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Sample records for biological rna molecules

  1. Engineering biological systems with synthetic RNA molecules

    PubMed Central

    Liang, Joe C.; Bloom, Ryan J.; Smolke, Christina D.

    2011-01-01

    RNA molecules play diverse functional roles in natural biological systems. There has been growing interest in designing synthetic RNA counterparts for programming biological function. The design of synthetic RNA molecules that exhibit diverse activities, including sensing, regulatory, information processing, and scaffolding activities, has highlighted the advantages of RNA as a programmable design substrate. Recent advances in implementing these engineered RNA molecules as key control elements in synthetic genetic networks are highlighting the functional relevance of this class of synthetic elements in programming cellular behaviors. PMID:21925380

  2. A chemical screen for biological small molecule-RNA conjugates reveals CoA-linked RNA.

    PubMed

    Kowtoniuk, Walter E; Shen, Yinghua; Heemstra, Jennifer M; Agarwal, Isha; Liu, David R

    2009-05-12

    Compared with the rapidly expanding set of known biological roles for RNA, the known chemical diversity of cellular RNA has remained limited primarily to canonical RNA, 3'-aminoacylated tRNAs, nucleobase-modified RNAs, and 5'-capped mRNAs in eukaryotes. We developed two methods to detect in a broad manner chemically labile cellular small molecule-RNA conjugates. The methods were validated by the detection of known tRNA and rRNA modifications. The first method analyzes small molecules cleaved from RNA by base or nucleophile treatment. Application to Escherichia coli and Streptomyces venezuelae RNA revealed an RNA-linked hydroxyfuranone or succinyl ester group, in addition to a number of other putative small molecule-RNA conjugates not previously reported. The second method analyzes nuclease-generated mononucleotides before and after treatment with base or nucleophile and also revealed a number of new putative small molecule-RNA conjugates, including 3'-dephospho-CoA and its succinyl-, acetyl-, and methylmalonyl-thioester derivatives. Subsequent experiments established that these CoA species are attached to E. coli and S. venezuelae RNA at the 5' terminus. CoA-linked RNA cannot be generated through aberrant transcriptional initiation by E. coli RNA polymerase in vitro, and CoA-linked RNA in E. coli is only found among smaller (approximately < 200 nucleotide) RNAs that have yet to be identified. These results provide examples of small molecule-RNA conjugates and suggest that the chemical diversity of cellular RNA may be greater than previously understood. PMID:19416889

  3. Identification of Biologically Active, HIV TAR RNA-Binding Small Molecules Using Small Molecule Microarrays

    PubMed Central

    2015-01-01

    Identifying small molecules that selectively bind to structured RNA motifs remains an important challenge in developing potent and specific therapeutics. Most strategies to find RNA-binding molecules have identified highly charged compounds or aminoglycosides that commonly have modest selectivity. Here we demonstrate a strategy to screen a large unbiased library of druglike small molecules in a microarray format against an RNA target. This approach has enabled the identification of a novel chemotype that selectively targets the HIV transactivation response (TAR) RNA hairpin in a manner not dependent on cationic charge. Thienopyridine 4 binds to and stabilizes the TAR hairpin with a Kd of 2.4 μM. Structure–activity relationships demonstrate that this compound achieves activity through hydrophobic and aromatic substituents on a heterocyclic core, rather than cationic groups typically required. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis was performed on a 365-nucleotide sequence derived from the 5′ untranslated region (UTR) of the HIV-1 genome to determine global structural changes in the presence of the molecule. Importantly, the interaction of compound 4 can be mapped to the TAR hairpin without broadly disrupting any other structured elements of the 5′ UTR. Cell-based anti-HIV assays indicated that 4 inhibits HIV-induced cytopathicity in T lymphocytes with an EC50 of 28 μM, while cytotoxicity was not observed at concentrations approaching 1 mM. PMID:24820959

  4. MicroRNA: a small molecule with a big biological impact.

    PubMed

    Zhou, Xiaofeng; Yang, Pan-Chyr

    2012-01-01

    One of the most significant achievements in biological science in the last decade is the discovery of RNA interference (RNAi), a process within living cells that regulates gene expression at post-transcriptional levels. Historically, this process was described by other more generic names, such as co-suppression and post transcriptional gene silencing. Only after the molecular mechanism underlying these apparently unrelated processes was fully understood did it become apparent that they all described the RNAi phenomenon. In 2006, Dr. Andrew Fire and Dr. Craig C. Mello were awarded the Nobel Prize in Physiology or Medicine for their work on RNAi interference. RNAi is an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by two types of small RNA molecules - microRNA (miRNA) and small interfering RNA (siRNA). However, the function of microRNA appears to be far beyond RNAi alone, including direct interaction with the gene promoter and epigenetic regulation of the DNA methylation and histone modification. By regulating gene expression, miRNAs are likely to be involved in diverse biological activities, such as tumorigenesis, immune response, insulin secretion, neurotransmitter synthesis, and circadian rhythm, to name a few. MicroRNAs are 21-23 nucleotide single stranded RNA molecules found in eukaryotic cells. The first miRNA, lin-4, was characterized in C. elegans in the early 1990s [1]. In the early years, the progress on microRNA research was slow and experienced substantial growing pains. The short length and uniqueness of each microRNA rendered many conventional hybridization based methods ineffective; very small RNAs are difficult to reliably amplify or label without introducing bias. In addition, hybridization-based methods for microRNA profiling relied on probes designed to detect known microRNAs or known microRNA species previously identified by sequencing or homology search. Recent evidence of

  5. Single Molecule Photobleaching (SMPB) Technology for Counting of RNA, DNA, Protein and Other Molecules in Nanoparticles and Biological Complexes by TIRF Instrumentation

    PubMed Central

    Zhang, Hui; Guo, Peixuan

    2014-01-01

    Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The Single Molecule Photobleaching (SMPB) technology for direct counting developed by our team (Shu et al, EMBO J, 2007, 26:527; Zhang et al, RNA, 2007, 13:1793) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described. PMID:24440482

  6. Reconstitution of biologically active 50S ribosomal subunits with artificial 5S RNA molecules carrying disturbances in the base pairing within the molecular stalk.

    PubMed Central

    Raué, H A; Lorenz, S; Erdmann, V A; Planta, R J

    1981-01-01

    Bacillus stearothermophilus 50S ribosomal subunits were reconstituted in vitro using artificial 5S RNA molecules constructed by combining parts of major and minor type (Raué et al. (1976) Europ. J. Biochem. 68, 169-176) B. licheniformis 5S RNA. The artificial 5S RNA molecules carry defined disturbances (A.C juxtapositions and extra G.U pairs) in the base pairing between the 5'- and 3'-terminal sequences of the molecule (the molecular stalk region). The biological activity of the reconstituted subunits was determined in an E. coli cell-free system programmed with poly-U. The results show that conservation of the base pairing within the molecular stalk is not required for biological activity of 5S RNA. Disturbances of the base pairing within this region do reduce the rate of reconstitution, however. Normal base pairing in the molecular stalk is thus required to ensure efficient ribosome assembly. PMID:6164987

  7. Synthetic biology with RNA motifs.

    PubMed

    Saito, Hirohide; Inoue, Tan

    2009-02-01

    Structural motifs in naturally occurring RNAs and RNPs can be employed as new molecular parts for synthetic biology to facilitate the development of novel devices and systems that modulate cellular functions. In this review, we focus on the following: (i) experimental evolution techniques of RNA molecules in vitro and (ii) their applications for regulating gene expression systems in vivo. For experimental evolution, new artificial RNA aptamers and RNA enzymes (ribozymes) have been selected in vitro. These functional RNA molecules are likely to be applicable in the reprogramming of existing gene regulatory systems. Furthermore, they may be used for designing hypothetical RNA-based living systems in the so-called RNA world. For the regulation of gene expressions in living cells, the development of new riboswitches allows us to modulate the target gene expression in a tailor-made manner. Moreover, recently RNA-based synthetic genetic circuits have been reported by employing functional RNA molecules, expanding the repertory of synthetic biology with RNA motifs. PMID:18775792

  8. Diversity in Biological Molecules

    ERIC Educational Resources Information Center

    Newbury, H. John

    2010-01-01

    One of the striking characteristics of fundamental biological processes, such as genetic inheritance, development and primary metabolism, is the limited amount of variation in the molecules involved. Natural selective pressures act strongly on these core processes and individuals carrying mutations and producing slightly sub-optimal versions of…

  9. Artemether Combined with shRNA Interference of Vascular Cell Adhesion Molecule-1 Significantly Inhibited the Malignant Biological Behavior of Human Glioma Cells

    PubMed Central

    Wang, Ping; Xue, Yi-Xue; Yao, Yi-Long; Yu, Bo; Liu, Yun-Hui

    2013-01-01

    Artemether is the derivative extracted from Chinese traditional herb and originally used for malaria. Artemether also has potential therapeutic effects against tumors. Vascular cell adhesion molecule-1 (VCAM-1) is an important cell surface adhesion molecule associated with malignancy of gliomas. In this work, we investigated the role and mechanism of artemether combined with shRNA interference of VCAM-1 (shRNA-VCAM-1) on the migration, invasion and apoptosis of glioma cells. U87 human glioma cells were treated with artemether at various concentrations and shRNA interfering technology was employed to silence the expression of VCAM-1. Cell viability, migration, invasiveness and apoptosis were assessed with MTT, wound healing, Transwell and Annexin V-FITC/PI staining. The expression of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and phosphorylated Akt (p-Akt) was checked by Western blot assay. Results showed that artemether and shRNA-VCAM-1 not only significantly inhibited the migration, invasiveness and expression of MMP-2/9 and p-Akt, but also promoted the apoptosis of U87 cells. Combined treatment of both displayed the maximum inhibitory effects on the malignant biological behavior of glioma cells. Our work revealed the potential therapeutic effects of artemether and antiVCAM-1 in the treatments of gliomas. PMID:23593320

  10. Biological mechanisms, one molecule at a time

    PubMed Central

    Tinoco, Ignacio; Gonzalez, Ruben L.

    2011-01-01

    The last 15 years have witnessed the development of tools that allow the observation and manipulation of single molecules. The rapidly expanding application of these technologies for investigating biological systems of ever-increasing complexity is revolutionizing our ability to probe the mechanisms of biological reactions. Here, we compare the mechanistic information available from single-molecule experiments with the information typically obtained from ensemble studies and show how these two experimental approaches interface with each other. We next present a basic overview of the toolkit for observing and manipulating biology one molecule at a time. We close by presenting a case study demonstrating the impact that single-molecule approaches have had on our understanding of one of life's most fundamental biochemical reactions: the translation of a messenger RNA into its encoded protein by the ribosome. PMID:21685361

  11. 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

  12. 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. PMID:25500006

  13. Aggregation and folding phase transitions of RNA molecules

    NASA Astrophysics Data System (ADS)

    Bundschuh, Ralf

    2007-03-01

    RNA is a biomolecule that is involved in nearly all aspects of cellular functions. In order to perform many of these functions, RNA molecules have to fold into specific secondary structures. This folding is driven by the tendency of the bases to form Watson-Crick base pairs. Beyond the biological importance of RNA, the relatively simple rules for structure formation of RNA make it a very interesting system from the statistical physics point of view. We will present examples of phase transitions in RNA secondary structure formation that are amenable to analytical descriptions. A special focus will be on aggregation between several RNA molecules which is important for some regulatory circuits based on RNA structure, triplet repeat diseases like Huntington's, and as a model for prion diseases. We show that depending on the relative strength of the intramolecular and the intermolecular base pairing, RNA molecules undergo a transition into an aggregated phase and quantitatively characterize this transition.

  14. Visualizing large RNA molecules in solution

    PubMed Central

    Gopal, Ajaykumar; Zhou, Z. Hong; Knobler, Charles M.; Gelbart, William M.

    2012-01-01

    Single-stranded RNAs (ssRNAs) longer than a few hundred nucleotides do not have a unique structure in solution. Their equilibrium properties therefore reflect the average of an ensemble of structures. We use cryo-electron microscopy to image projections of individual long ssRNA molecules and characterize the anisotropy of their ensembles in solution. A flattened prolate volume is found to best represent the shapes of these ensembles. The measured sizes and anisotropies are in good agreement with complementary determinations using small-angle X-ray scattering and coarse-grained molecular dynamics simulations. A long viral ssRNA is compared with shorter noncoding transcripts to demonstrate that prolate geometry and flatness are generic properties independent of sequence length and origin. The anisotropy persists under physiological as well as low-ionic-strength conditions, revealing a direct correlation between secondary structure asymmetry and 3D shape and size. We discuss the physical origin of the generic anisotropy and its biological implications. PMID:22190747

  15. Small Molecule-Mediated Cleavage of RNA in Living Cells

    PubMed Central

    Guan, Lirui

    2013-01-01

    Antisense oligonucleotides and small interfering RNAs (siRNAs) control gene expression by triggering the degradation of a mRNA via recruitment of RNase H or the RNA-induced silencing complex (RISC), respectively.[1] These approaches are hampered, however, by the poor cellular permeability of oligonucleotides. A small molecule approach to cleave RNA targets could obviate uptake issues. Several compounds can induce RNA cleavage in vitro,[2] however, to the best of our knowledge no small molecules have been previously described to cleave RNA in living cells. Herein, we describe the development of a potentially general approach to design small molecules that specifically cleave an RNA in a living cell, affecting biological function. Specifically, a designed, modularly assembled small molecule that binds the RNA that causes myotonic dystrophy type 1 (DM1)[3] was appended with a moiety that generates hydroxyl radicals upon irradiation. Cleavage of the transcript improves DM1-associated defects in cell culture, and compounds are non-toxic at an efficacious dose as determined by a MTT viability assay. This approach may allow for the site-specific cleavage and inactivation of other cellular RNAs.[4] Compounds that bind to and cleave RNA have the potential to serve as chemical genetics probes of function or lead therapeutics with spatial and temporal control. PMID:23280953

  16. Frameworks for programming biological function through RNA parts and devices

    PubMed Central

    Win, Maung Nyan; Liang, Joe C.; Smolke, Christina D.

    2009-01-01

    One of the long-term goals of synthetic biology is to reliably engineer biological systems that perform human-defined functions. Currently, researchers face several scientific and technical challenges in designing and building biological systems, one of which is associated with our limited ability to access, transmit, and control molecular information through the design of functional biomolecules exhibiting novel properties. The fields of RNA biology and nucleic acid engineering, along with the tremendous interdisciplinary growth of synthetic biology, are fueling advances in the emerging field of RNA programming in living systems. Researchers are designing functional RNA molecules that exhibit increasingly complex functions and integrating these molecules into cellular circuits to program higher-level biological functions. The continued integration and growth of RNA design and synthetic biology presents exciting potential to transform how we interact with and program biology. PMID:19318211

  17. tRNA--the golden standard in molecular biology.

    PubMed

    Barciszewska, Mirosława Z; Perrigue, Patrick M; Barciszewski, Jan

    2016-01-01

    Transfer RNAs (tRNAs) represent a major class of RNA molecules. Their primary function is to help decode a messenger RNA (mRNA) sequence in order to synthesize protein and thus ensures the precise translation of genetic information that is imprinted in DNA. The discovery of tRNA in the late 1950's provided critical insight into a genetic machinery when little was known about the central dogma of molecular biology. In 1965, Robert Holley determined the first nucleotide sequence of alanine transfer RNA (tRNA(Ala)) which earned him the 1968 Nobel Prize in Physiology or Medicine. Today, tRNA is one of the best described and characterized biological molecules. Here we review some of the key historical events in tRNA research which led to breakthrough discoveries and new developments in molecular biology. PMID:26549858

  18. Covalent small-molecule-RNA complex formation enables cellular profiling of small-molecule-RNA interactions

    PubMed Central

    Guan, Lirui

    2013-01-01

    Won’t let you go! A strategy is described to design small molecules that react with their cellular RNA targets. This approach not only improves the activity of compounds targeting RNA in cell culture by ≈2500-fold but also enables cell-wide profiling of its RNA targets. PMID:23913698

  19. Covalent small-molecule-RNA complex formation enables cellular profiling of small-molecule-RNA interactions.

    PubMed

    Guan, Lirui; Disney, Matthew D

    2013-09-16

    Won't let you go! A strategy is described to design small molecules that react with their cellular RNA targets. This approach not only improves the activity of compounds targeting RNA in cell culture by a factor of about 2500 but also enables cell-wide profiling of its RNA targets. PMID:23913698

  20. Geochemical Origin of Biological Molecules

    NASA Astrophysics Data System (ADS)

    Bassez, Marie-Paule

    2013-04-01

    A model for the geochemical origin of biological molecules is presented. Rocks such as peridotites and basalts, which contain ferromagnesian minerals, evolve in the presence of water. Their hydrolysis is an exothermic reaction which generates heat and a release of H2 and of minerals with modified structures. The hydrogen reacts with the CO2 embedded inside the rock or with the CO2 of the environment to form CO in an hydrothermal process. With the N2 of the environment, and with an activation source arising from cosmic radiation, ferromagnesian rocks might evolve towards the abiotic formation of biological molecules, such as peptide like macromolecules which produce amino acids after acid hydrolysis. The reactions concerned are described. The production of hydrothermal CO is discussed in geological sites containing ferromagnesian silicate minerals and the low intensity of the Earth's magnetic field during Paleoarchaean Era is also discussed. It is concluded that excitation sources arising from cosmic radiation were much more abundant during Paleoarchaean Era and that macromolecular structures of biological relevance might consequently form during Archaean Eon, as a product of the chemical evolution of the rocks and of their mineral contents. This synthesis of abiotically formed biological molecules is consecutively discussed for meteorites and other planets such as Mars. This model for the geochemical origin of biological molecules has first been proposed in 2008 in the context of reactions involving catalysers such as kaolinite [Bassez 2008a] and then presented in conferences and articles [Bassez 2008b, 2009, 2012; Bassez et al. 2009a to 2012b]. BASSEZ M.P. 2008a Synthèse prébiotique dans les conditions hydrothermales, CNRIUT'08, Lyon 29-30/05/2008, Conf. and open access article:http://liris.cnrs.fr/~cnriut08/actes/ 29 mai 11h-12h40. BASSEZ M.P. 2008b Prebiotic synthesis under hydrothermal conditions, ISSOL'08, P2-6, Firenze-Italy, 24-29/08/2008. Poster at the

  1. Combinatorial analysis of interacting RNA molecules.

    PubMed

    Li, Thomas J X; Reidys, Christian M

    2011-09-01

    Recently several minimum free energy (MFE) folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Their folding targets are interaction structures, that can be represented as diagrams with two backbones drawn horizontally on top of each other such that (1) intramolecular and intermolecular bonds are noncrossing and (2) there is no "zigzag" configuration. This paper studies joint structures with arc-length at least four in which both, interior and exterior stack-lengths are at least two (no isolated arcs). The key idea in this paper is to consider a new type of shape, based on which joint structures can be derived via symbolic enumeration. Our results imply simple asymptotic formulas for the number of joint structures with surprisingly small exponential growth rates. They are of interest in the context of designing prediction algorithms for RNA-RNA interactions. PMID:21689666

  2. Bringing RNA into View: RNA and Its Roles in Biology.

    ERIC Educational Resources Information Center

    Atkins, John F.; Ellington, Andrew; Friedman, B. Ellen; Gesteland, Raymond F.; Noller, Harry F.; Pasquale, Stephen M.; Storey, Richard D.; Uhlenbeck, Olke C.; Weiner, Alan M.

