule donors that are compatible with both fullerene and nonfullerene acceptors. Both molecules consist of an (E)-1,2-di(thiophen-2-yl)ethane-substituted (TVT-substituted) benzo[1,2-b:4,5-b']dithiophene (BDT) as the central unit, and two rhodanine units as the terminal electron-withdrawing groups. The central units are modified with either alkyl side chains (DRBDT-TVT) or alkylthio side chains (DRBDT-STVT). Both molecules exhibit a medium bandgap with complementary absorption and proper energy level offset with typical acceptors like PC 71 BM and IDIC. The optimized devices show a decent power conversion efficiency (PCE) of 6.87% for small-molecule organic solar cells and 6.63% for nonfullerene all small-molecule organic solar cells. Our results reveal that rationally designed medium-bandgap small-molecule donors can be applied in high-performance small-molecule organic solar cells with different types of acceptors.

Lucky Imaging: Improved Localization Accuracy for Single Molecule Imaging

PubMed Central

Cronin, Bríd; de Wet, Ben; Wallace, Mark I.

2009-01-01

We apply the astronomical data-analysis technique, Lucky imaging, to improve resolution in single molecule fluorescence microscopy. We show that by selectively discarding data points from individual single-molecule trajectories, imaging resolution can be improved by a factor of 1.6 for individual fluorophores and up to 5.6 for more complex images. The method is illustrated using images of fluorescent dye molecules and quantum dots, and the in vivo imaging of fluorescently labeled linker for activation of T cells. PMID:19348772

Inforna 2.0: A Platform for the Sequence-Based Design of Small Molecules Targeting Structured RNAs.

PubMed

Disney, Matthew D; Winkelsas, Audrey M; Velagapudi, Sai Pradeep; Southern, Mark; Fallahi, Mohammad; Childs-Disney, Jessica L

2016-06-17

The development of small molecules that target RNA is challenging yet, if successful, could advance the development of chemical probes to study RNA function or precision therapeutics to treat RNA-mediated disease. Previously, we described Inforna, an approach that can mine motifs (secondary structures) within target RNAs, which is deduced from the RNA sequence, and compare them to a database of known RNA motif-small molecule binding partners. Output generated by Inforna includes the motif found in both the database and the desired RNA target, lead small molecules for that target, and other related meta-data. Lead small molecules can then be tested for binding and affecting cellular (dys)function. Herein, we describe Inforna 2.0, which incorporates all known RNA motif-small molecule binding partners reported in the scientific literature, a chemical similarity searching feature, and an improved user interface and is freely available via an online web server. By incorporation of interactions identified by other laboratories, the database has been doubled, containing 1936 RNA motif-small molecule interactions, including 244 unique small molecules and 1331 motifs. Interestingly, chemotype analysis of the compounds that bind RNA in the database reveals features in small molecule chemotypes that are privileged for binding. Further, this updated database expanded the number of cellular RNAs to which lead compounds can be identified.

SERS microscopy: plasmonic nanoparticle probes and biomedical applications

NASA Astrophysics Data System (ADS)

Gellner, M.; Schütz, M.; Salehi, M.; Packeisen, J.; Ströbel, P.; Marx, A.; Schmuck, C.; Schlücker, S.

2010-08-01

Nanoparticle probes for use in targeted detection schemes and readout by surface-enhanced Raman scattering (SERS) comprise a metal core, Raman reporter molecules and a protective shell. One design of SERS labels specifically optimized for biomedical applications in conjunction with red laser excitation is based on tunable gold/silver nanoshells, which are completely covered by a self-assembled monolayer (SAM) of Raman reporters. A shell around the SAM-coated metal core stabilizes the colloid and prevents particle aggregation. The optical properties and SERS efficiencies of these plasmonic nanostructures are characterized both experimentally and theoretically. Subsequent bioconjugation of SERS probes to ligands such as antibodies is a prerequisite for the selective detection of the corresponding target molecule via the characteristic Raman signature of the label. Biomedical imaging applications of SERS-labeled antibodies for tumor diagnostics by SERS microscopy are presented, using the localization of the tumor suppressor p63 in prostate tissue sections as an example.

pyQms enables universal and accurate quantification of mass spectrometry data.

PubMed

Leufken, Johannes; Niehues, Anna; Sarin, L Peter; Wessel, Florian; Hippler, Michael; Leidel, Sebastian A; Fufezan, Christian

2017-10-01

Quantitative mass spectrometry (MS) is a key technique in many research areas (1), including proteomics, metabolomics, glycomics, and lipidomics. Because all of the corresponding molecules can be described by chemical formulas, universal quantification tools are highly desirable. Here, we present pyQms, an open-source software for accurate quantification of all types of molecules measurable by MS. pyQms uses isotope pattern matching that offers an accurate quality assessment of all quantifications and the ability to directly incorporate mass spectrometer accuracy. pyQms is, due to its universal design, applicable to every research field, labeling strategy, and acquisition technique. This opens ultimate flexibility for researchers to design experiments employing innovative and hitherto unexplored labeling strategies. Importantly, pyQms performs very well to accurately quantify partially labeled proteomes in large scale and high throughput, the most challenging task for a quantification algorithm. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis

PubMed Central

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-01-01

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots. PMID:28714873

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis.

PubMed

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-07-15

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots.

Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications

PubMed Central

Scheler, Ott; Glynn, Barry; Parkel, Sven; Palta, Priit; Toome, Kadri; Kaplinski, Lauris; Remm, Maido; Maher, Majella; Kurg, Ants

2009-01-01

Background Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. Results Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. Conclusion We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology. PMID:19445684

A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance

PubMed Central

Watterson, Daniel; Robinson, Jodie; Chappell, Keith J.; Butler, Mark S.; Edwards, David J.; Fry, Scott R.; Bermingham, Imogen M.; Cooper, Matthew A.; Young, Paul R.

2016-01-01

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus. PMID:26976324

Portable capillary electrophoresis-system for on-site food analysis with lab-on-a-chip based contactless conductivity detection

NASA Astrophysics Data System (ADS)

Gärtner, Claudia; Sewart, René; Klemm, Richard; Becker, Holger

2014-06-01

A portable analytical system for the characterization of liquid environmental samples and beverages in food control was realized. The key element is the implementation of contactless conductivity detection on lab-on-a-chip basis ensuring the system to be operated in a label free mode. Typical target molecules such as small ionic species like Li+, Na+, K+, SO4 2- or NO3-, organic acids in wine whose concentration and ratio to each other documents the wine quality, or caffeine or phosphate in coke were detected. Results from sample matrices like various beverages as water, cola, tea, wine and milk, water from heaters, environmental samples and blood will be presented.

Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

PubMed Central

Kurata, Shinya; Kanagawa, Takahiro; Yamada, Kazutaka; Torimura, Masaki; Yokomaku, Toyokazu; Kamagata, Yoichi; Kurane, Ryuichiro

2001-01-01

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples. PMID:11239011

The C(2)1pi(u) state of Na2 molecule studied by polarization labelling spectroscopy method.

PubMed

Jastrzebski, W; Kowalczyk, P; Camacho, J J; Pardo, A; Poyato, J M

2001-08-01

The C1pi(u) <-- X1sigma(g)+ system of Na2 is studied by the polarization labelling spectroscopy technique. Accurate molecular constants are derived for the observed levels nu = 0-12, J = 12-100 in the C1pi(u) state.

A Novel Method for Assigning R, S Labels to Enantiomers.

ERIC Educational Resources Information Center

Huheey, James E.

1986-01-01

Discusses ways of teaching students about how to assign R (rectus) and S (sinister) labels to enantiomers by using their hands as models. The chirality of the human hands follows the Cahn-Ingold-Prelog Rules for assigning enantiomers and infers the correct chirality of molecules shown in two-dimensional drawings. (TW)

Quantum dots as strain- and metabolism-specific microbiological labels

NASA Technical Reports Server (NTRS)

Kloepfer, J. A.; Mielke, R. E.; Wong, M. S.; Nealson, K. H.; Stucky, G.; Nadeau, J. L.

2003-01-01

Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate.

No-carrier-added labeling of the neuroprotective Ebselen with selenium-73 and selenium-75.

PubMed

Helfer, Andreas; Ermert, Johannes; Humpert, Sven; Coenen, Heinz H

2015-03-01

Selenium-73 is a positron emitting non-standard radionuclide, which is suitable for positron emission tomography. A copper-catalyzed reaction allowed no-carrier-added labeling of the anti-inflammatory seleno-organic compound Ebselen with (73) Se and (75) Se under addition of sulfur carrier in a one-step reaction. The new authentically labeled radioselenium molecule is thus available for preclinical evaluation and positron emission tomography studies. Copyright © 2015 John Wiley & Sons, Ltd.

Determination of dye/protein ratios in a labeling reaction between a cyanine dye and bovine serum albumin by micellar electrokinetic chromatography using a diode laser-induced fluorescence detection.

PubMed

Jing, Peng; Kaneta, Takashi; Imasaka, Totaro

2002-08-01

The degree of labeling, i.e., dye/protein ratio (D/P) is important for characterizing properties of dye labeling with proteins. A method for the determination of this ratio between a fluorescent cyanine dye and bovine serum albumin (BSA), based on the separation of the labeling mixture using micellar electrokinetic chromatography with diode laser-induced fluorescence detection, is described. Two methods for the determination of D/P were examined in this study. In these methods, a hydrolysis product and impurities, which are usually unfavorable compounds that are best excluded for protein analysis, were utilized to determine the amounts of dye bound to BSA. One is a direct method in which a ratio of the peak area of BSA to the total peak area of all the products produced in the labeling reaction was used for determining the average number of dye molecules bound to a single BSA molecule. The other is an indirect determination, which is based on diminution of all peak areas related to the products except for the labeled BSA. These methods were directly compared by means of a spectrophotometric method. The experimental results show that the indirect method is both reliable and sensitive. Therefore, D/P values can be determined at trace levels using the indirect method.

Small molecule chemical probes of microRNA function.

PubMed

Velagapudi, Sai Pradeep; Vummidi, Balayeshwanth R; Disney, Matthew D

2015-02-01

MicroRNAs (miRNAs) are small, non-coding RNAs that control protein expression. Aberrant miRNA expression has been linked to various human diseases, and thus miRNAs have been explored as diagnostic markers and therapeutic targets. Although it is challenging to target RNA with small molecules in general, there have been successful campaigns that have identified small molecule modulators of miRNA function by targeting various pathways. For example, small molecules that modulate transcription and target nuclease processing sites in miRNA precursors have been identified. Herein, we describe challenges in developing chemical probes that target miRNAs and highlight aspects of miRNA cellular biology elucidated by using small molecule chemical probes. We expect that this area will expand dramatically in the near future as progress is made in understanding small molecule recognition of RNA. Copyright © 2014. Published by Elsevier Ltd.

Probing adenine rings and backbone linkages using base specific isotope-edited Raman spectroscopy: application to group II intron ribozyme domain V.