    This guide presents a module for college students on ribonucleic acid (RNA) and its role in biology. The module aims to integrate the latest research and its findings into college-level biology and provide an opportunity for students to understand biological processes. Four activities are presented: (1) "RNA Structure: Tapes to Shapes"; (2) "RNA…

  3. RNA aptamers for an essential prebiotic molecule: adenine

    NASA Astrophysics Data System (ADS)

    Meli, M.; Vergne, J.; Josse, T.; Décout, J.-L.; Maurel, M.-C.

    2001-08-01

    Among all known bio-organic molecules within the living cells, RNA molecules are the only ones storing genetic information and performing catalysis. The RNA world hypthesis assumes that livings on earth are derived from an RNA molecular ancestor where RNA both stored the genetic information and catalyzed the first metabolic reactions. Among diverse RNA worlds proposed, it is thought that the invention of translation and encoded peptide synthesis took place with a "breakthrough organism", then giving rise to a ribonucleoprotein (RNP) world. Finally, modern biochemistry arose with the invention of DNA and the birth of modern molecular biology where the information flows from DNA to RNA which directs protein synthesis. Considering modern metabolism, it is possible to assign biochemical traits to the last common ancestor by simple parsimony rules, and assumptions about earlier metabolisms are possible using chemical criteria. According to this point of view, modern metabolism is considered as a palimpsest that has to be read and deciphered in ordered to understand its origin and evolution.

  4. Helicase-mediated changes in RNA structure at the single-molecule level

    PubMed Central

    König, Sebastian L.B.; Liyanage, Pramodha S.; Sigel, Roland K.O.; Rueda, David

    2013-01-01

    RNA helicases are a diverse group of RNA-dependent ATPases known to play a large number of biological roles inside the cell, such as RNA unwinding, remodeling, export and degradation. Understanding how helicases mediate changes in RNA structure is therefore of fundamental interest. The advent of single-molecule spectroscopic techniques has unveiled with unprecedented detail the interplay of RNA helicases with their substrates. In this review, we describe the characterization of helicase-RNA interactions by single-molecule approaches. State-of-the-art techniques are presented, followed by a discussion of recent advancements in this exciting field. PMID:23353571

  5. Single molecule nanometry for biological physics

    PubMed Central

    Kim, Hajin; Ha, Taekjip

    2013-01-01

    Precision measurement is a hallmark of physics but the small length scale (~ nanometer) of elementary biological components and thermal fluctuations surrounding them challenge our ability to visualize their action. Here, we highlight the recent developments in single molecule nanometry where the position of a single fluorescent molecule can be determined with nanometer precision, reaching the limit imposed by the shot noise, and the relative motion between two molecules can be determined with ~ 0.3 nm precision at ~ 1 millisecond time resolution, and how these new tools are providing fundamental insights on how motor proteins move on cellular highways. We will also discuss how interactions between three and four fluorescent molecules can be used to measure three and six coordinates, respectively, allowing us to correlate movements of multiple components. Finally, we will discuss recent progress in combining angstrom precision optical tweezers with single molecule fluorescent detection, opening new windows for multi-dimensional single molecule nanometry for biological physics. PMID:23249673

  6. 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. PMID:22185671

  7. Biology Today: Respect for RNA.

    ERIC Educational Resources Information Center

    Flannery, Maura C., Ed.

    1991-01-01

    The high points of the story of RNA are presented. The functions of RNA within the cell, how these functions are carried out, and how they evolved are described. The topics of splicing, self-splicing, RNA editing, transcription and translation, and antisense RNA are discussed. (KR)

  8. Halogen bonds in biological molecules

    PubMed Central

    Auffinger, Pascal; Hays, Franklin A.; Westhof, Eric; Ho, P. Shing

    2004-01-01

    Short oxygen–halogen interactions have been known in organic chemistry since the 1950s and recently have been exploited in the design of supramolecular assemblies. The present survey of protein and nucleic acid structures reveals similar halogen bonds as potentially stabilizing inter- and intramolecular interactions that can affect ligand binding and molecular folding. A halogen bond in biomolecules can be defined as a short CX···OY interaction (CX is a carbon-bonded chlorine, bromine, or iodine, and OY is a carbonyl, hydroxyl, charged carboxylate, or phosphate group), where the X···O distance is less than or equal to the sums of the respective van der Waals radii (3.27 Å for Cl···O, 3.37Å for Br···O, and 3.50 Å for I···O) and can conform to the geometry seen in small molecules, with the CX···O angle ≈165° (consistent with a strong directional polarization of the halogen) and the X···OY angle ≈120°. Alternative geometries can be imposed by the more complex environment found in biomolecules, depending on which of the two types of donor systems are involved in the interaction: (i) the lone pair electrons of oxygen (and, to a lesser extent, nitrogen and sulfur) atoms or (ii) the delocalized π -electrons of peptide bonds or carboxylate or amide groups. Thus, the specific geometry and diversity of the interacting partners of halogen bonds offer new and versatile tools for the design of ligands as drugs and materials in nanotechnology. PMID:15557000

  9. Halogen bonds in biological molecules.

    PubMed

    Auffinger, Pascal; Hays, Franklin A; Westhof, Eric; Ho, P Shing

    2004-11-30

    Short oxygen-halogen interactions have been known in organic chemistry since the 1950s and recently have been exploited in the design of supramolecular assemblies. The present survey of protein and nucleic acid structures reveals similar halogen bonds as potentially stabilizing inter- and intramolecular interactions that can affect ligand binding and molecular folding. A halogen bond in biomolecules can be defined as a short C-X...O-Y interaction (C-X is a carbon-bonded chlorine, bromine, or iodine, and O-Y is a carbonyl, hydroxyl, charged carboxylate, or phosphate group), where the X...O distance is less than or equal to the sums of the respective van der Waals radii (3.27 A for Cl...O, 3.37 A for Br...O, and 3.50 A for I...O) and can conform to the geometry seen in small molecules, with the C-X...O angle approximately 165 degrees (consistent with a strong directional polarization of the halogen) and the X...O-Y angle approximately 120 degrees . Alternative geometries can be imposed by the more complex environment found in biomolecules, depending on which of the two types of donor systems are involved in the interaction: (i) the lone pair electrons of oxygen (and, to a lesser extent, nitrogen and sulfur) atoms or (ii) the delocalized pi -electrons of peptide bonds or carboxylate or amide groups. Thus, the specific geometry and diversity of the interacting partners of halogen bonds offer new and versatile tools for the design of ligands as drugs and materials in nanotechnology. PMID:15557000

  10. Auxin biology revealed by small molecules.

    PubMed

    Ma, Qian; Robert, Stéphanie

    2014-05-01

    The plant hormone auxin regulates virtually every aspect of plant growth and development and unraveling its molecular and cellular modes of action is fundamental for plant biology research. Chemical genomics is the use of small molecules to modify protein functions. This approach currently rises as a powerful technology for basic research. Small compounds with auxin-like activities or affecting auxin-mediated biological processes have been widely used in auxin research. They can serve as a tool complementary to genetic and genomic methods, facilitating the identification of an array of components modulating auxin metabolism, transport and signaling. The employment of high-throughput screening technologies combined with informatics-based chemical design and organic chemical synthesis has since yielded many novel small molecules with more instantaneous, precise and specific functionalities. By applying those small molecules, novel molecular targets can be isolated to further understand and dissect auxin-related pathways and networks that otherwise are too complex to be elucidated only by gene-based methods. Here, we will review examples of recently characterized molecules used in auxin research, highlight the strategies of unraveling the mechanisms of these small molecules and discuss future perspectives of small molecule applications in auxin biology. PMID:24252105

  11. Linking RNA biology to lncRNAs

    PubMed Central

    Goff, Loyal A.; Rinn, John L.

    2015-01-01

    The regulatory potential of RNA has never ceased to amaze: from RNA catalysis, to RNA-mediated splicing, to RNA-based silencing of an entire chromosome during dosage compensation. More recently, thousands of long noncoding RNA (lncRNA) transcripts have been identified, the majority with unknown function. Thus, it is tempting to think that these lncRNAs represent a cadre of new factors that function through ribonucleic mechanisms. Some evidence points to several lncRNAs with tantalizing physiological contributions and thought-provoking molecular modalities. However, dissecting the RNA biology of lncRNAs has been difficult, and distinguishing the independent contributions of functional RNAs from underlying DNA elements, or the local act of transcription, is challenging. Here, we aim to survey the existing literature and highlight future approaches that will be needed to link the RNA-based biology and mechanisms of lncRNAs in vitro and in vivo. PMID:26430155

  12. Low energy positron interactions with biological molecules

    NASA Astrophysics Data System (ADS)

    Wanniarachchi, Indika L.

    Calculations of the positron density distribution which can be used for positrons bound to midsize and larger molecules have been tested for smaller molecules and subsequently applied to investigate the most likely e +e-- annihilation sites for positrons interacting with biological molecules containing C, H, O, and N. In order to allow consideration of positrons bound to extended molecules with regions of different character and no particular symmetry, atom-centered positron basis sets of Gaussian-type functions were developed for positrons bound to molecules containing O, N, C, H, Li, Na, and Be. Testing shows that there is no need to scale the positron basis functions to take into account different effective charges on the atoms in different molecules. Even at the HF level of theory the calculated positron and the contact density of e+LiH system is in qualitative agreement with the most accurate calculation was done in ECG method. Also it has been found that for larger biological molecules such as derivation of formaldehyde can leave out positron basis sets centered on H atoms and still get qualitatively acceptable contact density distribution. According to our results, the electronic and positronic wavefunctions have the most overlap in the regions of most negative electrostatic potential in the parent molecule, and we can expect that a positron bound to the molecule will be more likely to annihilate with one of the electrons in these regions. Also we find that the highest energy occupied electronic orbital often does not make the largest contribution to e+e -- annihilation, and that the energy liberated by subsequent electronic relaxation is sufficient to break the backbone in several places in di-peptides and other organic molecules.

  13. Toward reprogramming bacteria with small molecules and RNA.

    PubMed

    Gallivan, Justin P

    2007-12-01

    A major goal of synthetic biology is to reprogram bacteria to carry out complex tasks, such as synthesizing and delivering drugs, and seeking and destroying environmental pollutants. Advances in molecular biology and bacterial genetics have made it straightforward to modify, insert, or delete genes in many bacterial strains, and advances in gene synthesis have opened the door to replacing entire genomes. However, rewriting the underlying genetic code is only part of the challenge of reprogramming cellular behavior. A remaining challenge is to control how and when the modified genes are expressed. Several recent studies have highlighted how synthetic riboswitches, which are RNA sequences that undergo a ligand-induced conformational change to alter gene expression, can be used to reprogram how bacteria respond to small molecules. PMID:17967431

  14. Toward Reprogramming Bacteria with Small Molecules and RNA

    PubMed Central

    Gallivan, Justin P.

    2007-01-01

    Summary A major goal of synthetic biology is to reprogram bacteria to carry out complex tasks, such as synthesizing and delivering drugs, and seeking and destroying environmental pollutants. Advances in molecular biology and bacterial genetics have made it straightforward to modify, insert, or delete genes in many bacterial strains, and advances in gene synthesis have opened the door to replacing entire genomes. However, rewriting the underlying genetic code is only part of the challenge of reprogramming cellular behavior. A remaining challenge is to control how and when the modified genes are expressed. Several recent studies have highlighted how synthetic riboswitches, which are RNA sequences that undergo a ligand-induced conformational change to alter gene expression, can be used to reprogram how bacteria respond to small molecules. PMID:17967431

  15. Transfer RNA: From pioneering crystallographic studies to contemporary tRNA biology.

    PubMed

    Fernández-Millán, Pablo; Schelcher, Cédric; Chihade, Joseph; Masquida, Benoît; Giegé, Philippe; Sauter, Claude

    2016-07-15

    Transfer RNAs (tRNAs) play a key role in protein synthesis as adaptor molecules between messenger RNA and protein sequences on the ribosome. Their discovery in the early sixties provoked a worldwide infatuation with the study of their architecture and their function in the decoding of genetic information. tRNAs are also emblematic molecules in crystallography: the determination of the first tRNA crystal structures represented a milestone in structural biology and tRNAs were for a long period the sole source of information on RNA folding, architecture, and post-transcriptional modifications. Crystallographic data on tRNAs in complex with aminoacyl-tRNA synthetases (aaRSs) also provided the first insight into protein:RNA interactions. Beyond the translation process and the history of structural investigations on tRNA, this review also illustrates the renewal of tRNA biology with the discovery of a growing number of tRNA partners in the cell, the involvement of tRNAs in a variety of regulatory and metabolic pathways, and emerging applications in biotechnology and synthetic biology. PMID:26968773

  16. A chemical screen for biological small molecule–RNA conjugates reveals CoA-linked RNA

    PubMed Central

    Kowtoniuk, Walter E.; Shen, Yinghua; Heemstra, Jennifer M.; Agarwal, Isha; Liu, David R.

    2009-01-01

    Compared with the rapidly expanding set of known biological roles for RNA, the known chemical diversity of cellular RNA has remained limited primarily to canonical RNA, 3′-aminoacylated tRNAs, nucleobase-modified RNAs, and 5′-capped mRNAs in eukaryotes. We developed two methods to detect in a broad manner chemically labile cellular small molecule–RNA conjugates. The methods were validated by the detection of known tRNA and rRNA modifications. The first method analyzes small molecules cleaved from RNA by base or nucleophile treatment. Application to Escherichia coli and Streptomyces venezuelae RNA revealed an RNA-linked hydroxyfuranone or succinyl ester group, in addition to a number of other putative small molecule–RNA conjugates not previously reported. The second method analyzes nuclease-generated mononucleotides before and after treatment with base or nucleophile and also revealed a number of new putative small molecule–RNA conjugates, including 3′-dephospho-CoA and its succinyl-, acetyl-, and methylmalonyl-thioester derivatives. Subsequent experiments established that these CoA species are attached to E. coli and S. venezuelae RNA at the 5′ terminus. CoA-linked RNA cannot be generated through aberrant transcriptional initiation by E. coli RNA polymerase in vitro, and CoA-linked RNA in E. coli is only found among smaller (≲200 nucleotide) RNAs that have yet to be identified. These results provide examples of small molecule-RNA conjugates and suggest that the chemical diversity of cellular RNA may be greater than previously understood. PMID:19416889

  17. Synthesis of biological molecules on molecular sieves.

    PubMed

    Poncelet, G; Van Assche, A T; Fripiat, J J

    1975-07-01

    Catalytic properties of aluminosilicates may play a role in the synthesis of biological molecules from simple gaseous molecules commonly found in planetary atmospheres. Urea, amino acids and UV absorbing substances have been obtained by heating CO and NH3 with Linde molecular sieves saturated with Ca+2, NH4+ or Fe+3. The yields of amino acids produced have been determined by an amino acid analyzer. The quantity of urea produced largely depends on the nature of the saturating cation. Experiments using 14CO confirm that the amino acids are not due to contaminants adsorbed on the surface of the molecular sieves. PMID:171609

  18. Energy landscape exploration of the folding processes of biological molecules

    NASA Astrophysics Data System (ADS)

    Engel, Megan Clare

    For decades, scientists from every discipline have struggled to understand the mechanism of biological self-assembly, which allows proteins and nucleic acids to fold reliably into functional three-dimensional structures. Such an understanding may hold the key to eliminating diseases such as Alzheimer's and Parkinson's and to effective protein engineering. The current best framework for describing biological folding processes is that of statistical mechanical energy landscape theory, and one of the most promising experimental techniques for exploring molecular energy landscapes is single molecule force spectroscopy (SMFS), in which molecules are mechanically denatured. Theoretical advances have enabled the extraction of complete energy landscape profiles from SMFS data. Here, SMFS experiments performed using laser optical tweezers are analyzed to yield the first ever full landscape profile for an RNA pseudoknot. Further, a promising novel landscape reconstruction technique is validated for the first time using experimental data from a DNA hairpin.