PubMed

Chen, Yuanyuan; Eldho, Nadukkudy V; Dayie, T Kwaku; Carey, Paul R

2010-04-27

Raman difference spectroscopy is used to probe the properties of a 36-nt RNA molecule, "D5", which lies at the heart of the catalytic apparatus in group II introns. For D5 that has all of its adenine residues labeled with (13)C and (15)N and utilizing Raman difference spectroscopy, we identify the conformationally sensitive -C-O-P-O-C- stretching modes of the unlabeled bonds adjacent to adenine bases, as well as the adenine ring modes themselves. The phosphodiester modes can be assigned to individual adenine residues based on earlier NMR data. The effect of Mg(2+) binding was explored by analyzing the Raman difference spectra for [D5 + Mg(2+)] minus [D5 no Mg(2+)], for D5 unlabeled, or D5 labeled with (13)C/(15)N-enriched adenine. In both sets of data we assign differential features to G ring modes perturbed by Mg(2+) binding at the N7 position. In the A-labeled spectra we attribute a Raman differential near 1450 cm(-1) and changes of intensity at 1296 cm(-1) to Mg binding at the N7 position of adenine bases. The A and G bases involved in Mg(2+) binding again can be identified using earlier NMR results. For the unlabeled D5, a change in the C-O-P-O-C stretch profile at 811 cm(-1) upon magnesium binding is due to a "tightening up" (in the sense of a more rigid molecule with less dynamic interchange among competing ribose conformers) of the D5 structure. For adenine-labeled D5, small changes in the adenine backbone bond signatures in the 810-830 cm(-1) region suggest that small conformational changes occur in the tetraloop and bulge regions upon binding of Mg(2+). The PO(2)(-) stretching vibration, near 1100 cm(-1), from the nonbridging phosphate groups, probes the effect of Mg(2+)-hydrate inner-sphere interactions that cause an upshift. In turn, the upshift is modulated by the presence of monovalent cations since in the presence of Na(+) and Li(+) the upshift is 23 +/- 2 cm(-1) while in the presence of K(+) and Cs(+) it is 13 +/- 3 cm(-1), a finding that correlates with the differences in hydration radii. These subtle differences in electrostatic interactions may be related to observed variations in catalytic activity. For a reconstructed ribozyme comprising domains 1-3 (D123) connected in cis plus domain 5 (D5) supplied in trans, cleavage of spliced exon substrates in the presence of magnesium and K(+) or Cs(+) is more efficient than that in the presence of magnesium with Na(+) or Li(+).

Development of a Unique Small Molecule Modulator of CXCR4

PubMed Central

Yoon, Younghyoun; Lin, Songbai; Sasaki, Maiko; Klapproth, Jan-Michael A.; Yang, Hua; Grossniklaus, Hans E.; Xu, Jianguo; Rojas, Mauricio; Voll, Ronald J.; Goodman, Mark M.; Arrendale, Richard F.; Liu, Jin; Yun, C. Chris; Snyder, James P.; Liotta, Dennis C.; Shim, Hyunsuk

2012-01-01

Background Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites. Methodology/Principal Findings We describe the actions of N,N′-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using 18F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles. Conclusions/Significance We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4 antagonists. PMID:22485156

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

PubMed Central

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system. PMID:26222440

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

PubMed

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR

PubMed Central

Macpherson, Alex; Smith-Penzel, Susanne; Basse, Nicolas; Lecomte, Fabien; Deboves, Hervé; Taylor, Richard D.; Norman, Tim; Porter, John; Waters, Lorna C.; Westwood, Marta; Cossins, Ben; Cain, Katharine; White, James; Griffin, Robert; Prosser, Christine; Kelm, Sebastian; Sullivan, Amy H.; Fox, David; Carr, Mark D.; Henry, Alistair; Taylor, Richard; Meier, Beat H.; Oschkinat, Hartmut; Lawson, Alastair D.

2018-01-01

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)–Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD–IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns. PMID:29782488

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR.

PubMed

Stöppler, Daniel; Macpherson, Alex; Smith-Penzel, Susanne; Basse, Nicolas; Lecomte, Fabien; Deboves, Hervé; Taylor, Richard D; Norman, Tim; Porter, John; Waters, Lorna C; Westwood, Marta; Cossins, Ben; Cain, Katharine; White, James; Griffin, Robert; Prosser, Christine; Kelm, Sebastian; Sullivan, Amy H; Fox, David; Carr, Mark D; Henry, Alistair; Taylor, Richard; Meier, Beat H; Oschkinat, Hartmut; Lawson, Alastair D

2018-05-01

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)-Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD-IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns.

Synthesis of carbon-11, fluorine-18, and nitrogen-13 labeled radiotracers for biomedical applications. Nuclear Science Series: Nuclear Medicine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowler, J.S.; Wolf, A.P.

1982-09-01

Carbon 11, Fluorine 18, and Nitrogen 13-labeled radiotracers are reviewed from the standpoint of synthetic organic chemistry while keeping in perspective the necessity of integrating the organic chemistry with the design and ultimate application of the radiotracer. The reactions used, the principles used to adapt these reactions to labeling with short-lived radionuclides, and the concepts of chemical reactivity form the framework upon which synthetic strategies for short-lived radiotracers are developed. Potentially new routes are suggested which may be applied to problems in labeling organic molecules. (ACR)

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

In vivo targeting of HER2-positive tumor using 2-helix affibody molecules.

PubMed

Ren, Gang; Webster, Jack M; Liu, Zhe; Zhang, Rong; Miao, Zheng; Liu, Hongguang; Gambhir, Sanjiv S; Syud, Faisal A; Cheng, Zhen

2012-07-01

Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, 68Ga and 18F, with relatively short half-life (t1/2<2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with 64Cu or 111In and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA-MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I2 oxidation. DOTA-MUT-DS was then radiolabeled with 64Cu or 111In to prepare the HER2 imaging probe (64Cu/111In-DOTA-MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA-MUT-DS could be successfully synthesized and radiolabeled with 64Cu or 111In. Biodistribution study showed that tumor uptake value of 64Cu or 111In-labeled DOTA-MUT-DS was 4.66±0.38 or 2.17±0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n=3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for 64Cu-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for 111In-DOTA-MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule ZHER2:342 with 64Cu-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. 111In-DOTA-MUT-DS displayed lower liver uptake at all the time points investigated and higher tumor to blood ratios at 4 and 20 h p.i., when compared with 64Cu-DOTA-MUT-DS. This study demonstrates that the 2-helix protein based probes, 64Cu/111In DOTA-MUT-DS, are promising molecular probes for imaging HER2-positive tumor. Two-helix small protein scaffold holds great promise as a novel and robust platform for imaging and therapy applications.

Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

PubMed Central

McQuilken, Molly; La Riviere, Patrick J.; Occhipinti, Patricia; Verma, Amitabh; Oldenbourg, Rudolf; Gladfelter, Amy S.; Tani, Tomomi

2016-01-01

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells. PMID:27679846

Defining RNA-Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA.

PubMed

Velagapudi, Sai Pradeep; Luo, Yiling; Tran, Tuan; Haniff, Hafeez S; Nakai, Yoshio; Fallahi, Mohammad; Martinez, Gustavo J; Childs-Disney, Jessica L; Disney, Matthew D

2017-03-22

RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif-small molecule interactions identified via selection. Named High Throughput Structure-Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif-small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule-RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs.

Metabolic response of Candida albicans to phenylethyl alcohol under hyphae-inducing conditions.

PubMed

Han, Ting-Li; Tumanov, Sergey; Cannon, Richard D; Villas-Boas, Silas G

2013-01-01

Phenylethyl alcohol was one of the first quorum sensing molecules (QSMs) identified in C. albicans. This extracellular signalling molecule inhibits the hyphal formation of C. albicans at high cell density. Little is known, however, about the underlying mechanisms by which this QSM regulates the morphological switches of C. albicans. Therefore, we have applied metabolomics and isotope labelling experiments to investigate the metabolic changes that occur in C. albicans in response to phenylethyl alcohol under defined hyphae-inducing conditions. Our results showed a global upregulation of central carbon metabolism when hyphal development was suppressed by phenylethyl alcohol. By comparing the metabolic changes in response to phenylethyl alcohol to our previous metabolomic studies, we were able to short-list 7 metabolic pathways from central carbon metabolism that appear to be associated with C. albicans morphogenesis. Furthermore, isotope-labelling data showed that phenylethyl alcohol is indeed taken up and catabolised by yeast cells. Isotope-labelled carbon atoms were found in the majority of amino acids as well as in lactate and glyoxylate. However, isotope-labelled carbon atoms from phenylethyl alcohol accumulated mainly in the pyridine ring of NAD(+)/NADH and NADP(-/)NADPH molecules, showing that these nucleotides were the main products of phenylethyl alcohol catabolism. Interestingly, two metabolic pathways where these nucleotides play an important role, nitrogen metabolism and nicotinate/nicotinamide metabolism, were also short-listed through our previous metabolomics works as metabolic pathways likely to be closely associated with C. albicans morphogenesis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kardash, Maria E.; Dzuba, Sergei A., E-mail: dzuba@kinetics.nsc.ru

Lipid-cholesterol interactions are responsible for different properties of biological membranes including those determining formation in the membrane of spatial inhomogeneities (lipid rafts). To get new information on these interactions, electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR), was applied to study 3β-doxyl-5α-cholestane (DCh), a spin-labeled analog of cholesterol, in phospholipid bilayer consisted of equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. DCh concentration in the bilayer was between 0.1 mol.% and 4 mol.%. For comparison, a reference system containing a spin-labeled 5-doxyl-stearic acid (5-DSA) instead of DCh was studied as well. The effects of “instantaneousmore » diffusion” in ESE decay and in echo-detected (ED) EPR spectra were explored for both systems. The reference system showed good agreement with the theoretical prediction for the model of spin labels of randomly distributed orientations, but the DCh system demonstrated remarkably smaller effects. The results were explained by assuming that neighboring DCh molecules are oriented in a correlative way. However, this correlation does not imply the formation of clusters of cholesterol molecules, because conventional continuous wave EPR spectra did not show the typical broadening due to aggregation of spin labels and the observed ESE decay was not faster than in the reference system. So the obtained data evidence that cholesterol molecules at low concentrations in biological membranes can interact via large distances of several nanometers which results in their orientational self-ordering.« less

Sequence-specific inhibition of Dicer measured with a force-based microarray for RNA ligands.

PubMed

Limmer, Katja; Aschenbrenner, Daniela; Gaub, Hermann E

2013-04-01

Malfunction of protein translation causes many severe diseases, and suitable correction strategies may become the basis of effective therapies. One major regulatory element of protein translation is the nuclease Dicer that cuts double-stranded RNA independently of the sequence into pieces of 19-22 base pairs starting the RNA interference pathway and activating miRNAs. Inhibiting Dicer is not desirable owing to its multifunctional influence on the cell's gene regulation. Blocking specific RNA sequences by small-molecule binding, however, is a promising approach to affect the cell's condition in a controlled manner. A label-free assay for the screening of site-specific interference of small molecules with Dicer activity is thus needed. We used the Molecular Force Assay (MFA), recently developed in our lab, to measure the activity of Dicer. As a model system, we used an RNA sequence that forms an aptamer-binding site for paromomycin, a 615-dalton aminoglycoside. We show that Dicer activity is modulated as a function of concentration and incubation time: the addition of paromomycin leads to a decrease of Dicer activity according to the amount of ligand. The measured dissociation constant of paromomycin to its aptamer was found to agree well with literature values. The parallel format of the MFA allows a large-scale search and analysis for ligands for any RNA sequence.

Targeting prostate cancer: Prostate-specific membrane antigen based diagnosis and therapy.

PubMed

Wüstemann, Till; Haberkorn, Uwe; Babich, John; Mier, Walter

2018-05-17

The high incidence rates of prostate cancer (PCa) raise demand for improved therapeutic strategies. Prostate tumors specifically express the prostate-specific membrane antigen (PSMA), a membrane-bound protease. As PSMA is highly overexpressed on malignant prostate tumor cells and as its expression rate correlates with the aggressiveness of the disease, this tumor-associated biomarker provides the possibility to develop new strategies for diagnostics and therapy of PCa. Major advances have been made in PSMA targeting, ranging from immunotherapeutic approaches to therapeutic small molecules. This review elaborates the diversity of PSMA targeting agents while focusing on the radioactively labeled tracers for diagnosis and endoradiotherapy. A variety of radionuclides have been shown to either enable precise diagnosis or efficiently treat the tumor with minimal effects to nontargeted organs. Most small molecules with affinity for PSMA are based on either a phosphonate or a urea-based binding motif. Based on these pharmacophores, major effort has been made to identify modifications to achieve ideal pharmacokinetics while retaining the specific targeting of the PSMA binding pocket. Several tracers have now shown excellent clinical usability in particular for molecular imaging and therapy as proven by the efficiency of theranostic approaches in current studies. The archetypal expression profile of PSMA may be exploited for the treatment with alpha emitters to break radioresistance and thus to bring the power of systemic therapy to higher levels. © 2018 Wiley Periodicals, Inc.

Molecular Architecture of Contactin-associated Protein-like 2 (CNTNAP2) and Its Interaction with Contactin 2 (CNTN2)*

PubMed Central

Lu, Zhuoyang; Reddy, M. V. V. V. Sekhar; Liu, Jianfang; Kalichava, Ana; Liu, Jiankang; Zhang, Lei; Chen, Fang; Wang, Yun; Holthauzen, Luis Marcelo F.; White, Mark A.; Seshadrinathan, Suchithra; Zhong, Xiaoying; Ren, Gang; Rudenko, Gabby

2016-01-01

Contactin-associated protein-like 2 (CNTNAP2) is a large multidomain neuronal adhesion molecule implicated in a number of neurological disorders, including epilepsy, schizophrenia, autism spectrum disorder, intellectual disability, and language delay. We reveal here by electron microscopy that the architecture of CNTNAP2 is composed of a large, medium, and small lobe that flex with respect to each other. Using epitope labeling and fragments, we assign the F58C, L1, and L2 domains to the large lobe, the FBG and L3 domains to the middle lobe, and the L4 domain to the small lobe of the CNTNAP2 molecular envelope. Our data reveal that CNTNAP2 has a very different architecture compared with neurexin 1α, a fellow member of the neurexin superfamily and a prototype, suggesting that CNTNAP2 uses a different strategy to integrate into the synaptic protein network. We show that the ectodomains of CNTNAP2 and contactin 2 (CNTN2) bind directly and specifically, with low nanomolar affinity. We show further that mutations in CNTNAP2 implicated in autism spectrum disorder are not segregated but are distributed over the whole ectodomain. The molecular shape and dimensions of CNTNAP2 place constraints on how CNTNAP2 integrates in the cleft of axo-glial and neuronal contact sites and how it functions as an organizing and adhesive molecule. PMID:27621318

Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

PubMed

Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

2014-08-15

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

Label-Free Direct Electronic Detection of Biomolecules with Amorphous Silicon Nanostructures

PubMed Central

Lund, John; Mehta, Ranjana; Parviz, Babak A.

2007-01-01

We present the fabrication and characterization of a nano-scale sensor made of amorphous silicon for the label-free, electronic detection of three classes of biologically important molecules: ions, oligonucleotides, and proteins. The sensor structure has an active element which is a 50 nm wide amorphous silicon semicircle and has a total footprint of less than 4 μm2. We demonstrate the functionalization of the sensor with receptor molecules and the electronic detection of three targets: H+ ions, short single-stranded DNAs, and streptavidin. The sensor is able to reliably distinguish single base-pair mismatches in 12 base long strands of DNA and monitor the introduction and identification of straptavidin in real-time. The versatile sensor structure can be readily functionalized with a wide range of receptor molecules and is suitable for integration with high-speed electronic circuits as a post-process on an integrated circuit chip. PMID:17292148

Current-voltage characteristics of double stranded versus single stranded DNA molecules

NASA Astrophysics Data System (ADS)

Hartzell, B.; Chen, Hong; Heremans, J. J.; McCord, B.; Soghomonian, V.

2004-03-01

Investigation of DNA conductivity has focused on the native, duplex structure, with controversial results. Here, we present the influence of the double-helical structure on charge transport through lambda DNA molecules. The current-voltage (I-V) characteristics of both disulfide-labeled double stranded DNA (dsDNA) and disulfide-labeled single stranded DNA (ssDNA) were measured. The ssDNA was formed from the dsDNA using two different methods for comparison purposes: a thermal/chemical denaturation and enzymatic digestion utilizing lambda exonuclease. Resulting I-V characteristics of both the double stranded and single stranded samples were close-to-linear when measured at room temperature. However, the ssDNA samples consistently gave conductivity values about two orders of magnitude smaller in amplitude. Our results suggest an integral relationship between the native structure of DNA with its stacked base pairs and the molecule's ability to support charge transport.(NSF NIRT 0103034)

High-throughput, dual probe biological assays based on single molecule detection

DOEpatents

Hollars, Christopher W [Brentwood, CA; Huser, Thomas R [Livermore, CA; Lane, Stephen M [Oakland, CA; Balhorn, Rodney L [Livermore, CA; Bakajin, Olgica [San Leandro, CA; Darrow, Christopher [Pleasanton, CA; Satcher, Jr., Joe H.

2006-07-11

A method and apparatus with the sensitivity to detect and identify single target molecules through the localization of dual, fluorescently labeled probe molecules. This can be accomplished through specific attachment of the taget to a surface or in a two-dimensional (2D) flowing fluid sheet having approximate dimensions of 0.5 .mu.m.times.100 .mu.m.times.100 .mu.m. A device using these methods would have 10.sup.3 10.sup.4 greater throughput than previous one-dimensional (1D) micro-stream devices having 1 .mu.m.sup.3 interrogation volumes and would for the first time allow immuno- and DNA assays at ultra-low (femtomolar) concentrations to be performed in short time periods (.about.10 minutes). The use of novel labels (such as metal or semiconductor nanoparticles) may be incorporated to further extend the sensitivity possibly into the attomolar range.

Analysis of cholesterol trafficking with fluorescent probes

PubMed Central

Maxfield, Frederick R.; Wüstner, Daniel

2013-01-01

Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly. PMID:22325611

Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone.

PubMed Central

Nelson, P S; Kent, M; Muthini, S

1992-01-01

Novel CE-phosphoramidite (7a-e) and CPG (8a, c, d, e) reagents have been prepared from a unique 2-aminobutyl-1,3-propanediol backbone. The reagents have been used to directly label oligonucleotides with fluorescein, acridine, and biotin via automated DNA synthesis. The versatile 2-aminobutyl-1,3-propanediol backbone allows for labeling at any position (5', internal, and 3') during solid phase oligonucleotide synthesis. Multiple labels can be achieved by repetitive coupling cycles. Furthermore, the 3-carbon atom internucleotide phosphate distance is retained when inserted internally. Using this method, individual oligonucleotides possessing two and three different reporter molecules have been prepared. PMID:1475185

"Warning: This image has been digitally altered": The effect of disclaimer labels added to fashion magazine shoots on women's body dissatisfaction.

PubMed

Tiggemann, Marika; Brown, Zoe; Zaccardo, Mia; Thomas, Nicole

2017-06-01

The present experiment aimed to investigate the impact of the addition of disclaimer labels to fashion magazine shoots on women's body dissatisfaction. Participants were 320 female undergraduate students who viewed fashion shoots containing a thin and attractive model with no disclaimer label, or a small, large, or very large disclaimer label, or product images. Although thin-ideal fashion shoot images resulted in greater body dissatisfaction than product images, there was no significant effect of disclaimer label. Internalisation of the thin ideal was found to moderate the effect of disclaimer label, such that internalisation predicted increased body dissatisfaction in the no label and small label conditions, but not in the larger label conditions. Overall, the results showed no benefit for any size of disclaimer label in ameliorating the negative effect of viewing thin-ideal media images. It was concluded that more extensive research is required before the effective implementation of disclaimer labels. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vivo study of lipid synthesis and lipolysis dynamics by stimulated Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Li, Xuesong; Li, Yan; He, Sicong; Chen, Congping; Qin, Zhongya; Mak, Ho Yi; Qu, Jianan Y.

2018-02-01

To understand the mechanisms of important lipid-related biological processes and diseases, it is highly demanded to study the dynamics of lipids in living biological system with high spatiotemporal resolution. However, in vivo quantitative analysis of lipid synthesis and lipolysis has been technically difficult to achieve by conventional lipid extraction and fluorescent staining methods. Recently, SRS microscopy has emerged as a powerful tool to probe small molecules with alkyne (C≡C) or deuterium (C-D) labeling in cell-silent region. The Raman tags have been used for the quantitative study of lipids in cells. In this study, we investigated metabolic dynamics of lipid droplets (LDs) by tracing the alkyne-tagged fatty acid 17-ODYA and deuterium-labeled saturated and unsaturated fatty acids PA-D31 & OA-D34 in living C. elegans. Specifically, we developed a hyperspectral SRS microscope system for LDs characterization. The system can sequentially excite and probe the stimulated Raman scattering-induced CH2 stretching of endogenous lipids information (2863 cm-1), C≡C stretching from 17-ODYA (2125 cm-1) and C-D stretching from deuterium-labeled fatty acids (2117 cm-1). We first examined the concentration levels of fatty acids in E. coli OP50. Two major lipid metabolic processes, namely uptake and turnover, were further studied in adult C. elegans. We imaged alkyne-tagged and deuterated fatty acids using SRS and traced their uptake, transportation, incorporation and turnover over time. Additionally, several other treatments including starvation were also conducted to study their effects on metabolic dynamics of pulse labeled 17-ODYA, PA-D31 and OA-D34.

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Guitot, Karine; Scarabelli, Silvia; Drujon, Thierry; Bolbach, Gérard; Amoura, Mehdi; Burlina, Fabienne; Jeltsch, Albert; Sagan, Sandrine; Guianvarc'h, Dominique

2014-07-01

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors. Copyright © 2014 Elsevier Inc. All rights reserved.

Genomic Mapping of Human DNA provides Evidence of Difference in Stretch between AT and GC rich regions

NASA Astrophysics Data System (ADS)

Reifenberger, Jeffrey; Dorfman, Kevin; Cao, Han

Human DNA is a not a polymer consisting of a uniform distribution of all 4 nucleic acids, but rather contains regions of high AT and high GC content. When confined, these regions could have different stretch due to the extra hydrogen bond present in the GC basepair. To measure this potential difference, human genomic DNA was nicked with NtBspQI, labeled with a cy3 like fluorophore at the nick site, stained with YOYO, loaded into a device containing an array of nanochannels, and imaged. Over 473,000 individual molecules of DNA, corresponding to roughly 30x coverage of a human genome, were collected and aligned to the human reference. Based on the known AT/GC content between aligned pairs of labels, the stretch was measured for regions of similar size but different AT/GC content. We found that regions of high GC content were consistently more stretched than regions of high AT content between pairs of labels varying in size between 2.5 kbp and 500 kbp. We measured that for every 1% increase in GC content there was roughly a 0.06% increase in stretch. While this effect is small, it is important to take into account differences in stretch between AT and GC rich regions to improve the sensitivity of detection of structural variations from genomic variations. NIH Grant: R01-HG006851.