  19. Characterizing 3D RNA structure by single molecule FRET.

    PubMed

    Stephenson, James D; Kenyon, Julia C; Symmons, Martyn F; Lever, Andrew M L

    2016-07-01

    The importance of elucidating the three dimensional structures of RNA molecules is becoming increasingly clear. However, traditional protein structural techniques such as NMR and X-ray crystallography have several important drawbacks when probing long RNA molecules. Single molecule Förster resonance energy transfer (smFRET) has emerged as a useful alternative as it allows native sequences to be probed in physiological conditions and allows multiple conformations to be probed simultaneously. This review serves to describe the method of generating a three dimensional RNA structure from smFRET data from the biochemical probing of the secondary structure to the computational refinement of the final model. PMID:26853327

  20. 5-methylcytosine in RNA: detection, enzymatic formation and biological functions

    PubMed Central

    Motorin, Yuri; Lyko, Frank; Helm, Mark

    2010-01-01

    The nucleobase modification 5-methylcytosine (m5C) is widespread both in DNA and different cellular RNAs. The functions and enzymatic mechanisms of DNA m5C-methylation were extensively studied during the last decades. However, the location, the mechanism of formation and the cellular function(s) of the same modified nucleobase in RNA still remain to be elucidated. The recent development of a bisulfite sequencing approach for efficient m5C localization in various RNA molecules puts ribo-m5C in a highly privileged position as one of the few RNA modifications whose detection is amenable to PCR-based amplification and sequencing methods. Additional progress in the field also includes the characterization of several specific RNA methyltransferase enzymes in various organisms, and the discovery of a new and unexpected link between DNA and RNA m5C-methylation. Numerous putative RNA:m5C-MTases have now been identified and are awaiting characterization, including the identification of their RNA substrates and their related cellular functions. In order to bring these recent exciting developments into perspective, this review provides an ordered overview of the detection methods for RNA methylation, of the biochemistry, enzymology and molecular biology of the corresponding modification enzymes, and discusses perspectives for the emerging biological functions of these enzymes. PMID:20007150

  1. Did the Pre-RNA World Rest Upon DNA Molecules?

    NASA Technical Reports Server (NTRS)

    Lazcano, Antonio; Dworkin, Jason P.; Miller, Stanley L.

    2004-01-01

    The isolation of a DNA sequence that catalyzes the ligation of oligodeoxynucleotides via the formation of 3' - 5' phosphodiester linkage significance in selection experiments has been reported. Ball recently used this to discuss the possibility that natural DNA molecules may have formed in the primitive Earth leading to the origin of life. As noted by Ferris and Usher, if metabolic pathways evolved backwards, it could be argued that the biosynthesis of 2-deoxyribose from ribose suggests that RNA came from DNA. As summarized elsewhere, there are several properties of deoxyribose which could be interpreted to support the possibility that DNA-like molecules arose prior to the RNA world. For example, 2-deoxyribose is slightly more soluble than ribose (which may have been an advantage in a drying pool scenario), may have been more reactive under possible prebiotic conditions (it forms a nucleoside approx. 150 times faster than ribose with the alternative base urazole at 25 C), while it decomposes in solution (approximately 2.6 times more slowly than ribose at 100 C). Other advantages of DNA over RNA are that it has one fewer chiral center, has a greater stability at the 8.2 pH value of the current oceans, and does not has the 2'5' and 3'5' ambiguity in polymerizations. Yet, there is strong molecular biological and biochemical evidence that RNA was featured in the biology well before the last common ancestor. The presence of sugar acids, including both ribo- and deoxysugar acids, in the 4.6 Ga old Murchison meteorite suggest that both may have been available in the primitive Earth, derived from the accretion of extraterrestrial sources and/or from endogenous processes involving formaldehyde and its derivatives. However, the abiotic synthesis of deoxyribose, ribose, and other sugars from glyceraldehyde and acetaldehyde under alkaline conditions is inefficient and unespecific. Although sugars are labile compounds, the role of cyanamide or borate minerals in the

  2. Single Molecule Approaches in RNA-Protein Interactions.

    PubMed

    Serebrov, Victor; Moore, Melissa J

    2016-01-01

    RNA-protein interactions govern every aspect of RNA metabolism, and aberrant RNA-binding proteins are the cause of hundreds of genetic diseases. Quantitative measurements of these interactions are necessary in order to understand mechanisms leading to diseases and to develop efficient therapies. Existing methods of RNA-protein interactome capture can afford a comprehensive snapshot of RNA-protein interaction networks but lack the ability to characterize the dynamics of these interactions. As all ensemble methods, their resolution is also limited by statistical averaging. Here we discuss recent advances in single molecule techniques that have the potential to tackle these challenges. We also provide a thorough overview of single molecule colocalization microscopy and the essential protein and RNA tagging and detection techniques. PMID:27256383

  3. Small Molecule Ligands for Bulged RNA Secondary Structures

    PubMed Central

    Meyer, S. Todd; Hergenrother, Paul J.

    2016-01-01

    A class of wedge-shaped small molecules has been designed, synthesized, and shown to bind bulged RNA secondary structures. These minimally cationic ligands exhibit good affinity and selectivity for certain RNA bulges as demonstrated in a fluorescent intercalator displacement assay. PMID:19678613

  4. Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device

    PubMed Central

    Smith, Andrew M.; Abu-Shumays, Robin; Akeson, Mark; Bernick, David L.

    2015-01-01

    Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which acts as a brake on the translocating tRNA. This increases the dwell time of adapted tRNA in the nanopore, allowing us to identify the region of the nanopore signal that is produced by the translocating tRNA itself. Using adapter-modified Escherichia coli tRNAfMet and tRNALys, we show that the nanopore signal during controlled translocation is dependent on the identity of the tRNA. This confirms that adapter-modified tRNA can translocate end-to-end through nanopores and provide the foundation for future work in direct sequencing of individual transfer RNA with a nanopore-based device. PMID:26157798

  5. RNA and RNP as Building Blocks for Nanotechnology and Synthetic Biology.

    PubMed

    Ohno, Hirohisa; Saito, Hirohide

    2016-01-01

    Recent technologies that aimed to elucidate cellular function have revealed essential roles for RNA molecules in living systems. Our knowledge concerning functional and structural information of naturally occurring RNA and RNA-protein (RNP) complexes is increasing rapidly. RNA and RNP interaction motifs are structural units that function as building blocks to constitute variety of complex structures. RNA-central synthetic biology and nanotechnology are constructive approaches that employ the accumulated information and build synthetic RNA (RNP)-based circuits and nanostructures. Here, we describe how to design and construct synthetic RNA (RNP)-based devices and structures at the nanometer-scale for biological and future therapeutic applications. RNA/RNP nanostructures can also be utilized as the molecular scaffold to control the localization or interactions of target molecule(s). Moreover, RNA motifs recognized by RNA-binding proteins can be applied to make protein-responsive translational "switches" that can turn gene expression "on" or "off" depending on the intracellular environment. This "synthetic RNA and RNP world" will expand tools for nanotechnology and synthetic biology. In addition, these reconstructive approaches would lead to a greater understanding of building principle in naturally occurring RNA/RNP molecules and systems. PMID:26970194

  6. Carotenoids as signaling molecules in cardiovascular biology

    PubMed Central

    Barzegari, Abolfazl; Pavon-Djavid, Graciela

    2014-01-01

    Oxidative stress and inflammation play important roles in the etiology of cardiovascular disease (CVD). Thus, natural antioxidant carotenoids existing in fruits and vegetables could have a significant role in the prevention of CVD. Nevertheless, clinical data are conflicting about the positive effect of some antioxidant carotenoids in reducing cardiovascular morbidity and mortality. Many biological actions of carotenoids have been attributed to their antioxidant effect; however, the precise mechanism by which carotenoids produce their beneficial effects is still under discussion. They might modulate molecular pathways involved in cell proliferation, acting at Akt, tyrosine kinases, mitogen activated protein kinase (MAP kinase) and growth factor signaling cascades. Screening for a promising cardiovascular protective carotenoids therefore might be performed in vitro and in vivo with caution in cross-interaction with other molecules involved in signaling pathways especially those affecting microRNAs, performing a role in molecular modulation of cardiovascular cells. PMID:25337462

  7. Mean-field theory of planar absorption of RNA molecules

    NASA Astrophysics Data System (ADS)

    Nguyen, Toan; Bruinsma, Robijn; Gelbart, William

    2006-03-01

    Interaction between the viral RNA and the protective protein capsid plays a very important role in the cell infection and self-assembly process of a virus. To better understand this interaction, we study a similar problem of absorption of RNA on an attractive wall. It is known that the secondary structure of a folded RNA molecules without pseudo-knots has the same topology as that of a branched polymer. We use a mean-field theory for branched polymers to analytically calculate the RNA concentration profile. The results are compared to known exact scaling calculations and computer simulations.

  8. Design of a small molecule against an oncogenic noncoding RNA.

    PubMed

    Velagapudi, Sai Pradeep; Cameron, Michael D; Haga, Christopher L; Rosenberg, Laura H; Lafitte, Marie; Duckett, Derek R; Phinney, Donald G; Disney, Matthew D

    2016-05-24

    The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm. PMID:27170187

  9. Origins and Early Evolution of the tRNA Molecule

    PubMed Central

    Tamura, Koji

    2015-01-01

    Modern transfer RNAs (tRNAs) are composed of ~76 nucleotides and play an important role as “adaptor” molecules that mediate the translation of information from messenger RNAs (mRNAs). Many studies suggest that the contemporary full-length tRNA was formed by the ligation of half-sized hairpin-like RNAs. A minihelix (a coaxial stack of the acceptor stem on the T-stem of tRNA) can function both in aminoacylation by aminoacyl tRNA synthetases and in peptide bond formation on the ribosome, indicating that it may be a vestige of the ancestral tRNA. The universal CCA-3′ terminus of tRNA is also a typical characteristic of the molecule. “Why CCA?” is the fundamental unanswered question, but several findings give a comprehensive picture of its origin. Here, the origins and early evolution of tRNA are discussed in terms of various perspectives, including nucleotide ligation, chiral selectivity of amino acids, genetic code evolution, and the organization of the ribosomal peptidyl transferase center (PTC). The proto-tRNA molecules may have evolved not only as adaptors but also as contributors to the composition of the ribosome. PMID:26633518

  10. Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules.

    PubMed

    McDonald, James E; Larsen, Niels; Pennington, Andrea; Connolly, John; Wallis, Corrin; Rooks, David J; Hall, Neil; McCarthy, Alan J; Allison, Heather E

    2016-01-01

    PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, 'universal' SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample. In addition, we have developed BIONmeta, a novel, open-source, computer package for the processing and taxonomic classification of the randomly fragmented RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that 16S rRNA molecules belonging to the bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome (92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detected greater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera) when compared with general bacterial 16S rRNA amplicons from the same sample, simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya, and detected significant numbers of sequences not recognised by 'universal' primer sets. Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology. PMID:27276347

  11. Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules

    PubMed Central

    McDonald, James E.; Larsen, Niels; Pennington, Andrea; Connolly, John; Wallis, Corrin; Rooks, David J.; Hall, Neil; McCarthy, Alan J.; Allison, Heather E.

    2016-01-01

    PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, ‘universal’ SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample. In addition, we have developed BIONmeta, a novel, open-source, computer package for the processing and taxonomic classification of the randomly fragmented RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that 16S rRNA molecules belonging to the bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome (92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detected greater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera) when compared with general bacterial 16S rRNA amplicons from the same sample, simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya, and detected significant numbers of sequences not recognised by ‘universal’ primer sets. Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology. PMID:27276347

  12. The secondary structure of guide RNA molecules from Trypanosoma brucei.

    PubMed Central

    Schmid, B; Riley, G R; Stuart, K; Göringer, H U

    1995-01-01

    RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is characterized by the insertion and deletion of uridine nucleotides into incomplete mRNAs. Key molecules in the process are guide RNAs which direct the editing reaction by virtue of their primary sequences in an RNA-RNA interaction with the pre-edited mRNAs. To understand the molecular details of this reaction, especially potential RNA folding and unfolding processes as well as assembly phenomena with mitochondrial proteins, we analyzed the secondary structure of four different guide RNAs from Trypanosoma brucei at physiological conditions. By using structure-sensitive chemical and enzymatic probes in combination with spectroscopic techniques we found that the four molecules despite their different primary sequences, fold into similar structures consisting of two imperfect hairpin loops of low thermodynamic stability. The molecules melt in two-state monomolecular transitions with Tms between 33 and 39 degrees C and transition enthalpies of -32 to -38 kcal/mol. Both terminal ends of the RNAs are single-stranded with the 3' ends possibly adopting a single-stranded, helical conformation. Thus, it appears that the gRNA structures are fine tuned to minimize stability for an optimal annealing reaction to the pre-mRNAs while at the same time maximizing higher order structural features to permit the assembly with other mitochondrial components into the editing machinery. Images PMID:7667084

  13. Studying a Drug-like, RNA-Focused Small Molecule Library Identifies Compounds That Inhibit RNA Toxicity in Myotonic Dystrophy.

    PubMed

    Rzuczek, Suzanne G; Southern, Mark R; Disney, Matthew D

    2015-12-18

    There are many RNA targets in the transcriptome to which small molecule chemical probes and lead therapeutics are desired. However, identifying compounds that bind and modulate RNA function in cellulo is difficult. Although rational design approaches have been developed, they are still in their infancies and leave many RNAs "undruggable". In an effort to develop a small molecule library that is biased for binding RNA, we computationally identified "drug-like" compounds from screening collections that have favorable properties for binding RNA and for suitability as lead drugs. As proof-of-concept, this collection was screened for binding to and modulating the cellular dysfunction of the expanded repeating RNA (r(CUG)(exp)) that causes myotonic dystrophy type 1. Hit compounds bind the target in cellulo, as determined by the target identification approach Competitive Chemical Cross-Linking and Isolation by Pull-down (C-ChemCLIP), and selectively improve several disease-associated defects. The best compounds identified from our 320-member library are more potent in cellulo than compounds identified by high-throughput screening (HTS) campaigns against this RNA. Furthermore, the compound collection has a higher hit rate (9% compared to 0.01-3%), and the bioactive compounds identified are not charged; thus, RNA can be "drugged" with compounds that have favorable pharmacological properties. Finally, this RNA-focused small molecule library may serve as a useful starting point to identify lead "drug-like" chemical probes that affect the biological (dys)function of other RNA targets by direct target engagement. PMID:26414664

  14. tRNA Biology in Mitochondria

    PubMed Central

    Salinas-Giegé, Thalia; Giegé, Richard; Giegé, Philippe

    2015-01-01

    Mitochondria are the powerhouses of eukaryotic cells. They are considered as semi-autonomous because they have retained genomes inherited from their prokaryotic ancestor and host fully functional gene expression machineries. These organelles have attracted considerable attention because they combine bacterial-like traits with novel features that evolved in the host cell. Among them, mitochondria use many specific pathways to obtain complete and functional sets of tRNAs as required for translation. In some instances, tRNA genes have been partially or entirely transferred to the nucleus and mitochondria require precise import systems to attain their pool of tRNAs. Still, tRNA genes have also often been maintained in mitochondria. Their genetic arrangement is more diverse than previously envisaged. The expression and maturation of mitochondrial tRNAs often use specific enzymes that evolved during eukaryote history. For instance many mitochondria use a eukaryote-specific RNase P enzyme devoid of RNA. The structure itself of mitochondrial encoded tRNAs is also very diverse, as e.g., in Metazoan, where tRNAs often show non canonical or truncated structures. As a result, the translational machinery in mitochondria evolved adapted strategies to accommodate the peculiarities of these tRNAs, in particular simplified identity rules for their aminoacylation. Here, we review the specific features of tRNA biology in mitochondria from model species representing the major eukaryotic groups, with an emphasis on recent research on tRNA import, maturation and aminoacylation. PMID:25734984

  15. Engineering modular 'ON' RNA switches using biological components.

    PubMed

    Ceres, Pablo; Trausch, Jeremiah J; Batey, Robert T

    2013-12-01

    Riboswitches are cis-acting regulatory elements broadly distributed in bacterial mRNAs that control a wide range of critical metabolic activities. Expression is governed by two distinct domains within the mRNA leader: a sensory 'aptamer domain' and a regulatory 'expression platform'. Riboswitches have also received considerable attention as important tools in synthetic biology because of their conceptually simple structure and the ability to obtain aptamers that bind almost any conceivable small molecule using in vitro selection (referred to as SELEX). In the design of artificial riboswitches, a significant hurdle has been to couple the two domains enabling their efficient communication. We previously demonstrated that biological transcriptional 'OFF' expression platforms are easily coupled to diverse aptamers, both biological and SELEX-derived, using simple design rules. Here, we present two modular transcriptional 'ON' riboswitch expression platforms that are also capable of hosting foreign aptamers. We demonstrate that these biological parts can be used to facilely generate artificial chimeric riboswitches capable of robustly regulating transcription both in vitro and in vivo. We expect that these modular expression platforms will be of great utility for various synthetic biological applications that use RNA-based biosensors. PMID:23999097

  16. Engineering modular ‘ON’ RNA switches using biological components

    PubMed Central

    Ceres, Pablo; Trausch, Jeremiah J.; Batey, Robert T.