Analysis of Molecular Movement Reveals Latticelike Obstructions to Diffusion in Heart Muscle Cells

PubMed Central

Illaste, Ardo; Laasmaa, Martin; Peterson, Pearu; Vendelin, Marko

2012-01-01

Intracellular diffusion in muscle cells is known to be restricted. Although characteristics and localization of these restrictions is yet to be elucidated, it has been established that ischemia-reperfusion injury reduces the overall diffusion restriction. Here we apply an extended version of raster image correlation spectroscopy to determine directional anisotropy and coefficients of diffusion in rat cardiomyocytes. Our experimental results indicate that diffusion of a smaller molecule (1127 MW fluorescently labeled ATTO633-ATP) is restricted more than that of a larger one (10,000 MW Alexa647-dextran), when comparing diffusion in cardiomyocytes to that in solution. We attempt to provide a resolution to this counterintuitive result by applying a quantitative stochastic model of diffusion. Modeling results suggest the presence of periodic intracellular barriers situated ∼1 μm apart having very low permeabilities and a small effect of molecular crowding in volumes between the barriers. Such intracellular structuring could restrict diffusion of molecules of energy metabolism, reactive oxygen species, and apoptotic signals, enacting a significant role in normally functioning cardiomyocytes as well as in pathological conditions of the heart. PMID:22385844

Mobile Phone Sensing of Cocaine in a Lateral Flow Assay Combined with a Biomimetic Material.

PubMed

Guler, Emine; Yilmaz Sengel, Tulay; Gumus, Z Pinar; Arslan, Mustafa; Coskunol, Hakan; Timur, Suna; Yagci, Yusuf

2017-09-19

Lateral flow assays (LFAs) are an ideal choice for drug abuse testing favored by their practicability, portability, and rapidity. LFA based on-site rapid screening devices provide positive/negative judgment in a short response time. The conventionally applied competitive assay format used for small molecule analysis such as abused drugs restricts the quantitation ability of LFA strips. We report herein, for the first time, a new strategy using the noncompetitive assay format via a biomimetic material, namely, poly(p-phenylene) β-cyclodextrin poly(ethylene glycol) (PPP-CD-g-PEG) combined with gold nanoparticle (AuNP) conjugates as the labeling agent to recognize the target cocaine molecule in the test zone. The intensities of the visualized red color in the test line indicate that the cocaine concentrations were analyzed via a smartphone application. Significantly, a combination of this platform with a smartphone application provides quantitative data on the cocaine amount, making it a very inventive and attractive approach especially for on-site applications at critical points such as traffic stops and the workplace.

Small Molecule Inhibitors of AI-2 Signaling in Bacteria: State-of-the-Art and Future Perspectives for Anti-Quorum Sensing Agents

PubMed Central

Guo, Min; Gamby, Sonja; Zheng, Yue; Sintim, Herman O.

2013-01-01

Bacteria respond to different small molecules that are produced by other neighboring bacteria. These molecules, called autoinducers, are classified as intraspecies (i.e., molecules produced and perceived by the same bacterial species) or interspecies (molecules that are produced and sensed between different bacterial species). AI-2 has been proposed as an interspecies autoinducer and has been shown to regulate different bacterial physiology as well as affect virulence factor production and biofilm formation in some bacteria, including bacteria of clinical relevance. Several groups have embarked on the development of small molecules that could be used to perturb AI-2 signaling in bacteria, with the ultimate goal that these molecules could be used to inhibit bacterial virulence and biofilm formation. Additionally, these molecules have the potential to be used in synthetic biology applications whereby these small molecules are used as inputs to switch on and off AI-2 receptors. In this review, we highlight the state-of-the-art in the development of small molecules that perturb AI-2 signaling in bacteria and offer our perspective on the future development and applications of these classes of molecules. PMID:23994835

Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences.

PubMed

van Gijlswijk, R P; Wiegant, J; Vervenne, R; Lasan, R; Tanke, H J; Raap, A K

1996-01-01

We present a sensitive and rapid fluorescence in situ hybridization (FISH) strategy for detecting chromosome-specific repeat sequences. It uses horseradish peroxidase (HRP)-labeled oligonucleotide sequences in combination with fluorescent tyramide-based detection. After in situ hybridization, the HRP conjugated to the oligonucleotide probe is used to deposit fluorescently labeled tyramide molecules at the site of hybridization. The method features full chemical synthesis of probes, strong FISH signals, and short processing periods, as well as multicolor capabilities.

Method for rapid base sequencing in DNA and RNA with two base labeling

DOEpatents

Jett, J.H.; Keller, R.A.; Martin, J.C.; Posner, R.G.; Marrone, B.L.; Hammond, M.L.; Simpson, D.J.

1995-04-11

A method is described for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand. 4 figures.

A Radiochemical Biotechnological Approach: Preliminary Study of Lactose Uptake Rate by Kefir Cells, Using 14C-labeled Lactose, in Anaerobic Fermentation

NASA Astrophysics Data System (ADS)

Golfinopoulos, A.; Soupioni, M.; Kanellaki, M.; Koutinas, A. A.

2008-08-01

The effect of initial lactose concentration on lactose uptake rate by kefir free cells, during the lactose fermentation, was studied in this work. For the investigation 14C-labelled lactose was used due to the fact that labeled and unlabeled molecules are fermented in the same way. The results illustrated lactose uptake rates are about up to two fold higher at lower initial ∘Bé densities as compared with higher initial ∘Bé densities.

Method for rapid base sequencing in DNA and RNA with two base labeling

DOEpatents

Jett, James H.; Keller, Richard A.; Martin, John C.; Posner, Richard G.; Marrone, Babetta L.; Hammond, Mark L.; Simpson, Daniel J.

1995-01-01

Method for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand.

Progress of new label-free techniques for biosensors: a review.

PubMed

Sang, Shengbo; Wang, Yajun; Feng, Qiliang; Wei, Ye; Ji, Jianlong; Zhang, Wendong

2016-01-01

The detection techniques used in biosensors can be broadly classified into label-based and label-free. Label-based detection relies on the specific properties of labels for detecting a particular target. In contrast, label-free detection is suitable for the target molecules that are not labeled or the screening of analytes which are not easy to tag. Also, more types of label-free biosensors have emerged with developments in biotechnology. The latest developed techniques in label-free biosensors, such as field-effect transistors-based biosensors including carbon nanotube field-effect transistor biosensors, graphene field-effect transistor biosensors and silicon nanowire field-effect transistor biosensors, magnetoelastic biosensors, optical-based biosensors, surface stress-based biosensors and other type of biosensors based on the nanotechnology are discussed. The sensing principles, configurations, sensing performance, applications, advantages and restriction of different label-free based biosensors are considered and discussed in this review. Most concepts included in this survey could certainly be applied to the development of this kind of biosensor in the future.

HIV Virions as Nanoscopic Test Tubes for Probing Oligomerization of the Integrase Enzyme

PubMed Central

2015-01-01

Employing viruses as nanoscopic lipid-enveloped test tubes allows the miniaturization of protein–protein interaction (PPI) assays while preserving the physiological environment necessary for particular biological processes. Applied to the study of the human immunodeficiency virus type 1 (HIV-1), viral biology and pathology can also be investigated in novel ways, both in vitro as well as in infected cells. In this work we report on an experimental strategy that makes use of engineered HIV-1 viral particles, to allow for probing PPIs of the HIV-1 integrase (IN) inside viruses with single-molecule Förster resonance energy transfer (FRET) using fluorescent proteins (FP). We show that infectious fluorescently labeled viruses can be obtained and that the quantity of labels can be accurately measured and controlled inside individual viral particles. We demonstrate, with proper control experiments, the formation of IN oligomers in single viral particles and inside viral complexes in infected cells. Finally, we show a clear effect on IN oligomerization of small molecule inhibitors of interactions of IN with its natural human cofactor LEDGF/p75, corroborating that IN oligomer enhancing drugs are active already at the level of the virus and strongly suggesting the presence of a dynamic, enhanceable equilibrium between the IN dimer and tetramer in viral particles. Although applied to the HIV-1 IN enzyme, our methodology for utilizing HIV virions as nanoscopic test tubes for probing PPIs is generic, i.e., other PPIs targeted into the HIV-1, or PPIs targeted into other viruses, can potentially be studied with a similar strategy. PMID:24654558

FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

PubMed

Kuipers, Jeroen; van Ham, Tjakko J; Kalicharan, Ruby D; Veenstra-Algra, Anneke; Sjollema, Klaas A; Dijk, Freark; Schnell, Ulrike; Giepmans, Ben N G

2015-04-01

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM.

Raman Spectroscopic Imaging of the Whole Ciona intestinalis Embryo during Development

PubMed Central

Nakamura, Mitsuru J.; Hotta, Kohji; Oka, Kotaro

2013-01-01

Intracellular composition and the distribution of bio-molecules play central roles in the specification of cell fates and morphogenesis during embryogenesis. Consequently, investigation of changes in the expression and distribution of bio-molecules, especially mRNAs and proteins, is an important challenge in developmental biology. Raman spectroscopic imaging, a non-invasive and label-free technique, allows simultaneous imaging of the intracellular composition and distribution of multiple bio-molecules. In this study, we explored the application of Raman spectroscopic imaging in the whole Ciona intestinalis embryo during development. Analysis of Raman spectra scattered from C. intestinalis embryos revealed a number of localized patterns of high Raman intensity within the embryo. Based on the observed distribution of bio-molecules, we succeeded in identifying the location and structure of differentiated muscle and endoderm within the whole embryo, up to the tailbud stage, in a label-free manner. Furthermore, during cell differentiation, we detected significant differences in cell state between muscle/endoderm daughter cells and daughter cells with other fates that had divided from the same mother cells; this was achieved by focusing on the Raman intensity of single Raman bands at 1002 or 1526 cm−1, respectively. This study reports the first application of Raman spectroscopic imaging to the study of identifying and characterizing differentiating tissues in a whole chordate embryo. Our results suggest that Raman spectroscopic imaging is a feasible label-free technique for investigating the developmental process of the whole embryo of C. intestinalis. PMID:23977129

Rapid Characterization of Candidate Biomarkers for Pancreatic Cancer Using Cell Microarrays (CMAs)

PubMed Central

Kim, Min-Sik; Kuppireddy, Sarada V.; Sakamuri, Sruthi; Singal, Mukul; Getnet, Derese; Harsha, H. C.; Goel, Renu; Balakrishnan, Lavanya; Jacob, Harrys K. C.; Kashyap, Manoj K.; Tankala, Shantal G.; Maitra, Anirban; Iacobuzio-Donahue, Christine A.; Jaffee, Elizabeth; Goggins, Michael G.; Velculescu, Victor E.; Hruban, Ralph H.; Pandey, Akhilesh

2013-01-01

Tissue microarrays have become a valuable tool for high-throughput analysis using immunohistochemical labeling. However, the large majority of biochemical studies are carried out in cell lines to further characterize candidate biomarkers or therapeutic targets with subsequent studies in animals or using primary tissues. Thus, cell line-based microarrays could be a useful screening tool in some situations. Here, we constructed a cell microarray (CMA) containing a panel of 40 pancreatic cancer cell lines available from American Type Culture Collection in addition to those locally available at Johns Hopkins. As proof of principle, we performed immunocytochemical labeling of an epithelial cell adhesion molecule (Ep-CAM), a molecule generally expressed in the epithelium, on this pancreatic cancer CMA. In addition, selected molecules that have been previously shown to be differentially expressed in pancreatic cancer in the literature were validated. For example, we observed strong labeling of CA19-9 antigen, a prognostic and predictive marker for pancreatic cancer. We also carried out a bioinformatics analysis of a literature curated catalog of pancreatic cancer biomarkers developed previously by our group and identified two candidate biomarkers, HLA class I and transmembrane protease, serine 4 (TMPRSS4), and examined their expression in the cell lines represented on the pancreatic cancer CMAs. Our results demonstrate the utility of CMAs as a useful resource for rapid screening of molecules of interest and suggest that CMAs can become a universal standard platform in cancer research. PMID:22985314

Organic small molecule semiconducting chromophores for use in organic electronic devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Welch, Gregory C.; Hoven, Corey V.; Nguyen, Thuc-Quyen

Small organic molecule semi-conducting chromophores containing a pyridalthiadiazole, pyridaloxadiazole, or pyridaltriazole core structure are disclosed. Such compounds can be used in organic heterojunction devices, such as organic small molecule solar cells and transistors.