    2013-01-01

    Riboswitches are cis-acting regulatory elements broadly distributed in bacterial mRNAs that control a wide range of critical metabolic activities. Expression is governed by two distinct domains within the mRNA leader: a sensory ‘aptamer domain’ and a regulatory ‘expression platform’. Riboswitches have also received considerable attention as important tools in synthetic biology because of their conceptually simple structure and the ability to obtain aptamers that bind almost any conceivable small molecule using in vitro selection (referred to as SELEX). In the design of artificial riboswitches, a significant hurdle has been to couple the two domains enabling their efficient communication. We previously demonstrated that biological transcriptional ‘OFF’ expression platforms are easily coupled to diverse aptamers, both biological and SELEX-derived, using simple design rules. Here, we present two modular transcriptional ‘ON’ riboswitch expression platforms that are also capable of hosting foreign aptamers. We demonstrate that these biological parts can be used to facilely generate artificial chimeric riboswitches capable of robustly regulating transcription both in vitro and in vivo. We expect that these modular expression platforms will be of great utility for various synthetic biological applications that use RNA-based biosensors. PMID:23999097

  17. Constructing and characterizing a bioactive small molecule and microRNA association network for Alzheimer's disease

    PubMed Central

    Meng, Fanlin; Dai, Enyu; Yu, Xuexin; Zhang, Yan; Chen, Xiaowen; Liu, Xinyi; Wang, Shuyuan; Wang, Lihua; Jiang, Wei

    2014-01-01

    Alzheimer's disease (AD) is an incurable neurodegenerative disorder. Much effort has been devoted to developing effective therapeutic agents. Recently, targeting microRNAs (miRNAs) with small molecules has become a novel therapy for human diseases. In this study, we present a systematic computational approach to construct a bioactive Small molecule and miRNA association Network in AD (SmiRN-AD), which is based on the gene expression signatures of bioactive small molecule perturbation and AD-related miRNA regulation. We also performed topological and functional analysis of the SmiRN-AD from multiple perspectives. At the significance level of p ≤ 0.01, 496 small molecule–miRNA associations, including 25 AD-related miRNAs and 275 small molecules, were recognized and used to construct the SmiRN-AD. The drugs that were connected with the same miRNA tended to share common drug targets (p = 1.72 × 10−4) and belong to the same therapeutic category (p = 4.22 × 10−8). The miRNAs that were linked to the same small molecule regulated more common miRNA targets (p = 6.07 × 10−3). Further analysis of the positive connections (quinostatin and miR-148b, amantadine and miR-15a) and the negative connections (melatonin and miR-30e-5p) indicated that our large-scale predictions afforded specific biological insights into AD pathogenesis and therapy. This study proposes a holistic strategy for deciphering the associations between small molecules and miRNAs in AD, which may be helpful for developing a novel effective miRNA-associated therapeutic strategy for AD. A comprehensive database for the SmiRN-AD and the differential expression patterns of the miRNA targets in AD is freely available at http://bioinfo.hrbmu.edu.cn/SmiRN-AD/. PMID:24352679

  18. Single-molecule correlated chemical probing of RNA.

    PubMed

    Homan, Philip J; Favorov, Oleg V; Lavender, Christopher A; Kursun, Olcay; Ge, Xiyuan; Busan, Steven; Dokholyan, Nikolay V; Weeks, Kevin M

    2014-09-23

    Complex higher-order RNA structures play critical roles in all facets of gene expression; however, the through-space interaction networks that define tertiary structures and govern sampling of multiple conformations are poorly understood. Here we describe single-molecule RNA structure analysis in which multiple sites of chemical modification are identified in single RNA strands by massively parallel sequencing and then analyzed for correlated and clustered interactions. The strategy thus identifies RNA interaction groups by mutational profiling (RING-MaP) and makes possible two expansive applications. First, we identify through-space interactions, create 3D models for RNAs spanning 80-265 nucleotides, and characterize broad classes of intramolecular interactions that stabilize RNA. Second, we distinguish distinct conformations in solution ensembles and reveal previously undetected hidden states and large-scale structural reconfigurations that occur in unfolded RNAs relative to native states. RING-MaP single-molecule nucleic acid structure interrogation enables concise and facile analysis of the global architectures and multiple conformations that govern function in RNA. PMID:25205807

  19. A small molecule enhances RNA interference and promotes microRNA processing

    PubMed Central

    Shan, Ge; Li, Yujing; Zhang, Junliang; Li, Wendi; Szulwach, Keith E; Duan, Ranhui; Faghihi, Mohammad A; Khalil, Ahmad M; Lu, Lianghua; Paroo, Zain; Chan, Anthony W S; Shi, Zhangjie; Liu, Qinghua; Wahlestedt, Claes; He, Chuan; Jin, Peng

    2010-01-01

    Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are sequence-specific post-transcriptional regulators of gene expression. Although major components of the RNA interference (RNAi) pathway have been identified, regulatory mechanisms for this pathway remain largely unknown. Here we demonstrate that the RNAi pathway can be modulated intracellularly by small molecules. We have developed a cell-based assay to monitor the activity of the RNAi pathway and find that the small-molecule enoxacin (Penetrex) enhances siRNA-mediated mRNA degradation and promotes the biogenesis of endogenous miRNAs. We show that this RNAi-enhancing activity depends on the trans-activation-responsive region RNA-binding protein. Our results provide a proof-of-principle demonstration that small molecules can be used to modulate the activity of the RNAi pathway. RNAi enhancers may be useful in the development of research tools and therapeutics. PMID:18641635

  20. Computer Modelling of Biological Molecules: Free Resources on the Internet.

    ERIC Educational Resources Information Center

    Millar, Neil

    1996-01-01

    Describes a three-dimensional computer modeling system for biological molecules which is suitable for sixth-form teaching. Consists of the modeling program "RasMol" together with structure files of proteins, DNA, and small biological molecules. Describes how the whole system can be downloaded from various sites on the Internet. (Author/JRH)

  1. Suppressing RNA silencing with small molecules and the viral suppressor of RNA silencing protein p19.

    PubMed

    Danielson, Dana C; Filip, Roxana; Powdrill, Megan H; O'Hara, Shifawn; Pezacki, John P

    2015-08-01

    RNA silencing is a gene regulatory and host defense mechanism whereby small RNA molecules are engaged by Argonaute (AGO) proteins, which facilitate gene knockdown of complementary mRNA targets. Small molecule inhibitors of AGO represent a convenient method for reversing this effect and have applications in human therapy and biotechnology. Viral suppressors of RNA silencing, such as p19, can also be used to suppress the pathway. Here we assess the compatibility of these two approaches, by examining whether synthetic inhibitors of AGO would inhibit p19-siRNA interactions. We observe that aurintricarboxylic acid (ATA) is a potent inhibitor of p19's ability to bind siRNA (IC50 = 0.43 μM), oxidopamine does not inhibit p19:siRNA interactions, and suramin is a mild inhibitor of p19:siRNA interactions (IC50 = 430 μM). We observe that p19 and suramin are compatible inhibitors of RNA silencing in human hepatoma cells. Our data suggests that at least some inhibitors of AGO may be used in combination with p19 to inhibit RNA silencing at different points in the pathway. PMID:26079891

  2. 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

  3. Single-molecule Studies of RNA Polymerase: Motoring Along

    PubMed Central

    Herbert, Kristina M.; Greenleaf, William J.; Block, Steven M.

    2010-01-01

    Single-molecule techniques have advanced our understanding of transcription by RNA polymerase. A new arsenal of approaches, including single-molecule fluorescence, atomic-force microscopy, magnetic tweezers, and optical traps have been employed to probe the many facets of the transcription cycle. These approaches supply fresh insights into the means by which RNA polymerase identifies a promoter; initiates transcription, translocates and pauses along the DNA template, proofreads errors, and ultimately terminates transcription. Results from single-molecule experiments complement knowledge gained from biochemical and genetic assays by facilitating the observation of states that are otherwise obscured by ensemble averaging, such as those resulting from heterogeneity in molecular structure, elongation rate, or pause propensity. Most studies to date have been performed with bacterial RNA polymerase, but work is also being carried out with eukaryotic polymerase (Pol II) and single-subunit polymerases from bacteriophages. We discuss recent progress achieved by single-molecule studies, highlighting some of the unresolved questions and ongoing debates. PMID:18410247

  4. 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.

  5. Tracking single mRNA molecules in live cells

    NASA Astrophysics Data System (ADS)

    Moon, Hyungseok C.; Lee, Byung Hun; Lim, Kiseong; Son, Jae Seok; Song, Minho S.; Park, Hye Yoon

    2016-06-01

    mRNAs inside cells interact with numerous RNA-binding proteins, microRNAs, and ribosomes that together compose a highly heterogeneous population of messenger ribonucleoprotein (mRNP) particles. Perhaps one of the best ways to investigate the complex regulation of mRNA is to observe individual molecules. Single molecule imaging allows the collection of quantitative and statistical data on subpopulations and transient states that are otherwise obscured by ensemble averaging. In addition, single particle tracking reveals the sequence of events that occur in the formation and remodeling of mRNPs in real time. Here, we review the current state-of-the-art techniques in tagging, delivery, and imaging to track single mRNAs in live cells. We also discuss how these techniques are applied to extract dynamic information on the transcription, transport, localization, and translation of mRNAs. These studies demonstrate how single molecule tracking is transforming the understanding of mRNA regulation in live cells.

  6. Intramolecular phenotypic capacitance in a modular RNA molecule.

    PubMed

    Hayden, Eric J; Bendixsen, Devin P; Wagner, Andreas

    2015-10-01

    Phenotypic capacitance refers to the ability of a genome to accumulate mutations that are conditionally hidden and only reveal phenotype-altering effects after certain environmental or genetic changes. Capacitance has important implications for the evolution of novel forms and functions, but experimentally studied mechanisms behind capacitance are mostly limited to complex, multicomponent systems often involving several interacting protein molecules. Here we demonstrate phenotypic capacitance within a much simpler system, an individual RNA molecule with catalytic activity (ribozyme). This naturally occurring RNA molecule has a modular structure, where a scaffold module acts as an intramolecular chaperone that facilitates folding of a second catalytic module. Previous studies have shown that the scaffold module is not absolutely required for activity, but dramatically decreases the concentration of magnesium ions required for the formation of an active site. Here, we use an experimental perturbation of magnesium ion concentration that disrupts the folding of certain genetic variants of this ribozyme and use in vitro selection followed by deep sequencing to identify genotypes with altered phenotypes (catalytic activity). We identify multiple conditional mutations that alter the wild-type ribozyme phenotype under a stressful environmental condition of low magnesium ion concentration, but preserve the phenotype under more relaxed conditions. This conditional buffering is confined to the scaffold module, but controls the catalytic phenotype, demonstrating how modularity can enable phenotypic capacitance within a single macromolecule. RNA's ancient role in life suggests that phenotypic capacitance may have influenced evolution since life's origins. PMID:26401020

  7. Folding and unfolding single RNA molecules under tension

    PubMed Central

    Woodside, Michael T; García-García, Cuauhtémoc; Block, Steven M

    2010-01-01

    Single-molecule force spectroscopy constitutes a powerful method for probing RNA folding: it allows the kinetic, energetic, and structural properties of intermediate and transition states to be determined quantitatively, yielding new insights into folding pathways and energy landscapes. Recent advances in experimental and theoretical methods, including fluctuation theorems, kinetic theories, novel force clamps, and ultrastable instruments, have opened new avenues for study. These tools have been used to probe folding in simple model systems, for example, RNA and DNA hairpins. Knowledge gained from such systems is helping to build our understanding of more complex RNA structures composed of multiple elements, as well as how nucleic acids interact with proteins involved in key cellular activities, such as transcription and translation. PMID:18786653

  8. Single-Molecule Electrical Random Resequencing of DNA and RNA

    NASA Astrophysics Data System (ADS)

    Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

    2012-07-01

    Two paradigm shifts in DNA sequencing technologies--from bulk to single molecules and from optical to electrical detection--are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5'-UGAGGUA-3' from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

  9. Single-Molecule Electrical Random Resequencing of DNA and RNA

    PubMed Central

    Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

    2012-01-01

    Two paradigm shifts in DNA sequencing technologies—from bulk to single molecules and from optical to electrical detection—are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5′-UGAGGUA-3′ from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes. PMID:22787559

  10. Mathematical models in biology: from molecules to life

    PubMed Central

    Kaznessis, Yiannis N.

    2011-01-01

    A vexing question in the biological sciences is the following: can biological phenotypes be explained with mathematical models of molecules that interact according to physical laws? At the crux of the matter lies the doubt that humans can develop physically faithful mathematical representations of living organisms. We discuss advantages that synthetic biological systems confer that may help us describe life’s distinctiveness with tractable mathematics that are grounded on universal laws of thermodynamics and molecular biology. PMID:21472998

  11. Computer display and manipulation of biological molecules

    NASA Technical Reports Server (NTRS)

    Coeckelenbergh, Y.; Macelroy, R. D.; Hart, J.; Rein, R.

    1978-01-01

    This paper describes a computer model that was designed to investigate the conformation of molecules, macromolecules and subsequent complexes. Utilizing an advanced 3-D dynamic computer display system, the model is sufficiently versatile to accommodate a large variety of molecular input and to generate data for multiple purposes such as visual representation of conformational changes, and calculation of conformation and interaction energy. Molecules can be built on the basis of several levels of information. These include the specification of atomic coordinates and connectivities and the grouping of building blocks and duplicated substructures using symmetry rules found in crystals and polymers such as proteins and nucleic acids. Called AIMS (Ames Interactive Molecular modeling System), the model is now being used to study pre-biotic molecular evolution toward life.

  12. Biased and unbiased strategies to identify biologically active small molecules.

    PubMed

    Abet, Valentina; Mariani, Angelica; Truscott, Fiona R; Britton, Sébastien; Rodriguez, Raphaël

    2014-08-15

    Small molecules are central players in chemical biology studies. They promote the perturbation of cellular processes underlying diseases and enable the identification of biological targets that can be validated for therapeutic intervention. Small molecules have been shown to accurately tune a single function of pluripotent proteins in a reversible manner with exceptional temporal resolution. The identification of molecular probes and drugs remains a worthy challenge that can be addressed by the use of biased and unbiased strategies. Hypothesis-driven methodologies employs a known biological target to synthesize complementary hits while discovery-driven strategies offer the additional means of identifying previously unanticipated biological targets. This review article provides a general overview of recent synthetic frameworks that gave rise to an impressive arsenal of biologically active small molecules with unprecedented cellular mechanisms. PMID:24811300

  13. Intramolecular phenotypic capacitance in a modular RNA molecule

    PubMed Central

    Hayden, Eric J.; Bendixsen, Devin P.; Wagner, Andreas

    2015-01-01

    Phenotypic capacitance refers to the ability of a genome to accumulate mutations that are conditionally hidden and only reveal phenotype-altering effects after certain environmental or genetic changes. Capacitance has important implications for the evolution of novel forms and functions, but experimentally studied mechanisms behind capacitance are mostly limited to complex, multicomponent systems often involving several interacting protein molecules. Here we demonstrate phenotypic capacitance within a much simpler system, an individual RNA molecule with catalytic activity (ribozyme). This naturally occurring RNA molecule has a modular structure, where a scaffold module acts as an intramolecular chaperone that facilitates folding of a second catalytic module. Previous studies have shown that the scaffold module is not absolutely required for activity, but dramatically decreases the concentration of magnesium ions required for the formation of an active site. Here, we use an experimental perturbation of magnesium ion concentration that disrupts the folding of certain genetic variants of this ribozyme and use in vitro selection followed by deep sequencing to identify genotypes with altered phenotypes (catalytic activity). We identify multiple conditional mutations that alter the wild-type ribozyme phenotype under a stressful environmental condition of low magnesium ion concentration, but preserve the phenotype under more relaxed conditions. This conditional buffering is confined to the scaffold module, but controls the catalytic phenotype, demonstrating how modularity can enable phenotypic capacitance within a single macromolecule. RNA’s ancient role in life suggests that phenotypic capacitance may have influenced evolution since life’s origins. PMID:26401020

  14. Theory of sequence-dependent scaling and confinement of viral RNA molecules

    NASA Astrophysics Data System (ADS)

    Kelly, Joshua

    We present a general theory for the fractal dimensions of the genomic viral RNA molecules of small RNA viruses and apply the theory as well to RNA encapsidation competition experiments. Work supported by NSF.