Structure-guided Discovery of Dual-recognition Chemibodies.

PubMed

Cheng, Alan C; Doherty, Elizabeth M; Johnstone, Sheree; DiMauro, Erin F; Dao, Jennifer; Luthra, Abhinav; Ye, Jay; Tang, Jie; Nixey, Thomas; Min, Xiaoshan; Tagari, Philip; Miranda, Les P; Wang, Zhulun

2018-05-15

Small molecules and antibodies each have advantages and limitations as therapeutics. Here, we present for the first time to our knowledge, the structure-guided design of "chemibodies" as small molecule-antibody hybrids that offer dual recognition of a single target by both a small molecule and an antibody, using DPP-IV enzyme as a proof of concept study. Biochemical characterization demonstrates that the chemibodies present superior DPP-IV inhibition compared to either small molecule or antibody component alone. We validated our design by successfully solving a co-crystal structure of a chemibody in complex with DPP-IV, confirming specific binding of the small molecule portion at the interior catalytic site and the Fab portion at the protein surface. The discovery of chemibodies presents considerable potential for novel therapeutics that harness the power of both small molecule and antibody modalities to achieve superior specificity, potency, and pharmacokinetic properties.

Rational design of chemical genetic probes of RNA function and lead therapeutics targeting repeating transcripts.

PubMed

Disney, Matthew D

2013-12-01

RNA is an important yet vastly underexploited target for small molecule chemical probes or lead therapeutics. Small molecules have been used successfully to modulate the function of the bacterial ribosome, viral RNAs and riboswitches. These RNAs are either highly expressed or can be targeted using substrate mimicry, a mainstay in the design of enzyme inhibitors. However, most cellular RNAs are neither highly expressed nor have a lead small molecule inhibitor, a significant challenge for drug discovery efforts. Herein, I describe the design of small molecules targeting expanded repeating transcripts that cause myotonic muscular dystrophy (DM). These test cases illustrate the challenges of designing small molecules that target RNA and the advantages of targeting repeating transcripts. Lastly, I discuss how small molecules might be more advantageous than oligonucleotides for targeting RNA. Copyright © 2013 Elsevier Ltd. All rights reserved.

Advancing Biological Understanding and Therapeutics Discovery with Small Molecule Probes

PubMed Central

Schreiber, Stuart L.; Kotz, Joanne D.; Li, Min; Aubé, Jeffrey; Austin, Christopher P.; Reed, John C.; Rosen, Hugh; White, E. Lucile; Sklar, Larry A.; Lindsley, Craig W.; Alexander, Benjamin R.; Bittker, Joshua A.; Clemons, Paul A.; de Souza, Andrea; Foley, Michael A.; Palmer, Michelle; Shamji, Alykhan F.; Wawer, Mathias J.; McManus, Owen; Wu, Meng; Zou, Beiyan; Yu, Haibo; Golden, Jennifer E.; Schoenen, Frank J.; Simeonov, Anton; Jadhav, Ajit; Jackson, Michael R.; Pinkerton, Anthony B.; Chung, Thomas D.Y.; Griffin, Patrick R.; Cravatt, Benjamin F.; Hodder, Peter S.; Roush, William R.; Roberts, Edward; Chung, Dong-Hoon; Jonsson, Colleen B.; Noah, James W.; Severson, William E.; Ananthan, Subramaniam; Edwards, Bruce; Oprea, Tudor I.; Conn, P. Jeffrey; Hopkins, Corey R.; Wood, Michael R.; Stauffer, Shaun R.; Emmitte, Kyle A.

2015-01-01

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the U.S. National Institutes of Health launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines, but also highlight the need to innovate the science of therapeutic discovery. PMID:26046436

High mobility high efficiency organic films based on pure organic materials

DOEpatents

Salzman, Rhonda F [Ann Arbor, MI; Forrest, Stephen R [Ann Arbor, MI

2009-01-27

A method of purifying small molecule organic material, performed as a series of operations beginning with a first sample of the organic small molecule material. The first step is to purify the organic small molecule material by thermal gradient sublimation. The second step is to test the purity of at least one sample from the purified organic small molecule material by spectroscopy. The third step is to repeat the first through third steps on the purified small molecule material if the spectroscopic testing reveals any peaks exceeding a threshold percentage of a magnitude of a characteristic peak of a target organic small molecule. The steps are performed at least twice. The threshold percentage is at most 10%. Preferably the threshold percentage is 5% and more preferably 2%. The threshold percentage may be selected based on the spectra of past samples that achieved target performance characteristics in finished devices.

Nucleic acid analysis using terminal-phosphate-labeled nucleotides

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2008-04-22

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

A pretargeting system for tumor PET imaging and radioimmunotherapy

PubMed Central

Kraeber-Bodéré, Françoise; Rousseau, Caroline; Bodet-Milin, Caroline; Frampas, Eric; Faivre-Chauvet, Alain; Rauscher, Aurore; Sharkey, Robert M.; Goldenberg, David M.; Chatal, Jean-François; Barbet, Jacques

2015-01-01

Labeled antibodies, as well as their fragments and antibody-derived recombinant constructs, have long been proposed as general vectors to target radionuclides to tumor lesions for imaging and therapy. They have indeed shown promise in both imaging and therapeutic applications, but they have not fulfilled the original expectations of achieving sufficient image contrast for tumor detection or sufficient radiation dose delivered to tumors for therapy. Pretargeting was originally developed for tumor immunoscintigraphy. It was assumed that directly-radiolabled antibodies could be replaced by an unlabeled immunoconjugate capable of binding both a tumor-specific antigen and a small molecular weight molecule. The small molecular weight molecule would carry the radioactive payload and would be injected after the bispecific immunoconjugate. It has been demonstrated that this approach does allow for both antibody-specific recognition and fast clearance of the radioactive molecule, thus resulting in improved tumor-to-normal tissue contrast ratios. It was subsequently shown that pretargeting also held promise for tumor therapy, translating improved tumor-to-normal tissue contrast ratios into more specific delivery of absorbed radiation doses. Many technical approaches have been proposed to implement pretargeting, and two have been extensively documented. One is based on the avidin-biotin system, and the other on bispecific antibodies binding a tumor-specific antigen and a hapten. Both have been studied in preclinical models, as well as in several clinical studies, and have shown improved targeting efficiency. This article reviews the historical and recent preclinical and clinical advances in the use of bispecific-antibody-based pretargeting for radioimmunodetection and radioimmunotherapy of cancer. The results of recent evaluation of pretargeting in PET imaging also are discussed. PMID:25873896

The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule.

PubMed

Garousi, Javad; Andersson, Ken G; Dam, Johan H; Olsen, Birgitte B; Mitran, Bogdan; Orlova, Anna; Buijs, Jos; Ståhl, Stefan; Löfblom, John; Thisgaard, Helge; Tolmachev, Vladimir

2017-07-20

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The 111 In-labelled DOTA-conjugated Z EGFR:2377 Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-Z EGFR:2377 . DOTA-Z EGFR:2377 was labelled with 57 Co (T 1/2  = 271.8 d), 55 Co (T 1/2  = 17.5 h), and, for comparison, with the positron-emitting radionuclide 68 Ga (T 1/2  = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope 57 Co was used in animal studies. Both 57 Co-DOTA-Z EGFR:2377 and 68 Ga-DOTA-Z EGFR:2377 demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-Z EGFR:2377 were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, 57 Co-DOTA-Z EGFR:2377 demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for 68 Ga-DOTA-Z EGFR:2377 . The results of this study suggest that the positron-emitting cobalt isotope 55 Co would be an optimal label for DOTA-Z EGFR:2377 and further development should concentrate on this radionuclide as a label.

Characterization of D-glucaric acid using NMR, x-ray crystal structure, and MM3 molecular modeling analyses

USDA-ARS?s Scientific Manuscript database

D-glucaric acid was characterized in solution by comparing NMR spectra from the isotopically unlabeled molecule with those from D-glucaric acid labeled with deuterium or carbon-13 atoms. The NMR studies provided unequivocal assignments for all carbon atoms and non-hydroxyl protons of the molecule. ...

Fe3O4-in-silica super crystal of defined interstices for single protein molecules entrapment under magnetic flux.

PubMed

Ye, Lin; Yu, Chih Hao; Jiang, PengJu; Qiu, Lin; Ng, Olivia T W; Yung, Ken K L; He, Heyong; Tsang, Shik Chi

2010-09-28

Confocal fluorescence demonstrates that single molecules of dye-labelled Cytochrome C or B5 containing paramagnetic Fe(III) can be magnetically placed into the interstices of super-crystal which is composed of three dimensional regular arrays of Fe(3)O(4) nanoparticles.

NMR spectroscopic study of organic phosphate esters coprecipitated with calcite

NASA Astrophysics Data System (ADS)

Phillips, Brian L.; Zhang, Zelong; Kubista, Laura; Frisia, Silvia; Borsato, Andrea

2016-06-01

Organic phosphorus incorporated in calcite during laboratory precipitation experiments and in natural cave deposits was investigated by solid-state NMR spectroscopy. For calcite precipitated in the presence of organic phosphoesters of varying size and functionality, solid-state 31P{1H} CP/MAS NMR shows that the phosphoesters were incorporated intact into the solid. Systematic changes in the 31P NMR chemical shift of the phosphate group were observed between the solid phosphoester and that incorporated in the solid precipitate, yielding 31P NMR chemical shifts of the coprecipitates in the range of +1.8 to -2.2 ppm. These chemical shifts are distinct from that of similarly prepared calcite coprecipitated with inorganic phosphate, 3.5 ppm. Only minor changes were noted in the phosphoester 31P chemical shift anisotropy (CSA) which suggests no significant change in the local structure of the phosphate group, which is dominated by C-O-P bonding. Close spatial proximity of the organic phosphate group to calcite structural components was revealed by 31P/13C rotational echo double resonance (REDOR) experiments for coprecipitates prepared with 13C-labeled carbonate. All coprecipitates showed significant 31P dephasing effects upon 13C-irradiation, signaling atomic-scale proximity to carbonate carbon. The dephasing rate for smaller organophosphate molecules is similar to that observed for inorganic phosphate, whereas much slower dephasing was observed for larger molecules having long and/or bulky side-chains. This result suggests that small organic molecules can be tightly enclosed within the calcite structure, whereas significant structural disruption required to accommodate the larger organic molecules leads to longer phosphate-carbonate distances. Comparison of 31P NMR spectroscopic data from the synthetic coprecipitates with those from calcite moonmilk speleothems indicates that phosphorus occurs mainly as inorganic orthophosphate in the natural deposits, although small signals occur with characteristics consistent with phosphate monoesters. The results of this study indicate that trace- to minor concentrations of dissolved organic molecules can be effectively taken up during calcite precipitation and incorporated in the structure, leaving a resilient record of materials present during crystallization.

Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

NASA Astrophysics Data System (ADS)

Chandra, Subhash

2004-06-01

Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13C and 15N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13C15N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39K, 23Na and 40Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

PubMed Central

Thakur, Chandar S.; Sama, Jacob N.; Jackson, Melantha E.; Chen, Bin

2010-01-01

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems. PMID:21057854

Characterizing protein domain associations by Small-molecule ligand binding

PubMed Central

Li, Qingliang; Cheng, Tiejun; Wang, Yanli; Bryant, Stephen H.