  15. An in vitro selection for small molecule induced switching RNA molecules.

    PubMed

    Martini, Laura; Ellington, Andrew D; Mansy, Sheref S

    2016-08-15

    The selection of RNA and DNA aptamers now has a long history. However, the ability to directly select for conformational changes upon ligand binding has remained elusive. These difficulties have stymied attempts at making small molecule responsive strand displacement circuitry as well as synthetic riboswitches. Herein we present a detailed strand displacement based selection protocol to directly select for RNA molecules with switching activity. The library was based on a previously selected thiamine pyrophosphate riboswitch. The fully in vitro methodology gave sequences that showed strong strand displacement activity in the presence of thiamine pyrophosphate. Further, the selected sequences possessed riboswitch activity similar to that of natural riboswitches. The presented methodology should aid in the design of more complex, environmentally responsive strand displacement circuitry and in the selection of riboswitches responsive to toxic ligands. PMID:26899430

  16. Gas biology: tiny molecules controlling metabolic systems.

    PubMed

    Kajimura, Mayumi; Nakanishi, Tsuyoshi; Takenouchi, Toshiki; Morikawa, Takayuki; Hishiki, Takako; Yukutake, Yoshinori; Suematsu, Makoto

    2012-11-15

    It has been recognized that gaseous molecules and their signaling cascades play a vital role in alterations of metabolic systems in physiologic and pathologic conditions. Contrary to this awareness, detailed mechanisms whereby gases exert their actions, in particular in vivo, have been unclear because of several reasons. Gaseous signaling involves diverse reactions with metal centers of metalloproteins and thiol modification of cysteine residues of proteins. Both the multiplicity of gas targets and the technical limitations in accessing local gas concentrations make dissection of exact actions of any gas mediator a challenge. However, a series of advanced technologies now offer ways to explore gas-responsive regulatory processes in vivo. Imaging mass spectrometry combined with quantitative metabolomics by capillary-electrophoresis/mass spectrometry reveals spatio-temporal profiles of many metabolites. Comparing the metabolic footprinting of murine samples with a targeted deletion of a specific gas-producing enzyme makes it possible to determine sites of actions of the gas. In this review, we intend to elaborate on the ideas how small gaseous molecules interact with metabolic systems to control organ functions such as cerebral vascular tone and energy metabolism in vivo. PMID:22516267

  17. Synthetic biology devices and circuits for RNA-based 'smart vaccines': a propositional review.

    PubMed

    Andries, Oliwia; Kitada, Tasuku; Bodner, Katie; Sanders, Niek N; Weiss, Ron

    2015-02-01

    Nucleic acid vaccines have been gaining attention as an alternative to the standard attenuated pathogen or protein based vaccine. However, an unrealized advantage of using such DNA or RNA based vaccination modalities is the ability to program within these nucleic acids regulatory devices that would provide an immunologist with the power to control the production of antigens and adjuvants in a desirable manner by administering small molecule drugs as chemical triggers. Advances in synthetic biology have resulted in the creation of highly predictable and modular genetic parts and devices that can be composed into synthetic gene circuits with complex behaviors. With the recent advent of modified RNA gene delivery methods and developments in the RNA replicon platform, we foresee a future in which mammalian synthetic biologists will create genetic circuits encoded exclusively on RNA. Here, we review the current repertoire of devices used in RNA synthetic biology and propose how programmable 'smart vaccines' will revolutionize the field of RNA vaccination. PMID:25566800

  18. Steroid receptor RNA activator: Biologic function and role in disease.

    PubMed

    Liu, Chan; Wu, Hong-Tao; Zhu, Neng; Shi, Ya-Ning; Liu, Zheng; Ao, Bao-Xue; Liao, Duan-Fang; Zheng, Xi-Long; Qin, Li

    2016-08-01

    Steroid receptor RNA activator (SRA) is a type of long noncoding RNA (lncRNA) which coordinates the functions of various transcription factors, enhances steroid receptor-dependent gene expression, and also serves as a distinct scaffold. The novel, profound and expanded roles of SRA are emerging in critical aspects of coactivation of nuclear receptors (NRs). As a nuclear receptor coactivator, SRA can coactivate androgen receptor (AR), estrogen receptor α (ERα), ERβ, progesterone receptor (PR), glucocorticoid receptor (GR), thyroid hormone receptor and retinoic acid receptor (RAR). Although SRA is one of the least well-understood molecules, increasing studies have revealed that SRA plays a key role in both biological processes, such as myogenesis and steroidogenesis, and pathological changes, including obesity, cardiomyopathy, and tumorigenesis. Furthermore, the SRA-related signaling pathways, such as the mitogen-activated protein kinase (p38 MAPK), Notch and tumor necrosis factor α (TNFα) pathways, play critical roles in the pathogenesis of estrogen-dependent breast cancers. In addition, the most recent data demonstrates that SRA expression may serve as a new prognostic marker in patients with ER-positive breast cancer. Thus, elucidating the molecular mechanisms underlying SRA-mediated functions is important to develop proper novel strategies to target SRA in the diagnosis and treatment of human diseases. PMID:27282881

  19. SINGLE MOLECULE APPROACHES TO BIOLOGY, 2010 GORDON RESEARCH CONFERENCE, JUNE 27-JULY 2, 2010, ITALY

    SciTech Connect

    Professor William Moerner

    2010-07-09

    The 2010 Gordon Conference on Single-Molecule Approaches to Biology focuses on cutting-edge research in single-molecule science. Tremendous technical developments have made it possible to detect, identify, track, and manipulate single biomolecules in an ambient environment or even in a live cell. Single-molecule approaches have changed the way many biological problems are addressed, and new knowledge derived from these approaches continues to emerge. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of biomolecular machines: what they do, how they work individually, how they work together, and finally, how they work inside live cells. The burgeoning use of single-molecule methods to elucidate biological problems is a highly multidisciplinary pursuit, involving both force- and fluorescence-based methods, the most up-to-date advances in microscopy, innovative biological and chemical approaches, and nanotechnology tools. This conference seeks to bring together top experts in molecular and cell biology with innovators in the measurement and manipulation of single molecules, and will provide opportunities for junior scientists and graduate students to present their work in poster format and to exchange ideas with leaders in the field. A number of excellent poster presenters will be selected for short oral talks. Topics as diverse as single-molecule sequencing, DNA/RNA/protein interactions, folding machines, cellular biophysics, synthetic biology and bioengineering, force spectroscopy, new method developments, superresolution imaging in cells, and novel probes for single-molecule imaging will be on the program. Additionally, the collegial atmosphere of this Conference, with programmed discussion sessions as well as opportunities for informal gatherings in the afternoons and evenings in the beauty of the Il Ciocco site in

  20. tRNA biology charges to the front

    PubMed Central

    Phizicky, Eric M.; Hopper, Anita K.

    2010-01-01

    tRNA biology has come of age, revealing an unprecedented level of understanding and many unexpected discoveries along the way. This review highlights new findings on the diverse pathways of tRNA maturation, and on the formation and function of a number of modifications. Topics of special focus include the regulation of tRNA biosynthesis, quality control tRNA turnover mechanisms, widespread tRNA cleavage pathways activated in response to stress and other growth conditions, emerging evidence of signaling pathways involving tRNA and cleavage fragments, and the sophisticated intracellular tRNA trafficking that occurs during and after biosynthesis. PMID:20810645

  1. Perspective: Mechanochemistry of biological and synthetic molecules.

    PubMed

    Makarov, Dmitrii E

    2016-01-21

    Coupling of mechanical forces and chemical transformations is central to the biophysics of molecular machines, polymer chemistry, fracture mechanics, tribology, and other disciplines. As a consequence, the same physical principles and theoretical models should be applicable in all of those fields; in fact, similar models have been invoked (and often repeatedly reinvented) to describe, for example, cell adhesion, dry and wet friction, propagation of cracks, and action of molecular motors. This perspective offers a unified view of these phenomena, described in terms of chemical kinetics with rates of elementary steps that are force dependent. The central question is then to describe how the rate of a chemical transformation (and its other measurable properties such as the transition path) depends on the applied force. I will describe physical models used to answer this question and compare them with experimental measurements, which employ single-molecule force spectroscopy and which become increasingly common. Multidimensionality of the underlying molecular energy landscapes and the ensuing frequent misalignment between chemical and mechanical coordinates result in a number of distinct scenarios, each showing a nontrivial force dependence of the reaction rate. I will discuss these scenarios, their commonness (or its lack), and the prospects for their experimental validation. Finally, I will discuss open issues in the field. PMID:26801011

  2. Perspective: Mechanochemistry of biological and synthetic molecules

    NASA Astrophysics Data System (ADS)

    Makarov, Dmitrii E.

    2016-01-01

    Coupling of mechanical forces and chemical transformations is central to the biophysics of molecular machines, polymer chemistry, fracture mechanics, tribology, and other disciplines. As a consequence, the same physical principles and theoretical models should be applicable in all of those fields; in fact, similar models have been invoked (and often repeatedly reinvented) to describe, for example, cell adhesion, dry and wet friction, propagation of cracks, and action of molecular motors. This perspective offers a unified view of these phenomena, described in terms of chemical kinetics with rates of elementary steps that are force dependent. The central question is then to describe how the rate of a chemical transformation (and its other measurable properties such as the transition path) depends on the applied force. I will describe physical models used to answer this question and compare them with experimental measurements, which employ single-molecule force spectroscopy and which become increasingly common. Multidimensionality of the underlying molecular energy landscapes and the ensuing frequent misalignment between chemical and mechanical coordinates result in a number of distinct scenarios, each showing a nontrivial force dependence of the reaction rate. I will discuss these scenarios, their commonness (or its lack), and the prospects for their experimental validation. Finally, I will discuss open issues in the field.

  3. MicroRNA expression profiling reveals miRNA families regulating specific biological pathways in mouse frontal cortex and hippocampus.

    PubMed

    Juhila, Juuso; Sipilä, Tessa; Icay, Katherine; Nicorici, Daniel; Ellonen, Pekka; Kallio, Aleksi; Korpelainen, Eija; Greco, Dario; Hovatta, Iiris

    2011-01-01

    MicroRNAs (miRNAs) are small regulatory molecules that cause post-transcriptional gene silencing. Although some miRNAs are known to have region-specific expression patterns in the adult brain, the functional consequences of the region-specificity to the gene regulatory networks of the brain nuclei are not clear. Therefore, we studied miRNA expression patterns by miRNA-Seq and microarrays in two brain regions, frontal cortex (FCx) and hippocampus (HP), which have separate biological functions. We identified 354 miRNAs from FCx and 408 from HP using miRNA-Seq, and 245 from FCx and 238 from HP with microarrays. Several miRNA families and clusters were differentially expressed between FCx and HP, including the miR-8 family, miR-182|miR-96|miR-183 cluster, and miR-212|miR-312 cluster overexpressed in FCx and miR-34 family overexpressed in HP. To visualize the clusters, we developed support for viewing genomic alignments of miRNA-Seq reads in the Chipster genome browser. We carried out pathway analysis of the predicted target genes of differentially expressed miRNA families and clusters to assess their putative biological functions. Interestingly, several miRNAs from the same family/cluster were predicted to regulate specific biological pathways. We have developed a miRNA-Seq approach with a bioinformatic analysis workflow that is suitable for studying miRNA expression patterns from specific brain nuclei. FCx and HP were shown to have distinct miRNA expression patterns which were reflected in the predicted gene regulatory pathways. This methodology can be applied for the identification of brain region-specific and phenotype-specific miRNA-mRNA-regulatory networks from the adult and developing rodent brain. PMID:21731767

  4. MicroRNA Expression Profiling Reveals MiRNA Families Regulating Specific Biological Pathways in Mouse Frontal Cortex and Hippocampus

    PubMed Central

    Juhila, Juuso; Sipilä, Tessa; Icay, Katherine; Nicorici, Daniel; Ellonen, Pekka; Kallio, Aleksi; Korpelainen, Eija; Greco, Dario; Hovatta, Iiris

    2011-01-01

    MicroRNAs (miRNAs) are small regulatory molecules that cause post-transcriptional gene silencing. Although some miRNAs are known to have region-specific expression patterns in the adult brain, the functional consequences of the region-specificity to the gene regulatory networks of the brain nuclei are not clear. Therefore, we studied miRNA expression patterns by miRNA-Seq and microarrays in two brain regions, frontal cortex (FCx) and hippocampus (HP), which have separate biological functions. We identified 354 miRNAs from FCx and 408 from HP using miRNA-Seq, and 245 from FCx and 238 from HP with microarrays. Several miRNA families and clusters were differentially expressed between FCx and HP, including the miR-8 family, miR-182|miR-96|miR-183 cluster, and miR-212|miR-312 cluster overexpressed in FCx and miR-34 family overexpressed in HP. To visualize the clusters, we developed support for viewing genomic alignments of miRNA-Seq reads in the Chipster genome browser. We carried out pathway analysis of the predicted target genes of differentially expressed miRNA families and clusters to assess their putative biological functions. Interestingly, several miRNAs from the same family/cluster were predicted to regulate specific biological pathways. We have developed a miRNA-Seq approach with a bioinformatic analysis workflow that is suitable for studying miRNA expression patterns from specific brain nuclei. FCx and HP were shown to have distinct miRNA expression patterns which were reflected in the predicted gene regulatory pathways. This methodology can be applied for the identification of brain region-specific and phenotype-specific miRNA-mRNA-regulatory networks from the adult and developing rodent brain. PMID:21731767

  5. Deciphering Transcriptional Dynamics In Vivo by Counting Nascent RNA Molecules

    PubMed Central

    Choubey, Sandeep; Kondev, Jane; Sanchez, Alvaro

    2015-01-01

    Abstract Deciphering how the regulatory DNA sequence of a gene dictates its expression in response to intra and extracellular cues is one of the leading challenges in modern genomics. The development of novel single-cell sequencing and imaging techniques, as well as a better exploitation of currently available single-molecule imaging techniques, provides an avenue to interrogate the process of transcription and its dynamics in cells by quantifying the number of RNA polymerases engaged in the transcription of a gene (or equivalently the number of nascent RNAs) at a given moment in time. In this paper, we propose that measurements of the cell-to-cell variability in the number of nascent RNAs provide a mostly unexplored method for deciphering mechanisms of transcription initiation in cells. We propose a simple kinetic model of transcription initiation and elongation from which we calculate nascent RNA copy-number fluctuations. To demonstrate the usefulness of this approach, we test our theory against published nascent RNA data for twelve constitutively expressed yeast genes. Rather than transcription being initiated through a single rate limiting step, as it had been previously proposed, our single-cell analysis reveals the presence of at least two rate limiting steps. Surprisingly, half of the genes analyzed have nearly identical rates of transcription initiation, suggesting a common mechanism. Our analytical framework can be used to extract quantitative information about dynamics of transcription from single-cell sequencing data, as well as from single-molecule imaging and electron micrographs of fixed cells, and provides the mathematical means to exploit the quantitative power of these technologies. PMID:26544860

  6. A New Three-Dimensional Educational Model Kit for Building DNA and RNA Molecules: Development and Evaluation

    ERIC Educational Resources Information Center

    Beltramini, Leila Maria; Araujo, Ana Paula Ulian; de Oliveira, Tales Henrique Goncalves; dos Santos Abel, Luciano Douglas; da Silva, Aparecido Rodrigues; dos Santos, Neusa Fernandes

    2006-01-01

    International specialized literature focused on research in biology education is sadly scarce, especially regarding biochemical and molecular aspects. In this light, researchers from this Centre for Structural Molecular Biotechnology developed and evaluated a three-dimensional educational model named "Building Life Molecules DNA and RNA." The…

  7. Bottom-up design of small molecules that stimulate exon 10 skipping in mutant MAPT pre-mRNA.

    PubMed

    Luo, Yiling; Disney, Matthew D

    2014-09-22

    One challenge in chemical biology is to develop small molecules that control cellular protein content. The amount and identity of proteins are influenced by the RNAs that encode them; thus, protein content in a cell could be affected by targeting mRNA. However, RNA has been traditionally difficult to target with small molecules. In this report, we describe controlling the protein products of the mutated microtubule-associated protein tau (MAPT) mature mRNA with a small molecule. MAPT mutations in exon 10 are associated with inherited frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), an incurable disease that is directly caused by increased inclusion of exon 10 in MAPT mRNA. Recent studies have shown that mutations within a hairpin at the MAPT exon 10-intron junction decrease the thermodynamic stability of the RNA, increasing binding to U1 snRNP and thus exon 10 inclusion. Therefore, we designed small molecules that bind and stabilize a mutant MAPT by using Inforna, a computational approach based on information about RNA-small-molecule interactions. The optimal compound selectively bound the mutant MAPT hairpin and thermodynamically stabilized its folding, facilitating exon 10 exclusion. PMID:25115866

  8. Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules

    PubMed Central

    Rahong, Sakon; Yasui, Takao; Yanagida, Takeshi; Nagashima, Kazuki; Kanai, Masaki; Meng, Gang; He, Yong; Zhuge, Fuwei; Kaji, Noritada; Kawai, Tomoji; Baba, Yoshinobu

    2015-01-01

    Separation and analysis of biomolecules represent crucial processes for biological and biomedical engineering development; however, separation resolution and speed for biomolecules analysis still require improvements. To achieve separation and analysis of biomolecules in a short time, the use of highly-ordered nanostructures fabricated by top-down or bottom-up approaches have been proposed. Here, we reported on the use of three-dimensional (3D) nanowire structures embedded in microchannels fabricated by a bottom-up approach for ultrafast separation of small biomolecules, such as DNA, protein, and RNA molecules. The 3D nanowire structures could analyze a mixture of DNA molecules (50–1000 bp) within 50 s, a mixture of protein molecules (20–340 kDa) within 5 s, and a mixture of RNA molecules (100–1000 bases) within 25 s. And, we could observe the electrophoretic mobility difference of biomolecules as a function of molecular size in the 3D nanowire structures. Since the present methodology allows users to control the pore size of sieving materials by varying the number of cycles for nanowire growth, the 3D nanowire structures have a good potential for use as alternatives for other sieving materials. PMID:26073192

  9. On the biological activity of drug molecules: Busulfan and nabumetone

    NASA Astrophysics Data System (ADS)

    Novak, Igor; Kovač, Branka

    2010-10-01

    The electronic structures of drug molecules busulfan (BSU) and nabumetone (NAB) have been investigated by HeI and HeII UV photoelectron spectroscopy (UPS), quantum chemical calculations and virtual docking studies. Their biological activities are discussed in the framework of their electronic and molecular structures, reactivity and drug-enzyme binding.