2012-01-01

Background Protein domains are evolutionarily conserved building blocks for protein structure and function, which are conventionally identified based on protein sequence or structure similarity. Small molecule binding domains are of great importance for the recognition of small molecules in biological systems and drug development. Many small molecules, including drugs, have been increasingly identified to bind to multiple targets, leading to promiscuous interactions with protein domains. Thus, a large scale characterization of the protein domains and their associations with respect to small-molecule binding is of particular interest to system biology research, drug target identification, as well as drug repurposing. Methods We compiled a collection of 13,822 physical interactions of small molecules and protein domains derived from the Protein Data Bank (PDB) structures. Based on the chemical similarity of these small molecules, we characterized pairwise associations of the protein domains and further investigated their global associations from a network point of view. Results We found that protein domains, despite lack of similarity in sequence and structure, were comprehensively associated through binding the same or similar small-molecule ligands. Moreover, we identified modules in the domain network that consisted of closely related protein domains by sharing similar biochemical mechanisms, being involved in relevant biological pathways, or being regulated by the same cognate cofactors. Conclusions A novel protein domain relationship was identified in the context of small-molecule binding, which is complementary to those identified by traditional sequence-based or structure-based approaches. The protein domain network constructed in the present study provides a novel perspective for chemogenomic study and network pharmacology, as well as target identification for drug repurposing. PMID:23745168

Topological events in single molecules of E. coli DNA confined in nanochannels

PubMed Central

Reifenberger, Jeffrey G.; Dorfman, Kevin D.; Cao, Han

2015-01-01

We present experimental data concerning potential topological events such as folds, internal backfolds, and/or knots within long molecules of double-stranded DNA when they are stretched by confinement in a nanochannel. Genomic DNA from E. coli was labeled near the ‘GCTCTTC’ sequence with a fluorescently labeled dUTP analog and stained with the DNA intercalator YOYO. Individual long molecules of DNA were then linearized and imaged using methods based on the NanoChannel Array technology (Irys® System) available from BioNano Genomics. Data were collected on 189,153 molecules of length greater than 50 kilobases. A custom code was developed to search for abnormal intensity spikes in the YOYO backbone profile along the length of individual molecules. By correlating the YOYO intensity spikes with the aligned barcode pattern to the reference, we were able to correlate the bright intensity regions of YOYO with abnormal stretching in the molecule, which suggests these events were either a knot or a region of internal backfolding within the DNA. We interpret the results of our experiments involving molecules exceeding 50 kilobases in the context of existing simulation data for relatively short DNA, typically several kilobases. The frequency of these events is lower than the predictions from simulations, while the size of the events is larger than simulation predictions and often exceeds the molecular weight of the simulated molecules. We also identified DNA molecules that exhibit large, single folds as they enter the nanochannels. Overall, topological events occur at a low frequency (~7% of all molecules) and pose an easily surmountable obstacle for the practice of genome mapping in nanochannels. PMID:25991508

Classic maximum entropy recovery of the average joint distribution of apparent FRET efficiency and fluorescence photons for single-molecule burst measurements.

PubMed

DeVore, Matthew S; Gull, Stephen F; Johnson, Carey K

2012-04-05

We describe a method for analysis of single-molecule Förster resonance energy transfer (FRET) burst measurements using classic maximum entropy. Classic maximum entropy determines the Bayesian inference for the joint probability describing the total fluorescence photons and the apparent FRET efficiency. The method was tested with simulated data and then with DNA labeled with fluorescent dyes. The most probable joint distribution can be marginalized to obtain both the overall distribution of fluorescence photons and the apparent FRET efficiency distribution. This method proves to be ideal for determining the distance distribution of FRET-labeled biomolecules, and it successfully predicts the shape of the recovered distributions.

Ca(2+)-triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay.

PubMed

Krasitskaya, V V; Korneeva, S I; Kudryavtsev, A N; Markova, S V; Stepanyuk, G A; Frank, L A

2011-11-01

The recombinant Ca(2+)-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.

Proteoform-specific protein binding of small molecules in complex matrices

USDA-ARS?s Scientific Manuscript database

Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original ...

Connecting synthetic chemistry decisions to cell and genome biology using small-molecule phenotypic profiling

PubMed Central

Wagner, Bridget K.; Clemons, Paul A.

2009-01-01

Discovering small-molecule modulators for thousands of gene products requires multiple stages of biological testing, specificity evaluation, and chemical optimization. Many cellular profiling methods, including cellular sensitivity, gene-expression, and cellular imaging, have emerged as methods to assess the functional consequences of biological perturbations. Cellular profiling methods applied to small-molecule science provide opportunities to use complex phenotypic information to prioritize and optimize small-molecule structures simultaneously against multiple biological endpoints. As throughput increases and cost decreases for such technologies, we see an emerging paradigm of using more information earlier in probe- and drug-discovery efforts. Moreover, increasing access to public datasets makes possible the construction of “virtual” profiles of small-molecule performance, even when multiplexed measurements were not performed or when multidimensional profiling was not the original intent. We review some key conceptual advances in small-molecule phenotypic profiling, emphasizing connections to other information, such as protein-binding measurements, genetic perturbations, and cell states. We argue that to maximally leverage these measurements in probe and drug discovery requires a fundamental connection to synthetic chemistry, allowing the consequences of synthetic decisions to be described in terms of changes in small-molecule profiles. Mining such data in the context of chemical structure and synthesis strategies can inform decisions about chemistry procurement and library development, leading to optimal small-molecule screening collections. PMID:19825513

NMR studies of two spliced leader RNAs using isotope labeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lapham, J.; Crothers, D.M.

1994-12-01

Spliced leader RNAs are a class of RNA molecules (<200 nts) involved in the trans splicing of messenger RNA found in trypanosomes, nematodes, and other lower eukaryotes. The spliced leader RNA from the trypanosome Leptomonas Collosoma exists in two alternate structural forms with similar thermal stabilities. The 54 nucleotides on the 5{prime} end of the SL molecule is structurally independent from the 3{prime} half of the RNA, and displays the two structural forms. Furthermore, the favored of the two structures was shown to contain anomalous nuclease sensitivity and thermal stability features, which suggests that there may be tertiary interactions betweenmore » the splice site and other nucleotides in the 5{prime} end. Multidimensional NMR studies are underway to elucidate the structural elements present in the SL RNAs that give rise to their physical properties. Two spliced leader sequences have been studied. The first, the 54 nucleotides on the 5{prime} end of the L. Collosoma sequence, was selected because of earlier studies in our laboratory. The second sequence is the 5{prime} end of the trypanosome Crithidia Fasciculata, which was chosen because of its greater sequence homology to other SL sequences. Given the complexity of the NMR spectra for RNA molecules of this size, we have incorporated {sup 15}N/{sup 13}C-labeled nucleotides into the RNA. One of the techniques we have developed to simplify the spectra of these RNA molecules is isotope labeling of specific regions of the RNA. This has been especially helpful in assigning the secondary structure of molecules that may be able to adopt multiple conformations. Using this technique one can examine a part of the molecule without spectral interference from the unlabeled portion. We hope this approach will promote an avenue for studying the structure of larger RNAs in their native surroundings.« less

Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

NASA Astrophysics Data System (ADS)

Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

2011-10-01

Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

Small Molecule based Musculoskeletal Regenerative Engineering

PubMed Central

Lo, Kevin W.-H.; Jiang, Tao; Gagnon, Keith A.; Nelson, Clarke; Laurencin, Cato T.

2014-01-01

Clinicians and scientists working in the field of regenerative engineering are actively investigating a wide range of methods to promote musculoskeletal tissue regeneration. Small molecule-mediated tissue regeneration is emerging as a promising strategy for regenerating various musculoskeletal tissues and a large number of small molecule compounds have been recently discovered as potential bioactive molecules for musculoskeletal tissue repair and regeneration. In this review, we summarize the recent literature encompassing the past four years in the area of small bioactive molecule for promoting repair and regeneration of various musculoskeletal tissues including bone, muscle, cartilage, tendon, and nerve. PMID:24405851

Defining RNA–Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA

PubMed Central

2017-01-01

RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif–small molecule interactions identified via selection. Named High Throughput Structure–Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif–small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule–RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs. PMID:28386598

Differential processing of the two subunits of human choriogonadotropin (hCG) by granulosa cells. I. Preparation and characterization of selectively labeled hCG

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landefeld, T.D.; Byrne, M.D.; Campbell, K.L.

1981-12-01

The alpha- and beta-subunits of hCG were radioiodinated and recombined with unlabeled complementary subunits. The resultant recombined hormones, selectively labeled in either the alpha- or beta-subunit, were separated from unrecombined subunit by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, extracted with Triton X-100, and characterized by binding analysis. The estimates of maximum binding (active fraction) of the two resultant selectively labeled, recombined hCG preparations, determined with excess receptor were 0.41 and 0.59. These values are similar to those obtained when hCG is labeled as an intact molecule. The specific activities of the recombined preparations were estimated by four different methods, and themore » resulting values were used in combination with the active fraction estimates to determine the concentrations of active free and bound hormone. Binding analyses were run using varying concentrations of both labeled and unlabeled hormone. Estimates of the equilibrium dissociation binding constant (Kd) and receptor capacity were calculated in three different ways. The mean estimates of capacity (52.6 and 52.7 fmol/mg tissue) and Kd (66.6 and 65.7 pM) for the two preparations were indistinguishable. Additionally, these values were similar to values reported previously for hCG radioiodinated as an intact molecule. The availability of well characterized, selectively labeled hCG preparations provides new tools for studying the mechanism of action and the target cell processing of the subunits of this hormone.« less

Ovalbumin labeling with p-hydroxymercurybenzoate: The effect of different denaturing agents and the kinetics of reaction.

PubMed

Campanella, Beatrice; Onor, Massimo; Biancalana, Lorenzo; D'Ulivo, Alessandro; Bramanti, Emilia

2015-08-15

The aim of our study was to investigate how denaturing agents commonly used in protein analysis influence the labeling between a reactive molecule and proteins. For this reason, we investigated the labeling of ovalbumin (OVA) as a globular model protein with p-hydroxymercurybenzoate (pHMB) in its native state (phosphate buffer solution) and in different denaturing conditions (8 molL(-1) urea, 3 molL(-1) guanidinium thiocyanate, 6 molL(-1) guanidinium chloride, 0.2% sodium dodecyl sulfate, and 20% methanol). In addition to chemical denaturation, thermal denaturation was also tested. The protein was pre-column simultaneously denatured and derivatized, and the pHMB-labeled denatured OVA complexes were analyzed by size exclusion chromatography (SEC) coupled online with chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS). The number of -SH groups titrated greatly depends on the protein structure in solution. Indeed, we found that, depending on the adopted denaturing conditions, OVA gave different aggregate species that influence the complexation process. The results were compared with those obtained by a common alternative procedure for the titration of -SH groups that employs monobromobimane (mBBr) as tagging molecule and molecular fluorescence spectroscopy as detection technique. We also investigated the labeling kinetics for denatured OVA and pHMB, finding that the 4 thiolic groups of OVA have a very different reactivity toward mercury labeling, in agreement with previous studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of the procedure for separating barley from other spring small grains. [North Dakota, South Dakota, Minnesota and Montana

NASA Technical Reports Server (NTRS)

Magness, E. R. (Principal Investigator)

1980-01-01

The success of the Transition Year procedure to separate and label barley and the other small grains was assessed. It was decided that developers of the procedure would carry out the exercise in order to prevent compounding procedural problems with implementation problems. The evaluation proceeded by labeling the sping small grains first. The accuracy of this labeling was, on the average, somewhat better than that in the Transition Year operations. Other departures from the original procedure included a regionalization of the labeling process, the use of trend analysis, and the removal of time constraints from the actual processing. Segment selection, ground truth derivation, and data available for each segment in the analysis are discussed. Labeling accuracy is examined for North Dakota, South Dakota, Minnesota, and Montana as well as for the entire four-state area. Errors are characterized.