  10. Caenorhabditis elegans chemical biology: lessons from small molecules

    Technology Transfer Automated Retrieval System (TEKTRAN)

    How can we complement Caenorhabditis elegans genomics and proteomics with a comprehensive structural and functional annotation of its metabolome? Several lines of evidence indicate that small molecules of largely undetermined structure play important roles in C. elegans biology, including key pathw...

  11. Biologically-supported structural model for a viral satellite RNA

    PubMed Central

    Ashton, Peter; Wu, Baodong; D'Angelo, Jessica; Grigull, Jörg; White, K. Andrew

    2015-01-01

    Satellite RNAs (satRNAs) are a class of small parasitic RNA replicon that associate with different viruses, including plus-strand RNA viruses. Because satRNAs do not encode a polymerase or capsid subunit, they rely on a companion virus to provide these proteins for their RNA replication and packaging. SatRNAs recruit these and other required factors via their RNA sequences and structures. Here, through a combination of chemical probing analysis of RNA structure, phylogenetic structural comparisons, and viability assays of satRNA mutants in infected cells, the biological importance of a deduced higher-order structure for a 619 nt long tombusvirus satRNA was assessed. Functionally-relevant secondary and tertiary RNA structures were identified throughout the length of the satRNA. Notably, a 3′-terminal segment was found to adopt two mutually-exclusive RNA secondary structures, both of which were required for efficient satRNA accumulation. Accordingly, these alternative conformations likely function as a type of RNA switch. The RNA switch was also found to engage in a required long-range kissing-loop interaction with an upstream sequence. Collectively, these results establish a high level of conformational complexity within this small parasitic RNA and provide a valuable structural framework for detailed mechanistic studies. PMID:26384416

  12. Photodissociation mass spectrometry: New tools for characterization of biological molecules

    PubMed Central

    Brodbelt, Jennifer S.

    2014-01-01

    Photodissociation mass spectrometry combines the ability to activate and fragment ions using photons with the sensitive detection of the resulting product ions by mass spectrometry. The resulting combination affords a versatile tool for characterization of biological molecules. The scope and breadth of photodissociation mass spectrometry have increased substantially over the past decade as new research groups have entered the field and developed a number of innovative applications that illustrate the ability of photodissociation to produce rich fragmentation patterns, to cleave bonds selectively, and to target specific molecules based on incorporation of chromophores. This review focuses on many of the key developments in photodissociation mass spectrometry over the past decade with a particular emphasis on its applications to biological molecules. PMID:24481009

  13. Molecular biology Mediating transcription and RNA export

    PubMed Central

    Rubin, Jonathan D.; Taatjes, Dylan J.

    2016-01-01

    The finding that the Mediator protein complex contributes to messenger RNA export from the nucleus in yeast adds to a growing list of roles for the complex in regulating transcriptional processes. PMID:26450052

  14. The molecular biology of the positive strand RNA viruses

    SciTech Connect

    Rowlands, D.J.; Mayo, M.A.; Mahy, B.W.J.

    1987-01-01

    This book pulls together recent research findings on the molecular biology of the major families of positive strand RNA viruses infecting plants and animals. The topics covered include protein translation, processing and function, RNA replication, virus structure and antigenicity, mechanisms of infection and evolutionary relationships between the virus families.

  15. Reversible RNA adenosine methylation in biological regulation

    PubMed Central

    Jia, Guifang; Fu, Ye; He, Chuan

    2012-01-01

    N6-methyladenosine (m6A) is a ubiquitous modification in messenger RNA (mRNA) and other RNAs across most eukaryotes. For many years, however, the exact functions of m6A were not clearly understood. The discovery that the fat mass and obesity associated protein (FTO) is an m6A demethylase indicates that this modification is reversible and dynamically regulated, suggesting it has regulatory roles. In addition, it has been shown that m6A affects cell fate decisions in yeast and plant development. Recent affinity-based m6A profiling in mouse and human cells further showed that this modification is a widespread mark in coding and non-coding RNA transcripts and is likely dynamically regulated throughout developmental processes. Therefore, reversible RNA methylation, analogous to reversible DNA and histone modifications, may affect gene expression and cell fate decisions by modulating multiple RNA-related cellular pathways, which potentially provides rapid responses to various cellular and environmental signals, including energy and nutrient availability in mammals. PMID:23218460

  16. RNA triplexes: from structural principles to biological and biotech applications.

    PubMed

    Devi, Gitali; Zhou, Yuan; Zhong, Zhensheng; Toh, Desiree-Faye Kaixin; Chen, Gang

    2015-01-01

    The diverse biological functions of RNA are determined by the complex structures of RNA stabilized by both secondary and tertiary interactions. An RNA triplex is an important tertiary structure motif that is found in many pseudoknots and other structured RNAs. A triplex structure usually forms through tertiary interactions in the major or minor groove of a Watson-Crick base-paired stem. A major-groove RNA triplex structure is stable in isolation by forming consecutive major-groove base triples such as U·A-U and C(+) ·G-C. Minor-groove RNA triplexes, e.g., A-minor motif triplexes, are found in almost all large structured RNAs. As double-stranded RNA stem regions are often involved in biologically important tertiary triplex structure formation and protein binding, the ability to sequence specifically target any desired RNA duplexes by triplex formation would have great potential for biomedical applications. Programmable chemically modified triplex-forming oligonucleotides (TFOs) and triplex-forming peptide nucleic acids (PNAs) have been developed to form TFO·RNA2 and PNA·RNA2 triplexes, respectively, with enhanced binding affinity and sequence specificity at physiological conditions. Here, we (1) provide an overview of naturally occurring RNA triplexes, (2) summarize the experimental methods for studying triplexes, and (3) review the development of TFOs and triplex-forming PNAs for targeting an HIV-1 ribosomal frameshift-inducing RNA, a bacterial ribosomal A-site RNA, and a human microRNA hairpin precursor, and for inhibiting the RNA-protein interactions involving human RNA-dependent protein kinase and HIV-1 viral protein Rev. PMID:25146348

  17. Single-Molecule Force Spectroscopy Study on the Mechanism of RNA Disassembly in Tobacco Mosaic Virus

    PubMed Central

    Liu, Ningning; Chen, Ying; Peng, Bo; Lin, Yuan; Wang, Qian; Su, Zhaohui; Zhang, Wenke; Li, Hongbin; Shen, Jiacong

    2013-01-01

    To explore the disassembly mechanism of tobacco mosaic virus (TMV), a model system for virus study, during infection, we have used single-molecule force spectroscopy to mimic and follow the process of RNA disassembly from the protein coat of TMV by the replisome (molecular motor) in vivo, under different pH and Ca2+ concentrations. Dynamic force spectroscopy revealed the unbinding free-energy landscapes as that at pH 4.7 the disassembly process is dominated by one free-energy barrier, whereas at pH 7.0 the process is dominated by one barrier and that there exists a second barrier. The additional free-energy barrier at longer distance has been attributed to the hindrance of disordered loops within the inner channel of TMV, and the biological function of those protein loops was discussed. The combination of pH increase and Ca2+ concentration drop could weaken RNA-protein interactions so much that the molecular motor replisome would be able to pull and disassemble the rest of the genetic RNA from the protein coat in vivo. All these facts provide supporting evidence at the single-molecule level, to our knowledge for the first time, for the cotranslational disassembly mechanism during TMV infection under physiological conditions. PMID:24359751

  18. New Perspectives on DNA and RNA Triplexes As Effectors of Biological Activity.

    PubMed

    Bacolla, Albino; Wang, Guliang; Vasquez, Karen M

    2015-12-01

    Since the first description of the canonical B-form DNA double helix, it has been suggested that alternative DNA, DNA-RNA, and RNA structures exist and act as functional genomic elements. Indeed, over the past few years it has become clear that, in addition to serving as a repository for genetic information, genomic DNA elicits biological responses by adopting conformations that differ from the canonical right-handed double helix, and by interacting with RNA molecules to form complex secondary structures. This review focuses on recent advances on three-stranded (triplex) nucleic acids, with an emphasis on DNA-RNA and RNA-RNA interactions. Emerging work reveals that triplex interactions between noncoding RNAs and duplex DNA serve as platforms for delivering site-specific epigenetic marks critical for the regulation of gene expression. Additionally, an increasing body of genetic and structural studies demonstrates that triplex RNA-RNA interactions are essential for performing catalytic and regulatory functions in cellular nucleoprotein complexes, including spliceosomes and telomerases, and for enabling protein recoding during programmed ribosomal frameshifting. Thus, evidence is mounting that DNA and RNA triplex interactions are implemented to perform a range of diverse biological activities in the cell, some of which will be discussed in this review. PMID:26700634

  19. Methods to enable the design of bioactive small molecules targeting RNA

    PubMed Central

    Disney, Matthew D.; Yildirim, Ilyas; Childs-Disney, Jessica L.

    2014-01-01

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including Structure-Activity Relationships Through Sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome. PMID:24357181

  20. Methods to enable the design of bioactive small molecules targeting RNA.

    PubMed

    Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

    2014-02-21

    RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome. PMID:24357181

  1. Pulse requirements for electron diffraction imaging of single biological molecules

    SciTech Connect

    Hau-Riege, S; London, R; Chapman, H

    2004-10-20

    The pulse requirements for electron diffraction imaging of single biological molecules are calculated. We find that the electron fluence and pulse length requirements imposed by the damage limit and by the need to classify the diffraction patterns according to their angular orientation cannot be achieved with today's electron beam technology. A simple analytical model shows that the pulse requirements cannot be achieved due to beam broadening due to spacecharge effects.

  2. Method for imaging informational biological molecules on a semiconductor substrate

    NASA Technical Reports Server (NTRS)

    Coles, L. Stephen (Inventor)

    1994-01-01

    Imaging biological molecules such as DNA at rates several times faster than conventional imaging techniques is carried out using a patterned silicon wafer having nano-machined grooves which hold individual molecular strands and periodically spaced unique bar codes permitting repeatably locating all images. The strands are coaxed into the grooves preferably using gravity and pulsed electric fields which induce electric charge attraction to the molecular strands in the bottom surfaces of the grooves. Differential imaging removes substrate artifacts.

  3. Native purification and labeling of RNA for single molecule fluorescence studies

    PubMed Central

    Rinaldi, Arlie J.; Suddala, Krishna C.; Walter, Nils G.

    2012-01-01

    The recent discovery that non-coding RNAs are considerably more abundant and serve a much wider range of critical cellular functions than recognized over previous decades of research into molecular biology has sparked a renewed interest in the study of structure-function relationships of RNA. To perform their functions in the cell, RNAs must dominantly adopt their native conformations, avoiding deep, non-productive kinetic traps that may exist along a frustrated (rugged) folding free energy landscape. Intracellularly, RNAs are synthesized by RNA polymerase and fold co-transcriptionally starting from the 5’ end, sometimes with the aid of protein chaperones. By contrast, in the laboratory RNAs are commonly generated by in vitro transcription or chemical synthesis, followed by purification in a manner that includes the use of high concentrations of urea, heat and UV light (for detection), resulting in the denaturation and subsequent refolding of the entire RNA. Recent studies into the nature of heterogeneous RNA populations resulting from this process have underscored the need for non-denaturing (native) purification methods that maintain the co-transcriptional fold of an RNA. Here, we present protocols for the native purification of an RNA after its in vitro transcription and for fluorophore and biotin labeling methods designed to preserve its native conformation for use in single molecule fluorescence resonance energy transfer (smFRET) inquiries into its structure and function. Finally, we present methods for taking smFRET data and for analyzing them, as well as a description of plausible overall preparation schemes for the plethora of non-coding RNAs. PMID:25352138

  4. New Perspectives on DNA and RNA Triplexes As Effectors of Biological Activity

    PubMed Central

    Bacolla, Albino; Wang, Guliang; Vasquez, Karen M.

    2015-01-01

    Since the first description of the canonical B-form DNA double helix, it has been suggested that alternative DNA, DNA–RNA, and RNA structures exist and act as functional genomic elements. Indeed, over the past few years it has become clear that, in addition to serving as a repository for genetic information, genomic DNA elicits biological responses by adopting conformations that differ from the canonical right-handed double helix, and by interacting with RNA molecules to form complex secondary structures. This review focuses on recent advances on three-stranded (triplex) nucleic acids, with an emphasis on DNA–RNA and RNA–RNA interactions. Emerging work reveals that triplex interactions between noncoding RNAs and duplex DNA serve as platforms for delivering site-specific epigenetic marks critical for the regulation of gene expression. Additionally, an increasing body of genetic and structural studies demonstrates that triplex RNA–RNA interactions are essential for performing catalytic and regulatory functions in cellular nucleoprotein complexes, including spliceosomes and telomerases, and for enabling protein recoding during programmed ribosomal frameshifting. Thus, evidence is mounting that DNA and RNA triplex interactions are implemented to perform a range of diverse biological activities in the cell, some of which will be discussed in this review. PMID:26700634

  5. Synergistic self-assembly of RNA and DNA molecules

    NASA Astrophysics Data System (ADS)

    Ko, Seung Hyeon; Su, Min; Zhang, Chuan; Ribbe, Alexander E.; Jiang, Wen; Mao, Chengde

    2010-12-01

    DNA has recently been used as a programmable 'smart' building block for the assembly of a wide range of nanostructures. It remains difficult, however, to construct DNA assemblies that are also functional. Incorporating RNA is a promising strategy to circumvent this issue as RNA is structurally related to DNA but exhibits rich chemical, structural and functional diversities. However, only a few examples of rationally designed RNA structures have been reported. Herein, we describe a simple, general strategy for the de novo design of nanostructures in which the self-assembly of RNA strands is programmed by DNA strands. To demonstrate the versatility of this approach, we have designed and constructed three different RNA-DNA hybrid branched nanomotifs (tiles), which readily assemble into one-dimensional nanofibres, extended two-dimensional arrays and a discrete three-dimensional object. The current strategy could enable the integration of the precise programmability of DNA with the rich functionality of RNA.

  6. A simple backscattering microscope for fast tracking of biological molecules.

    PubMed

    Sowa, Yoshiyuki; Steel, Bradley C; Berry, Richard M

    2010-11-01

    Recent developments in techniques for observing single molecules under light microscopes have helped reveal the mechanisms by which molecular machines work. A wide range of markers can be used to detect molecules, from single fluorophores to micron sized markers, depending on the research interest. Here, we present a new and simple objective-type backscattering microscope to track gold nanoparticles with nanometer and microsecond resolution. The total noise of our system in a 55 kHz bandwidth is ~0.6 nm per axis, sufficient to measure molecular movement. We found our backscattering microscopy to be useful not only for in vitro but also for in vivo experiments because of lower background scattering from cells than in conventional dark-field microscopy. We demonstrate the application of this technique to measuring the motion of a biological rotary molecular motor, the bacterial flagellar motor, in live Escherichia coli cells. PMID:21133475

  7. Microarray-based technologies for the discovery of selective, RNA-binding molecules.

    PubMed

    Abulwerdi, Fardokht A; Schneekloth, John S

    2016-07-01

    The identification of small molecules that bind specifically to RNA is a challenge. However, the recent explosion in knowledge about the role RNA plays in a number of physiological processes apart from coding for protein sequences makes it a highly interesting target for chemical probes and therapeutics. One technology that has played an important role in the discovery of RNA-binding molecules is microarrays. Microarrays have been broadly employed to screen, profile, and quantify RNA interactions, and will likely play an important role in the discovery of new classes of ligands going forward. Here, we discuss the development of microarray technologies, including aminoglycoside, peptide, peptoid, and small molecule microarrays, and their use in studying RNA-interacting molecules. PMID:27109057

  8. Novel nuclear magnetic resonance techniques for studying biological molecules

    SciTech Connect

    Laws, David D.

    2000-06-01

    Over the fifty-five year history of Nuclear Magnetic Resonance (NMR), considerable progress has been made in the development of techniques for studying the structure, function, and dynamics of biological molecules. The majority of this research has involved the development of multi-dimensional NMR experiments for studying molecules in solution, although in recent years a number of groups have begun to explore NMR methods for studying biological systems in the solid-state. Despite this new effort, a need still exists for the development of techniques that improve sensitivity, maximize information, and take advantage of all the NMR interactions available in biological molecules. In this dissertation, a variety of novel NMR techniques for studying biomolecules are discussed. A method for determining backbone ({phi}/{psi}) dihedral angles by comparing experimentally determined {sup 13}C{sub a}, chemical-shift anisotropies with theoretical calculations is presented, along with a brief description of the theory behind chemical-shift computation in proteins and peptides. The utility of the Spin-Polarization Induced Nuclear Overhauser Effect (SPINOE) to selectively enhance NMR signals in solution is examined in a variety of systems, as are methods for extracting structural information from cross-relaxation rates that can be measured in SPINOE experiments. Techniques for the production of supercritical and liquid laser-polarized xenon are discussed, as well as the prospects for using optically pumped xenon as a polarizing solvent. In addition, a detailed study of the structure of PrP 89-143 is presented. PrP 89-143 is a 54 residue fragment of the prion proteins which, upon mutation and aggregation, can induce prion diseases in transgenic mice. Whereas the structure of the wild-type PrP 89-143 is a generally unstructured mixture of {alpha}-helical and {beta}-sheet conformers in the solid state, the aggregates formed from the PrP 89-143 mutants appear to be mostly {beta}-sheet.