Understanding the Halogenation Effects in Diketopyrrolopyrrole-Based Small Molecule Photovoltaics.

PubMed

Sun, Shi-Xin; Huo, Yong; Li, Miao-Miao; Hu, Xiaowen; Zhang, Hai-Jun; Zhang, You-Wen; Zhang, You-Dan; Chen, Xiao-Long; Shi, Zi-Fa; Gong, Xiong; Chen, Yongsheng; Zhang, Hao-Li

2015-09-16

Two molecules containing a central diketopyrrolopyrrole and two oligothiophene units have been designed and synthesized. Comparisons between the molecules containing terminal F (FDPP) and Cl (CDPP) atoms allowed us to evaluate the effects of halogenation on the photovoltaic properties of the small molecule organic solar cells (OSCs). The OSCs devices employing FDPP:PC71BM films showed power conversion efficiencies up to 4.32%, suggesting that fluorination is an efficient method for constructing small molecules for OSCs.

ChemBank: a small-molecule screening and cheminformatics resource database.

PubMed

Seiler, Kathleen Petri; George, Gregory A; Happ, Mary Pat; Bodycombe, Nicole E; Carrinski, Hyman A; Norton, Stephanie; Brudz, Steve; Sullivan, John P; Muhlich, Jeremy; Serrano, Martin; Ferraiolo, Paul; Tolliday, Nicola J; Schreiber, Stuart L; Clemons, Paul A

2008-01-01

ChemBank (http://chembank.broad.harvard.edu/) is a public, web-based informatics environment developed through a collaboration between the Chemical Biology Program and Platform at the Broad Institute of Harvard and MIT. This knowledge environment includes freely available data derived from small molecules and small-molecule screens and resources for studying these data. ChemBank is unique among small-molecule databases in its dedication to the storage of raw screening data, its rigorous definition of screening experiments in terms of statistical hypothesis testing, and its metadata-based organization of screening experiments into projects involving collections of related assays. ChemBank stores an increasingly varied set of measurements derived from cells and other biological assay systems treated with small molecules. Analysis tools are available and are continuously being developed that allow the relationships between small molecules, cell measurements, and cell states to be studied. Currently, ChemBank stores information on hundreds of thousands of small molecules and hundreds of biomedically relevant assays that have been performed at the Broad Institute by collaborators from the worldwide research community. The goal of ChemBank is to provide life scientists unfettered access to biomedically relevant data and tools heretofore available primarily in the private sector.

Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

PubMed Central

Ho, Dominik; Dose, Christian; Albrecht, Christian H.; Severin, Philip; Falter, Katja; Dervan, Peter B.; Gaub, Hermann E.

2009-01-01

Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA·ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA·ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA·ligand complexes. PMID:19486688

Synthesis and characterization of citrate-based fluorescent small molecules and biodegradable polymers.

PubMed

Xie, Zhiwei; Kim, Jimin P; Cai, Qing; Zhang, Yi; Guo, Jinshan; Dhami, Ranjodh S; Li, Li; Kong, Bin; Su, Yixue; Schug, Kevin A; Yang, Jian

2017-03-01

Novel citric acid based photoluminescent dyes and biodegradable polymers are synthesized via a facile "one-pot" reaction. A comprehensive understanding of the fluorescence mechanisms of the resulting citric acid-based fluorophores is reported. Two distinct types of fluorophores are identified: a thiozolopyridine family with high quantum yield, long lifetime, and exceptional photostability, and a dioxopyridine family with relatively lower quantum yield, multiple lifetimes, and solvent-dependent band shifting behavior. Applications in molecular labeling and cell imaging were demonstrated. The above discoveries contribute to the field of fluorescence chemistry and have laid a solid foundation for further development of new fluorophores and materials that show promise in a diversity of fluorescence-based applications. Photoluminescent materials are pivotal for fluorescence based imaging, labeling and sensing applications. Understanding their fluorescence mechanism is challenging and imperative. We develop a new class of citric acid-derived fluorescent materials in forms of polymers and small molecular dyes by a one-step solvent free reaction. We discovered two different classes of citric acid-derived fluorophores. A two-ring thiozolopyridine structure demonstrates strong fluorescence and exceptional resistance to photo-bleaching. A one-ring dioxopyridine exhibits relative weak fluorescence but with intriguing excitation and solvent-dependent emission wavelength shifting. Our methodology of synthesizing citric acid-derived fluorophores and the understanding on their luminescence are instrumental to the design and production of a large number of new photoluminescent materials for biological and biomedical applications. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability.

PubMed

Tsanov, Nikolay; Samacoits, Aubin; Chouaib, Racha; Traboulsi, Abdel-Meneem; Gostan, Thierry; Weber, Christian; Zimmer, Christophe; Zibara, Kazem; Walter, Thomas; Peter, Marion; Bertrand, Edouard; Mueller, Florian

2016-12-15

Single molecule FISH (smFISH) allows studying transcription and RNA localization by imaging individual mRNAs in single cells. We present smiFISH (single molecule inexpensive FISH), an easy to use and flexible RNA visualization and quantification approach that uses unlabelled primary probes and a fluorescently labelled secondary detector oligonucleotide. The gene-specific probes are unlabelled and can therefore be synthesized at low cost, thus allowing to use more probes per mRNA resulting in a substantial increase in detection efficiency. smiFISH is also flexible since differently labelled secondary detector probes can be used with the same primary probes. We demonstrate that this flexibility allows multicolor labelling without the need to synthesize new probe sets. We further demonstrate that the use of a specific acrydite detector oligonucleotide allows smiFISH to be combined with expansion microscopy, enabling the resolution of transcripts in 3D below the diffraction limit on a standard microscope. Lastly, we provide improved, fully automated software tools from probe-design to quantitative analysis of smFISH images. In short, we provide a complete workflow to obtain automatically counts of individual RNA molecules in single cells. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

48-spot single-molecule FRET setup with periodic acceptor excitation

NASA Astrophysics Data System (ADS)

Ingargiola, Antonino; Segal, Maya; Gulinatti, Angelo; Rech, Ivan; Labanca, Ivan; Maccagnani, Piera; Ghioni, Massimo; Weiss, Shimon; Michalet, Xavier

2018-03-01

Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature.

PubMed

Cheney, Philip P; Weisgerber, Alan W; Feuerbach, Alec M; Knowles, Michelle K

2017-03-15

The plasma membrane is a highly compartmentalized, dynamic material and this organization is essential for a wide variety of cellular processes. Nanoscale domains allow proteins to organize for cell signaling, endo- and exocytosis, and other essential processes. Even in the absence of proteins, lipids have the ability to organize into domains as a result of a variety of chemical and physical interactions. One feature of membranes that affects lipid domain formation is membrane curvature. To directly test the role of curvature in lipid sorting, we measured the accumulation of two similar lipids, 1,2-Dihexadecanoyl- sn -glycero-3-phosphoethanolamine (DHPE) and hexadecanoic acid (HDA), using a supported lipid bilayer that was assembled over a nanopatterned surface to obtain regions of membrane curvature. Both lipids studied contain 16 carbon, saturated tails and a head group tag for fluorescence microscopy measurements. The accumulation of lipids at curvatures ranging from 28 nm to 55 nm radii was measured and fluorescein labeled DHPE accumulated more than fluorescein labeled HDA at regions of membrane curvature. We then tested whether single biotinylated DHPE molecules sense curvature using single particle tracking methods. Similar to groups of fluorescein labeled DHPE accumulating at curvature, the dynamics of single molecules of biotinylated DHPE was also affected by membrane curvature and highly confined motion was observed.

Mini-titins in striated and smooth molluscan muscles: structure, location and immunological crossreactivity.

PubMed

Vibert, P; Edelstein, S M; Castellani, L; Elliott, B W

1993-12-01

Invertebrate mini-titins are members of a class of myosin-binding proteins belonging to the immunoglobulin superfamily that may have structural and/or regulatory properties. We have isolated mini-titins from three molluscan sources: the striated and smooth adductor muscles of the scallop, and the smooth catch muscles of the mussel. Electron microscopy reveals flexible rod-like molecules about 0.2 micron long and 30 A wide with a distinctive polarity. Antibodies to scallop mini-titin label the A-band and especially the A/I junction of scallop striated muscle myofibrils by indirect immunofluorescence and immuno-electron microscopy. This antibody crossreacts with mini-titins in scallop smooth and Mytilus catch muscles, as well as with proteins in striated muscles from Limulus, Lethocerus (asynchronous flight muscle), and crayfish. It labels the A/I junction (I-region in Lethocerus) in these striated muscles as well as in chicken skeletal muscle. Antibodies to the repetitive immunoglobulin-like regions and also to the kinase domain of nematode twitchin crossreact with scallop mini-titin and label the A-band of scallop myofibrils. Electron microscopy of single molecules shows that antibodies to twitchin kinase bind to scallop mini-titin near one end of the molecule, suggesting how the scallop structure might be aligned with the sequence of nematode twitchin.

Studies related to the development of the Viking 1975 labeled release experiment

NASA Technical Reports Server (NTRS)

Devincenzi, D. L.; Deal, P. H.

1976-01-01

The labeled release life detection experiment on the Viking 1975 Mars mission is based on the concept that microorganisms will metabolize radioactive organic substrates in a nutrient medium and release radioactive carbon dioxide. Several experiments, using laboratory equipment, were carried out to evaluate various aspects of the concept. Results indicate: (1) label is released by sterilization-treated soil, (2) substantial quantities of label are retained in solution under basic conditions, (3) the substrate used, as well as position of label in the molecule, affect release of label, (4) label release is depressed by radiolytic decomposition of substrates, and (5) About 100,000 organisms are required to produce a detectable response. These results, suggest additional areas for testing, add to the data base for interpretation of flight results, and have significance for broader application of this technique for assessing microbial activity.

Nanopipette Delivery of Individual Molecules to Cellular Compartments for Single-Molecule Fluorescence Tracking

PubMed Central

Bruckbauer, Andreas; James, Peter; Zhou, Dejian; Yoon, Ji Won; Excell, David; Korchev, Yuri; Jones, Roy; Klenerman, David

2007-01-01

We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 ± 0.04 μm2/s in the acrosomal region and 0.10 ± 0.02 μm2/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane. PMID:17631532

Harnessing Connectivity in a Large-Scale Small-Molecule Sensitivity Dataset | Office of Cancer Genomics

Cancer.gov

Identifying genetic alterations that prime a cancer cell to respond to a particular therapeutic agent can facilitate the development of precision cancer medicines. Cancer cell-line (CCL) profiling of small-molecule sensitivity has emerged as an unbiased method to assess the relationships between genetic or cellular features of CCLs and small-molecule response. Here, we developed annotated cluster multidimensional enrichment analysis to explore the associations between groups of small molecules and groups of CCLs in a new, quantitative sensitivity dataset.

A Nonfullerene Small Molecule Acceptor with 3D Interlocking Geometry Enabling Efficient Organic Solar Cells.