  9. Recoding Aminoacyl-tRNA Synthetases for Synthetic Biology by Rational Protein-RNA Engineering

    PubMed Central

    2015-01-01

    We have taken a rational approach to redesigning the amino acid binding and aminoacyl–tRNA pairing specificities of bacterial glutaminyl–tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNAGln by over 105-fold compared to the wild-type enzyme. Better optimized designs of the protein–RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl–tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology. PMID:25310879

  10. 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. PMID:27012177

  11. An RNA molecule copurifies with RNase P activity from Xenopus laevis oocytes.

    PubMed Central

    Doria, M; Carrara, G; Calandra, P; Tocchini-Valentini, G P

    1991-01-01

    Utilizing a procedure for the purification of RNase P from Xenopus laevis germinal vesicle (GV) extracts, according to which the contamination by a large, cytoplasmic, cylindrical structure (1) is avoided, we demonstrate that the X.laevis enzyme, like the HeLa RNase P, is precipitated by anti-Th antibodies and an RNA molecule (XL RNA), 320 nucleotides long, copurifies with the activity. The sequence of XL RNA is 60% homologous to HeLa H1 RNA, therefore the two molecules seem related. Images PMID:1710353

  12. Imaging biological molecules with single molecule sensitivity using near-field scanning optical microscopy

    SciTech Connect

    Ambrose, W.P.; Affleck, R.L.; Goodwin, P.M.; Keller, R.A.; Martin, J.C.; Petty, J.T.; Schecker, J.A.; Wu, Ming

    1995-12-01

    We have developed a near-field scanning optical microscope with the sensitivity to detect single fluorescent molecules. Our microscope is based on scanning a sample under a tapered and metal coated fiber optic probe and has an illumination-aperture diameter as small as 100 nm. The microscope simultaneously acquires a shear force image with a height noise of {approximately} 1 nm. We have used this system to demonstrate the detection of single molecules of Rhodamine-6G on silica. In this paper, we explore the use of NSOM for investigations of biological molecules. We have prepared and imaged double-stranded DNA intercalated with thiazole orange homodimer (TOTO); single chromosomes stained with propidium iodide; and {beta}-phycoerythrin proteins on dry, borosilicate-glass surfaces. At very dilute coverages, isolated fluorescent spots are observed for the un-intercalated TOTO dye and for {beta}-phycoerythrin. These fluorescent spots exhibit-emission intensity fluctuations and abrupt bleaching transitions, similar to the intensity behavior observed previously for single Rhodamine 6G molecules on silica.

  13. Structural analysis of RNA molecules involved in plasmid copy number control.

    PubMed Central

    Tamm, J; Polisky, B

    1983-01-01

    DNA replication of the plasmid ColE1 and its relatives is controlled mainly by a small plasmid-encoded transcript known as RNA11. We have studied the conformation of RNA1 molecules from two related but compatible plasmids, ColE1 and RSF1030, using T1 ribonuclease, S1 nuclease, cobra venom nuclease, and diethyl pyrocarbonate modification as probes for secondary structure. Both RNA1 molecules contain three double-stranded stems, three single-stranded loops, and an exposed 5' tail. All loops in both molecules show similar sensitivities to the probes used, as do stems 1 and 3. The region comprising stem 2 of each RNA1 molecule contains the most sequence differences as well as the most structural changes of any region in the two molecules. The structure of the RNA1 molecule encoded by a recessive high copy number mutant of ColE1 was also investigated. Structural alterations involving the first stem and loop of the molecule are proposed to be responsible for the inability of the mutant RNA1 to function in vivo. Images PMID:6194508

  14. Localization of single biological molecules out of the focal plane

    NASA Astrophysics Data System (ADS)

    Gardini, L.; Capitanio, M.; Pavone, F. S.

    2014-03-01

    Since the behaviour of proteins and biological molecules is tightly related to the cell's environment, more and more microscopy techniques are moving from in vitro to in living cells experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). We developed an apparatus that combines different microscopy techniques to satisfy all the technical requirements for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy. To account for the optical sectioning of thick samples we built up a HILO (Highly Inclined and Laminated Optical sheet) microscopy system through which we can excite the sample in a widefield (WF) configuration by a thin sheet of light that can follow the molecule up and down along the z axis spanning the entire thickness of the cell with a SNR much higher than traditional WF microscopy. Since protein dynamics inside a cell involve all three dimensions, we included a method to measure the x, y, and z coordinates with nanometer accuracy, exploiting the properties of the point-spread-function of out-of-focus quantum dots bound to the protein of interest. Finally, a feedback system stabilizes the microscope from thermal drifts, assuring accurate localization during the entire duration of the experiment.

  15. Interferometric observations of large biologically interesting interstellar and cometary molecules

    PubMed Central

    Snyder, Lewis E.

    2006-01-01

    Interferometric observations of high-mass regions in interstellar molecular clouds have revealed hot molecular cores that have substantial column densities of large, partly hydrogen-saturated molecules. Many of these molecules are of interest to biology and thus are labeled “biomolecules.” Because the clouds containing these molecules provide the material for star formation, they may provide insight into presolar nebular chemistry, and the biomolecules may provide information about the potential of the associated interstellar chemistry for seeding newly formed planets with prebiotic organic chemistry. In this overview, events are outlined that led to the current interferometric array observations. Clues that connect this interstellar hot core chemistry to the solar system can be found in the cometary detection of methyl formate and the interferometric maps of cometary methanol. Major obstacles to understanding hot core chemistry remain because chemical models are not well developed and interferometric observations have not been very sensitive. Differentiation in the molecular isomers glycolaldehdye, methyl formate, and acetic acid has been observed, but not explained. The extended source structure for certain sugars, aldehydes, and alcohols may require nonthermal formation mechanisms such as shock heating of grains. Major advances in understanding the formation chemistry of hot core species can come from observations with the next generation of sensitive, high-resolution arrays. PMID:16894168

  16. Chemo-enzymatic labeling for rapid assignment of RNA molecules.

    PubMed

    Longhini, Andrew P; LeBlanc, Regan M; Dayie, T Kwaku

    2016-07-01

    Even though Nuclear Magnetic Resonance (NMR) spectroscopy is one of the few techniques capable of determining atomic resolution structures of RNA, it is constrained by two major problems of chemical shift overlap of resonances and rapid signal loss due to line broadening. Emerging tools to tackle these problems include synthesis of atom specifically labeled or chemically modified nucleotides. Herein we review the synthesis of these nucleotides, the design and production of appropriate RNA samples, and the application and analysis of the NMR experiments that take advantage of these labels. PMID:27090003

  17. Target Biological Structures: The Cell, Organelles, DNA and RNA

    NASA Astrophysics Data System (ADS)

    van Holst, Marcelis; Grant, Maxine P.; Aldrich-Wright, Janice

    Living organisms are self replicating molecular factories of staggering complexity [1]. As a result, we are often overwhelmed when trying to identify potential targets for therapeutics. Water, inorganic ions and a large array of relatively small organic molecules (e.g., sugars, vitamins and fatty acids) account for approximately 80% of living matter, with water being the most abundant. Macromolecules such as proteins, polysaccharides, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) constitute the rest. The majority of potential therapeutic targets are found within the cell. Small molecules which are vital for cellular function are imported into the cell by a variety of mechanisms but unlike smaller molecules, macromolecules are assembled within the cell itself. Drugs are usually designed to target cellular macromolecules, as they perform very specific roles in the metabolic processes.

  18. Myricetin: A Dietary Molecule with Diverse Biological Activities.

    PubMed

    Semwal, Deepak Kumar; Semwal, Ruchi Badoni; Combrinck, Sandra; Viljoen, Alvaro

    2016-02-01

    Myricetin is a common plant-derived flavonoid and is well recognised for its nutraceuticals value. It is one of the key ingredients of various foods and beverages. The compound exhibits a wide range of activities that include strong anti-oxidant, anticancer, antidiabetic and anti-inflammatory activities. It displays several activities that are related to the central nervous system and numerous studies have suggested that the compound may be beneficial to protect against diseases such as Parkinson's and Alzheimer's. The use of myricetin as a preserving agent to extend the shelf life of foods containing oils and fats is attributed to the compound's ability to protect lipids against oxidation. A detailed search of existing literature revealed that there is currently no comprehensive review available on this important molecule. Hence, the present work includes the history, synthesis, pharmaceutical applications and toxicity studies of myricetin. This report also highlights structure-activity relationships and mechanisms of action for various biological activities. PMID:26891321

  19. Nanosecond and femtosecond laser spectroscopy of molecules of biological interest

    NASA Astrophysics Data System (ADS)

    Villani, P.; Orlando, S.; Santagata, A.; De Bonis, A.; Veronesi, S.; Giardini, A.

    2007-07-01

    This paper mainly concerns on nanosecond and femtosecond laser spectroscopy of aromatic organic compounds as neurotransmitters, and plume diagnostics of the ablated species, in order to characterize the plasma dynamics, i.e. the temporal and spatial evolution of the plume. Optical emission spectroscopy has been applied to characterize the transient species produced in the femtosecond (fs) and nanosecond (ns) regimes. The laser sources employed for optical emission spectroscopy are a frequency-doubled Nd:YAG Handy ( λ = 532 nm, τ = 5 ns) and a frequency-doubled Nd:glass ( λ = 527 nm, τ = 250 fs). These studies aim to detect and give information on the photoexcitation and photodissociation of these biological molecules and to compare the plasma characteristics in the two ablation regimes.

  20. Monte Carlo simulations to investigate the response of RNA molecules to applied tension forces

    NASA Astrophysics Data System (ADS)

    Le, Dan; Luettmer-Strathmann, Jutta

    2012-04-01

    In many viruses, genetic information is encoded in single-stranded ribonucleic acid (RNA) molecules. These molecules are very long chains with an interesting secondary structure that is still difficult to predict from the sequence of bases along the chain. In this work, we perform Monte Carlo Simulation of a simple model for RNA molecules under an applied tension force. We determine force-extension curves under equilibrium conditions for tension forces applied to different segments of the chain and investigate the relationship between the mechanical response and the secondary structure of the chain.

  1. A Bifurcated Proteoglycan Binding Small Molecule Carrier for siRNA Delivery

    PubMed Central

    Gooding, Matt; Adigbli, Derick; Edith Chan, A W; Melander, Roberta J; MacRobert, Alexander J; Selwood, David L

    2014-01-01

    A wider application of siRNA- and miRNA- based therapeutics is restricted by the currently available delivery systems. We have designed a new type of small molecule carrier (SMoC) system for siRNA modeled to interact with cell surface proteoglycans. This bifurcated SMoC has similar affinity for the model proteoglycan heparin to an equivalent polyarginine peptide and exhibits significant mRNA knockdown of protein levels comparable to lipofectamine and the previously reported linear SMoC. PMID:24472581

  2. Chromatography and mass spectrometry of prebiological and biological molecules

    NASA Astrophysics Data System (ADS)

    Navale, Vivek

    The detection and identification of prebiological and biological molecules are of importance for understanding chemical and biological processes occurring within the solar system. Molecular mass measurements, peptide mapping, and disulfide bond analysis of enzymes and recombinant proteins are important in the development of therapeutic drugs for human diseases. Separation of hydrocarbons (C1 to C6) and nitriles was achieved by 14%-cyanopropylphenyl-86%- dimethylpolysiloxane (CPPS-DMPS) stationary phase in a narrow bore metal capillary column. The calculation of modeling numbers enabled the differentiation of the C4 hydrocarbon isomers of 1-butene (cis and trans). The modeled retention time values for benzene, toluene, xylene, acetonitrile, propane, and propene nitriles were in good agreement with the measurements. The separation of C2 hydrocarbons (ethane and ethene) from predominantly N2 matrix was demonstrated for the first time on wall coated narrow bore low temperature glassy carbon column. Identification and accurate mass measurements of pepsin, an enzymatic protein with less number of basic amino acid residues were successfully demonstrated by matrix- assisted laser desorption ionization mass spectrometry (MALDI-MS). The molecular mass of pepsin was found to be 34,787 Da. Several decomposition products of pepsin, in m/z range of 3,500 to 4,700 were identified. Trypsin, an important endopeptidase enzyme had a mass of 46829.7 Da. Lower mass components with m/z 8047.5, 7776.6, 5722, 5446.2 and 5185 Da were also observed in trypsin spectrum. Both chemokine and growth factor recombinant proteins were mass analyzed as 8848.1 ± 3.5 and 16178.52 ± 4.1 Da, respectively. The accuracy of the measurements was in the range of 0.01 to 0.02%. Reduction and alkylation experiments on the chemokine showed the presence of six cysteines and three disulfide bonds. The two cysteines of the growth factor contained the free sulfhydryl groups and the accurate average mass of the

  3. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.

    PubMed

    Shah, Sheel; Lubeck, Eric; Schwarzkopf, Maayan; He, Ting-Fang; Greenbaum, Alon; Sohn, Chang Ho; Lignell, Antti; Choi, Harry M T; Gradinaru, Viviana; Pierce, Niles A; Cai, Long

    2016-08-01

    Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas - from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH. PMID:27342713

  4. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing

    PubMed Central

    Shah, Sheel; Lubeck, Eric; Schwarzkopf, Maayan; He, Ting-Fang; Greenbaum, Alon; Sohn, Chang Ho; Lignell, Antti; Choi, Harry M. T.; Gradinaru, Viviana; Pierce, Niles A.

    2016-01-01

    Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas – from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving ∼20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH. PMID:27342713

  5. RNA Systems Biology for Cancer: From Diagnosis to Therapy.

    PubMed

    Amirkhah, Raheleh; Farazmand, Ali; Wolkenhauer, Olaf; Schmitz, Ulf

    2016-01-01

    It is due to the advances in high-throughput omics data generation that RNA species have re-entered the focus of biomedical research. International collaborate efforts, like the ENCODE and GENCODE projects, have spawned thousands of previously unknown functional non-coding RNAs (ncRNAs) with various but primarily regulatory roles. Many of these are linked to the emergence and progression of human diseases. In particular, interdisciplinary studies integrating bioinformatics, systems biology, and biotechnological approaches have successfully characterized the role of ncRNAs in different human cancers. These efforts led to the identification of a new tool-kit for cancer diagnosis, monitoring, and treatment, which is now starting to enter and impact on clinical practice. This chapter is to elaborate on the state of the art in RNA systems biology, including a review and perspective on clinical applications toward an integrative RNA systems medicine approach. The focus is on the role of ncRNAs in cancer. PMID:26677189

  6. Myricetin: A Dietary Molecule with Diverse Biological Activities

    PubMed Central

    Semwal, Deepak Kumar; Semwal, Ruchi Badoni; Combrinck, Sandra; Viljoen, Alvaro

    2016-01-01

    Myricetin is a common plant-derived flavonoid and is well recognised for its nutraceuticals value. It is one of the key ingredients of various foods and beverages. The compound exhibits a wide range of activities that include strong anti-oxidant, anticancer, antidiabetic and anti-inflammatory activities. It displays several activities that are related to the central nervous system and numerous studies have suggested that the compound may be beneficial to protect against diseases such as Parkinson’s and Alzheimer’s. The use of myricetin as a preserving agent to extend the shelf life of foods containing oils and fats is attributed to the compound’s ability to protect lipids against oxidation. A detailed search of existing literature revealed that there is currently no comprehensive review available on this important molecule. Hence, the present work includes the history, synthesis, pharmaceutical applications and toxicity studies of myricetin. This report also highlights structure-activity relationships and mechanisms of action for various biological activities. PMID:26891321

  7. Progress in Small Molecule and Biologic Therapeutics Targeting Ghrelin Signaling.

    PubMed

    McGovern, Kayleigh R; Darling, Joseph E; Hougland, James L

    2016-01-01

    Ghrelin is a circulating peptide hormone involved in regulation of a wide array of physiological processes. As an endogenous ligand for growth hormone secretagogue receptor (GHSR1a), ghrelin is responsible for signaling involved in energy homeostasis, including appetite stimulation, glucose metabolism, insulin signaling, and adiposity. Ghrelin has also been implicated in modulation of several neurological processes. Dysregulation of ghrelin signaling is implicated in diseases related to these pathways, including obesity, type II diabetes, and regulation of appetite and body weight in patients with Prader-Willi syndrome. Multiple steps in the ghrelin signaling pathway are available for targeting in the development of therapeutics for these diseases. Agonists and antagonists of GHS-R1a have been widely studied and have shown varying levels of effectiveness within ghrelin-related physiological pathways. Agents targeting ghrelin directly, either through depletion of ghrelin levels in circulation or inhibitors of ghrelin O-acyltransferase whose action is required for ghrelin to become biologically active, are receiving increasing attention as potential therapeutic options. We discuss the approaches utilized to target ghrelin signaling and highlight the current challenges toward developing small-molecule agents as potential therapeutics for ghrelin-related diseases. PMID:26202202

  8. Voltammetric detection of biological molecules using chopped carbon fiber.

    PubMed

    Sugawara, Kazuharu; Yugami, Asako; Kojima, Akira

    2010-01-01

    Voltammetric detection of biological molecules was carried out using chopped carbon fibers produced from carbon fiber reinforced plastics that are biocompatible and inexpensive. Because chopped carbon fibers normally are covered with a sizing agent, they are difficult to use as an electrode. However, when the surface of a chopped carbon fiber was treated with ethanol and hydrochloric acid, it became conductive. To evaluate the functioning of chopped carbon fibers, voltammetric measurements of [Fe(CN)(6)](3-) were carried out. Redoxes of FAD, ascorbic acid and NADH as biomolecules were recorded using cyclic voltammetry. The sizing agents used to bundle the fibers were epoxy, polyamide and polyurethane resins. The peak currents were the greatest when using the chopped carbon fibers that were created with epoxy resins. When the electrode response of the chopped carbon fibers was compared with that of a glassy carbon electrode, the peak currents and the reversibility of the electrode reaction were sufficient. Therefore, the chopped carbon fibers will be useful as disposable electrodes for the sensing of biomolecules. PMID:20953048

  9. Applications of Two-Photon Absorption in Medicine and Biology Enabled by Specially Designed Biological Molecules

    NASA Astrophysics Data System (ADS)

    Drobizhev, M.