PubMed

Lee, Jaewon; Singh, Ranbir; Sin, Dong Hun; Kim, Heung Gyu; Song, Kyu Chan; Cho, Kilwon

2016-01-06

A new 3D nonfullerene small-molecule acceptor is reported. The 3D interlocking geometry of the small-molecule acceptor enables uniform molecular conformation and strong intermolecular connectivity, facilitating favorable nanoscale phase separation and electron charge transfer. By employing both a novel polymer donor and a nonfullerene small-molecule acceptor in the solution-processed organic solar cells, a high-power conversion efficiency of close to 6% is demonstrated. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A "roller-wheel" Pt-containing small molecule that outperforms its polymer analogs in organic solar cells

DOE PAGES

He, Wenhan; Wu, Qin; Livshits, Maksim Y.; ...

2016-05-23

A novel Pt-bisacetylide small molecule (Pt-SM) featuring “roller-wheel” geometry was synthesized and characterized. When compared with conventional Pt-containing polymers and small molecules having “dumbbell” shaped structures, Pt-SM displays enhanced crystallinity and intermolecular π–π interactions, as well as favorable panchromatic absorption behaviors. Furthermore, organic solar cells (OSCs) employing Pt-SM achieve power conversion efficiencies (PCEs) up to 5.9%, the highest reported so far for Pt-containing polymers and small molecules.

A "roller-wheel" Pt-containing small molecule that outperforms its polymer analogs in organic solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Wenhan; Wu, Qin; Livshits, Maksim Y.

A novel Pt-bisacetylide small molecule (Pt-SM) featuring “roller-wheel” geometry was synthesized and characterized. When compared with conventional Pt-containing polymers and small molecules having “dumbbell” shaped structures, Pt-SM displays enhanced crystallinity and intermolecular π–π interactions, as well as favorable panchromatic absorption behaviors. Furthermore, organic solar cells (OSCs) employing Pt-SM achieve power conversion efficiencies (PCEs) up to 5.9%, the highest reported so far for Pt-containing polymers and small molecules.

Biochemical Control of Marine Fouling

DTIC Science & Technology

1988-01-14

characterized directly. The receptors are specifically labeled with tritiated (-)- baclofen ( -chlorophenyl-GABA). This labeling is saturable, reversible (with...no change in the radioactive baclofen molecule), and stereochemically specific. Scatchard and log-logit analyses of binding data indicate that there...are approximately 1010 receptors per larva; the affinity of these receptors for (-)- baclofen is reflected by a KD = 3 x 10-7. [Our original results

Array biosensor: recent developments

NASA Astrophysics Data System (ADS)

Golden, Joel P.; Rowe-Taitt, Chris A.; Feldstein, Mark J.; Ligler, Frances S.

1999-05-01

A fluorescence-based immunosensor has been developed for simultaneous analyses of multiple samples for 1 to 6 different antigens. A patterned array of recognition antibodies immobilized on the surface of a planar waveguide is used to 'capture' analyte present in samples. Bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. A new design for a fluidics distribution system is shown, as well as results from assays for physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D- dimer, a marker of sepsis and thrombotic disorders.

Biofunctionalized Zinc Oxide Field Effect Transistors for Selective Sensing of Riboflavin with Current Modulation

PubMed Central

Hagen, Joshua A.; Kim, Sang N.; Bayraktaroglu, Burhan; Leedy, Kevin; Chávez, Jorge L.; Kelley-Loughnane, Nancy; Naik, Rajesh R.; Stone, Morley O.

2011-01-01

Zinc oxide field effect transistors (ZnO-FET), covalently functionalized with single stranded DNA aptamers, provide a highly selective platform for label-free small molecule sensing. The nanostructured surface morphology of ZnO provides high sensitivity and room temperature deposition allows for a wide array of substrate types. Herein we demonstrate the selective detection of riboflavin down to the pM level in aqueous solution using the negative electrical current response of the ZnO-FET by covalently attaching a riboflavin binding aptamer to the surface. The response of the biofunctionalized ZnO-FET was tuned by attaching a redox tag (ferrocene) to the 3′ terminus of the aptamer, resulting in positive current modulation upon exposure to riboflavin down to pM levels. PMID:22163977

Selective Transgenic Expression of Mutant Ubiquitin in Purkinje Cell Stripes in the Cerebellum.

PubMed

Verheijen, Bert M; Gentier, Romina J G; Hermes, Denise J H P; van Leeuwen, Fred W; Hopkins, David A

2017-06-01

The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B +1 (UBB +1 ), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB +1 -expressing transgenic mice display widespread labeling for UBB +1 in brain and exhibit behavioral deficits. Here, we show that UBB +1 is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB +1 -expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties.

A-π-D-π-A Electron-Donating Small Molecules for Solution-Processed Organic Solar Cells: A Review.

PubMed

Wang, Zhen; Zhu, Lingyun; Shuai, Zhigang; Wei, Zhixiang

2017-11-01

Organic solar cells based on semiconducting polymers and small molecules have attracted considerable attention in the last two decades. Moreover, the power conversion efficiencies for solution-processed solar cells containing A-π-D-π-A-type small molecules and fullerenes have reached 11%. However, the method for designing high-performance, photovoltaic small molecules still remains unclear. In this review, recent studies on A-π-D-π-A electron-donating small molecules for organic solar cells are introduced. Moreover, the relationships between molecular properties and device performances are summarized, from which inspiration for the future design of high performance organic solar cells may be obtained. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

76 FR 32215 - Agency Information Collection Activities; Announcement of Office of Management and Budget...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-03

...; Restaurant Menu and Vending Machine Labeling; Registration for Small Chains Under Section 4205 of the Patient... ``Restaurant Menu and Vending Machine Labeling: Registration for Small Chains Under Section 4205 of the Patient...

Convergent synthesis of 13N-labelled Peptidic structures using aqueous [13N]NH3.

PubMed

Blower, Julia E; Cousin, Samuel F; Gee, Antony D

2017-01-01

Nitrogen-13 has a 10-min half-life which places time constraints on the complexity of viable synthetic methods for its incorporation into PET imaging agents. In exploring ways to overcome this limitation, we have used the Ugi reaction to develop a rapid one-pot method for radiolabelling peptidic molecules using [ 13 N]NH 3 as a synthetic precursor. Carrier-added [ 13 N]NH 3 (50 μL) was added to a solution of carboxylic acid, aldehyde, and isocyanide in 2,2,2-TFE (200 μL). The mixture was heated in a microwave synthesiser at 120 °C for 10 min. Reactions were analysed by radio-HPLC and radio-LCMS. Isolation of the target 13 N-labelled peptidic Ugi compound was achieved via semi-preparative radio-HPLC as demonstrated for Ugi 1. Radio-HPLC analysis of each reaction confirmed the formation of radioactive products co-eluting with their respective reference standards with radiochemical yields of the crude products ranging from 11% to 23%. Two cyclic γ-lactam structures were also achieved via intra-molecular reactions. Additional radioactive by-products observed in the radio-chromatogram were identified as 13 N-labelled di-imines formed from the reaction of [ 13 N]NH 3 with two isocyanide molecules. The desired 13 N-labelled Ugi product was isolated using semi-preparative HPLC. We have developed a one-pot method that opens up new routes to radiolabel complex, peptidic molecules with 13 N using aqueous [ 13 N]NH 3 as a synthetic precursor.

TGF-β of lung cancer microenvironment upregulates B7H1 and GITRL expression in dendritic cells and is associated with regulatory T cell generation.

PubMed

Ni, Xiao Yan; Sui, Hua Xiu; Liu, Yao; Ke, Shi Zhong; Wang, Yi Nan; Gao, Feng Guang

2012-08-01

The effects of TGF-β on dendritic cells (DCs) on the tumor microenvironment are not well understood. We report, here, the establishment of an in vitro lung cancer microenvironment by co-incubation of seminaphtharhodafluor (SNARF) labeled Lewis lung cancer (LLC) cells, carboxyfluorescein succinimidyl ester (CFSE) labeled fibroblasts and 4-chloromethyl-7-hydroxycoumarin (CMHC) labeled DCs. Raw 264.7, EL4 and NCI-H446 cells were able to synthesize TGF-β which was determined by flow cyto-metry and western blotting, respectively. Furthermore, TGF-β efficiently increased regulatory T-cell (Treg) expansion and upregulated DC B7H1 and GITRL expression. TGF-β and the co-incubation of LLC cells, fibroblasts with DCs could augment the expression of B7H1 and GITRL molecules of DCs. The data presented here indicate that the B7H1 and GITRL molecules may play an important role in TGF-β-induced Treg expansion of lung cancer microenvironment.

Toward Generalization of Iterative Small Molecule Synthesis

PubMed Central

Lehmann, Jonathan W.; Blair, Daniel J.; Burke, Martin D.

2018-01-01

Small molecules have extensive untapped potential to benefit society, but access to this potential is too often restricted by limitations inherent to the customized approach currently used to synthesize this class of chemical matter. In contrast, the “building block approach”, i.e., generalized iterative assembly of interchangeable parts, has now proven to be a highly efficient and flexible way to construct things ranging all the way from skyscrapers to macromolecules to artificial intelligence algorithms. The structural redundancy found in many small molecules suggests that they possess a similar capacity for generalized building block-based construction. It is also encouraging that many customized iterative synthesis methods have been developed that improve access to specific classes of small molecules. There has also been substantial recent progress toward the iterative assembly of many different types of small molecules, including complex natural products, pharmaceuticals, biological probes, and materials, using common building blocks and coupling chemistry. Collectively, these advances suggest that a generalized building block approach for small molecule synthesis may be within reach. PMID:29696152

Choosing the right fluorophore for single-molecule fluorescence studies in a lipid environment.

PubMed

Zhang, Zhenfu; Yomo, Dan; Gradinaru, Claudiu

2017-07-01

Nonspecific interactions between lipids and fluorophores can alter the outcomes of single-molecule spectroscopy of membrane proteins in live cells, liposomes or lipid nanodiscs and of cytosolic proteins encapsulated in liposomes or tethered to supported lipid bilayers. To gain insight into these effects, we examined interactions between 9 dyes that are commonly used as labels for single-molecule fluorescence (SMF) and 6 standard lipids including cationic, zwitterionic and anionic types. The diffusion coefficients of dyes in the absence and presence of set amounts of lipid vesicles were measured by fluorescence correlation spectroscopy (FCS). The partition coefficients and the free energies of partitioning for different fluorophore-lipid pairs were obtained by global fitting of the titration FCS curves. Lipids with different charges, head groups and degrees of chain saturation were investigated, and interactions with dyes are discussed in terms of hydrophobic, electrostatic and steric contributions. Fluorescence imaging of individual fluorophores adsorbed on supported lipid bilayers provides visualization and additional quantification of the strength of dye-lipid interaction in the context of single-molecule measurements. By dissecting fluorophore-lipid interactions, our study provides new insights into setting up single-molecule fluorescence spectroscopy experiments with minimal interference from interactions between fluorescent labels and lipids in the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

How to measure separations and angles between intra-molecular fluorescent markers

NASA Astrophysics Data System (ADS)

Flyvbjerg, Henrik; Mortensen, Kim I.; Sung, Jongmin; Spudich, James A.

We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each color-separated microscope image in a time-lapse movie and using only simple means, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space with accuracy and precision. The positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA molecules internally labelled with two fixed fluorophores, we demonstrate the accuracy and precision of our method using the known structure of double-stranded DNA as a benchmark, resolve 10-base-pair differences in fluorophore separations, and determine the unique 3D orientation of each DNA molecule, thereby establishing short, double-labelled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly. This work was supported by a Lundbeck fellowship to K.I.M; a Stanford Bio-X fellowship to J.S. and Grants from the NIH (GM33289) to J.A.S. and the Human Frontier Science Program (GP0054/2009-C) to J.A.S. and H.F.