    2008-05-01

    We quantitatively study how the two-photon absorption (2PA) properties of biological molecules depend on their structure. 2PA is advantageous over regular one-photon absorption because of deeper penetration and more localized excitation in biological tissues. However, 2PA cross sections of biological chromophores are usually rather small to be useful in real life applications. Using quantum-mechanical few-level description of molecular electronic states, we interpret our data and predict new structures with considerably increased 2PA cross sections. These new materials either synthesized or genetically engineered make 2PA-based techniques applicable in medicine and biology. We show how our new porphyrin photosensitizers with drastically enhanced 2PA (˜1000 times compared to regular porphyrins) can be used for in vivo two-photon-induced closing of blood vessels in Age-Related Macular Degeneration. The second example describes the application of fluorescent proteins in two-photon laser microscopy of biological cells. We demonstrate how the 2PA properties of fluorescent proteins can be considerably improved by smart mutations of the environment of chromophore inside the protein.

  10. Single molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update

    PubMed Central

    Widom, Julia R.; Dhakal, Soma; Heinicke, Laurie A.; Walter, Nils G.

    2015-01-01

    Toxicology is the highly interdisciplinary field studying the adverse effects of chemicals on living organisms. It requires sensitive tools to detect such effects. After their initial implementation during the 1990s, single-molecule fluorescence detection tools were quickly recognized for their potential to contribute greatly to many different areas of scientific inquiry. In the intervening time, technical advances in the field have generated ever-improving spatial and temporal resolution, and have enabled the application of single-molecule fluorescence to increasingly complex systems, such as live cells. In this review, we give an overview of the optical components necessary to implement the most common versions of single-molecule fluorescence detection. We then discuss current applications to enzymology and structural studies, systems biology, and nanotechnology, presenting the technical considerations that are unique to each area of study, along with noteworthy recent results. We also highlight future directions that have the potential to revolutionize these areas of study by further exploiting the capabilities of single-molecule fluorescence microscopy. PMID:25212907

  11. Design of a bioactive small molecule that targets the myotonic dystrophy type 1 RNA via an RNA motif-ligand database and chemical similarity searching.

    PubMed

    Parkesh, Raman; Childs-Disney, Jessica L; Nakamori, Masayuki; Kumar, Amit; Wang, Eric; Wang, Thomas; Hoskins, Jason; Tran, Tuan; Housman, David; Thornton, Charles A; Disney, Matthew D

    2012-03-14

    Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5'CUG/3'GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, sarco(endo)plasmic reticulum Ca(2+) ATPase 1, and cardiac troponin T. Based on these observations, the development of small-molecule ligands that target specifically expanded DM1 repeats could be of use as therapeutics. In the present study, chemical similarity searching was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of in vitro inhibitors of the RNA-protein complex were identified with low micromolar IC(50)'s, which are >20-fold more potent than the query compounds. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with chemical similarity searching. PMID:22300544

  12. Design of a Bioactive Small Molecule that Targets the Myotonic Dystrophy Type 1 RNA Via an RNA Motif-Ligand Database & Chemical Similarity Searching

    PubMed Central

    Parkesh, Raman; Childs-Disney, Jessica L.; Nakamori, Masayuki; Kumar, Amit; Wang, Eric; Wang, Thomas; Hoskins, Jason; Tran, Tuan; Housman, David; Thornton, Charles A.; Disney, Matthew D.

    2012-01-01

    Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3′ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5′CUG/3′GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, Sarco(endo)plasmic reticulum Ca2+ ATPase 1 (Serca1/Atp2a1), and cardiac troponin T (cTNT). Based on these observations, the development of small molecule ligands that target specifically expanded DM1 repeats could serve as therapeutics. In the present study, computational screening was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of inhibitors of the RNA-protein complex with low micromolar IC50’s, which are >20-fold more potent than the query compounds, were identified. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with virtual screening. PMID:22300544

  13. Small-Molecule Inhibitors of Staphylococcus aureus RnpA-Mediated RNA Turnover and tRNA Processing

    PubMed Central

    Eidem, Tess M.; Lounsbury, Nicole; Emery, John F.; Bulger, Jeffrey; Smith, Andrew; Abou-Gharbia, Magid

    2015-01-01

    New agents are urgently needed for the therapeutic treatment of Staphylococcus aureus infections. In that regard, S. aureus RNase RnpA may represent a promising novel dual-function antimicrobial target that participates in two essential cellular processes, RNA degradation and tRNA maturation. Accordingly, we previously used a high-throughput screen to identify small-molecule inhibitors of the RNA-degrading activity of the enzyme and showed that the RnpA inhibitor RNPA1000 is an attractive antimicrobial development candidate. In this study, we used a series of in vitro and cellular assays to characterize a second RnpA inhibitor, RNPA2000, which was identified in our initial screening campaign and is structurally distinct from RNPA1000. In doing so, it was found that S. aureus RnpA does indeed participate in 5′-precursor tRNA processing, as was previously hypothesized. Further, we show that RNPA2000 is a bactericidal agent that inhibits both RnpA-associated RNA degradation and tRNA maturation activities both in vitro and within S. aureus. The compound appears to display specificity for RnpA, as it did not significantly affect the in vitro activities of unrelated bacterial or eukaryotic ribonucleases and did not display measurable human cytotoxicity. Finally, we show that RNPA2000 exhibits antimicrobial activity and inhibits tRNA processing in efflux-deficient Gram-negative pathogens. Taken together, these data support the targeting of RnpA for antimicrobial development purposes, establish that small-molecule inhibitors of both of the functions of the enzyme can be identified, and lend evidence that RnpA inhibitors may have broad-spectrum antimicrobial activities. PMID:25605356

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

    PubMed Central

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

    2014-01-01

    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. PMID:25428360

  15. Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.

    PubMed

    Costales, Matthew G; Rzuczek, Suzanne G; Disney, Matthew D

    2016-06-01

    Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example. PMID:27117425

  16. Sustainable production of biologically active molecules of marine based origin.

    PubMed

    Murray, Patrick M; Moane, Siobhan; Collins, Catherine; Beletskaya, Tanya; Thomas, Olivier P; Duarte, Alysson W F; Nobre, Fernando S; Owoyemi, Ifeloju O; Pagnocca, Fernando C; Sette, L D; McHugh, Edward; Causse, Eric; Pérez-López, Paula; Feijoo, Gumersindo; Moreira, Ma T; Rubiolo, Juan; Leirós, Marta; Botana, Luis M; Pinteus, Susete; Alves, Celso; Horta, André; Pedrosa, Rui; Jeffryes, Clayton; Agathos, Spiros N; Allewaert, Celine; Verween, Annick; Vyverman, Wim; Laptev, Ivan; Sineoky, Sergei; Bisio, Angela; Manconi, Renata; Ledda, Fabio; Marchi, Mario; Pronzato, Roberto; Walsh, Daniel J

    2013-09-25

    The marine environment offers both economic and scientific potential which are relatively untapped from a biotechnological point of view. These environments whilst harsh are ironically fragile and dependent on a harmonious life form balance. Exploitation of natural resources by exhaustive wild harvesting has obvious negative environmental consequences. From a European industry perspective marine organisms are a largely underutilised resource. This is not due to lack of interest but due to a lack of choice the industry faces for cost competitive, sustainable and environmentally conscientious product alternatives. Knowledge of the biotechnological potential of marine organisms together with the development of sustainable systems for their cultivation, processing and utilisation are essential. In 2010, the European Commission recognised this need and funded a collaborative RTD/SME project under the Framework 7-Knowledge Based Bio-Economy (KBBE) Theme 2 Programme 'Sustainable culture of marine microorganisms, algae and/or invertebrates for high value added products'. The scope of that project entitled 'Sustainable Production of Biologically Active Molecules of Marine Based Origin' (BAMMBO) is outlined. Although the Union is a global leader in many technologies, it faces increasing competition from traditional rivals and emerging economies alike and must therefore improve its innovation performance. For this reason innovation is placed at the heart of a European Horizon 2020 Strategy wherein the challenge is to connect economic performance to eco performance. This article provides a synopsis of the research activities of the BAMMBO project as they fit within the wider scope of sustainable environmentally conscientious marine resource exploitation for high-value biomolecules. PMID:23563183

  17. Three-dimensional model of a selective theophylline-binding RNA molecule

    SciTech Connect

    Tung, Chang-Shung; Oprea, T.I.; Hummer, G.; Garcia, A.E.

    1995-07-01

    We propose a three-dimensional (3D) model for an RNA molecule that selectively binds theophylline but not caffeine. This RNA, which was found using SELEX [Jenison, R.D., et al., Science (1994) 263:1425] is 10,000 times more specific for theophylline (Kd=320 nM) than for caffeine (Kd=3.5 mM), although the two ligands are identical except for a methyl group substituted at N7 (present only in caffeine). The binding affinity for ten xanthine-based ligands was used to derive a Comparative Molecular Field Analysis (CoMFA) model (R{sup 2} = 0.93 for 3 components, with cross-validated R{sup 2} of 0.73), using the SYBYL and GOLPE programs. A pharmacophoric map was generated to locate steric and electrostatic interactions between theophylline and the RNA binding site. This information was used to identify putative functional groups of the binding pocket and to generate distance constraints. Based on a model for the secondary structure (Jenison et al., idem), the 3D structure of this RNA was then generated using the following method: each helical region of the RNA molecule was treated as a rigid body; single-stranded loops with specific end-to-end distances were generated. The structures of RNA-xanthine complexes were studied using a modified Monte Carlo algorithm. The detailed structure of an RNA-ligand complex model, as well as possible explanations for the theophylline selectivity will be discussed.

  18. Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes.

    PubMed

    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

    2015-06-01

    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 NIH 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

  19. Single Fluorescent Molecules as Nano-Illuminators for Biological Structure and Function

    NASA Astrophysics Data System (ADS)

    Moerner, W. E.

    2011-03-01

    Since the first optical detection and spectroscopy of a single molecule in a solid (Phys. Rev. Lett. {62}, 2535 (1989)), much has been learned about the ability of single molecules to probe local nanoenvironments and individual behavior in biological and nonbiological materials in the absence of ensemble averaging that can obscure heterogeneity. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic imaging of individual fluorophores leads naturally to superlocalization, or determination of the position of the molecule with precision beyond the optical diffraction limit, simply by digitization of the point-spread function from the single emitter. For example, the shape of single filaments in a living cell can be extracted simply by allowing a single molecule to move through the filament (PNAS {103}, 10929 (2006)). The addition of photoinduced control of single-molecule emission allows imaging beyond the diffraction limit (super-resolution) and a new array of acronyms (PALM, STORM, F-PALM etc.) and advances have appeared. We have used the native blinking and switching of a common yellow-emitting variant of green fluorescent protein (EYFP) reported more than a decade ago (Nature {388}, 355 (1997)) to achieve sub-40 nm super-resolution imaging of several protein structures in the bacterium Caulobacter crescentus: the quasi-helix of the actin-like protein MreB (Nat. Meth. {5}, 947 (2008)), the cellular distribution of the DNA binding protein HU (submitted), and the recently discovered division spindle composed of ParA filaments (Nat. Cell Biol. {12}, 791 (2010)). Even with these advances, better emitters would provide more photons and improved resolution, and a new photoactivatable small-molecule emitter has recently been synthesized and targeted to specific structures in living cells to provide super-resolution images (JACS {132}, 15099 (2010)). Finally, a new optical method for extracting three-dimensional position information based on

  20. Probing the transcription mechanisms of reovirus cores with molecules that alter RNA duplex stability.

    PubMed

    Demidenko, Alexander A; Nibert, Max L

    2009-06-01

    The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transcription. Different mechanisms were identified for inhibition or activation by these molecules. With spermidine, one round of transcription occurred normally, but subsequent rounds were inhibited. Thus, inhibition occurred at the transition between the end of elongation in one round and initiation in the next round of transcription. Dimethyl sulfoxide or trimethylglycine, on the other hand, had no effect on transcription by a constitutively active fraction of cores in each preparation but activated transcription in another fraction that was otherwise silent for the production of elongated transcripts. Activation of this other fraction occurred at the transition between transcript initiation and elongation, i.e., at promoter escape. These results suggest that the relative stability of RNA duplexes is most important for certain steps in the particle-associated transcription cycles of dsRNA viruses and that small molecules are useful tools for probing these and probably other steps. PMID:19297468

  1. Biological chemistry of virus-encoded suppressors of RNA silencing: an overview.

    PubMed

    Omarov, Rustem T; Scholthof, Herman B

    2012-01-01

    RNA interference (RNAi) plays multiple biological roles in eukaryotic organisms to regulate gene expression. RNAi also operates as a conserved adaptive molecular immune mechanism against invading viruses. The antiviral RNAi pathway is initiated with the generation of virus-derived short-interfering RNAs (siRNAs) that are used for subsequent sequence-specific recognition and degradation of the cognate viral RNA molecules. As an efficient counter-defensive strategy, most plant viruses evolved the ability to encode specific proteins capable of interfering with RNAi, and this process is commonly known as RNA silencing suppression. Virus-encoded suppressors of RNAi (VSRs) operate at different steps in the RNAi pathway and display distinct biochemical properties that enable these proteins to efficiently interfere with the host-defense system. Recent molecular and biochemical studies of several VSRs significantly expanded our understanding of the complex nature of silencing suppression, and also remarkably advanced our overall knowledge on complex host-virus interactions. In this review, we describe the current knowledge on activities and biochemical mechanisms of selected VSRs with regard to their biological role of suppressing RNAi in plants. PMID:22678571

  2. Small molecules targeting microRNA for cancer therapy: Promises and obstacles.

    PubMed

    Wen, Di; Danquah, Michael; Chaudhary, Amit Kumar; Mahato, Ram I

    2015-12-10

    Aberrant expression of miRNAs is critically implicated in cancer initiation and progression. Therapeutic approaches focused on regulating miRNAs are therefore a promising approach for treating cancer. Antisense oligonucleotides, miRNA sponges, and CRISPR/Cas9 genome editing systems are being investigated as tools for regulating miRNAs. Despite the accruing insights in the use of these tools, delivery concerns have mitigated clinical application of such systems. In contrast, little attention has been given to the potential of small molecules to modulate miRNA expression for cancer therapy. In these years, many researches proved that small molecules targeting cancer-related miRNAs might have greater potential for cancer treatment. Small molecules targeting cancer related miRNAs showed significantly promising results in different cancer models. However, there are still several obstacles hindering the progress and clinical application in this area. This review discusses the development, mechanisms and application of small molecules for modulating oncogenic miRNAs (oncomiRs). Attention has also been given to screening technologies and perspectives aimed to facilitate clinical translation for small molecule-based miRNA therapeutics. PMID:26256260

  3. Approaches to Validate and Manipulate RNA Targets with Small Molecules in Cells.

    PubMed

    Childs-Disney, Jessica L; Disney, Matthew D

    2016-01-01

    RNA has become an increasingly important target for therapeutic interventions and for chemical probes that dissect and manipulate its cellular function. Emerging targets include human RNAs that have been shown to directly cause cancer, metabolic disorders, and genetic disease. In this review, we describe various routes to obtain bioactive compounds that target RNA, with a particular emphasis on the development of small molecules. We use these cases to describe approaches that are being developed for target validation, which include target-directed cleavage, classic pull-down experiments, and covalent cross-linking. Thus, tools are available to design small molecules to target RNA and to identify the cellular RNAs that are their targets. PMID:26514201

  4. Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive.

    PubMed

    Childs-Disney, Jessica L; Hoskins, Jason; Rzuczek, Suzanne G; Thornton, Charles A; Disney, Matthew D

    2012-05-18

    RNA is an important drug target, but it is difficult to design or discover small molecules that modulate RNA function. In the present study, we report that rationally designed, modularly assembled small molecules that bind the RNA that causes myotonic dystrophy type 1 (DM1) are potently bioactive in cell culture models. DM1 is caused when an expansion of r(CUG) repeats, or r(CUG)(exp), is present in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. r(CUG)(exp) folds into a hairpin with regularly repeating 5'CUG/3'GUC motifs and sequesters muscleblind-like 1 protein (MBNL1). A variety of defects are associated with DM1, including (i) formation of nuclear foci, (ii) decreased translation of DMPK mRNA due to its nuclear retention, and (iii) pre-mRNA splicing defects due to inactivation of MBNL1, which controls the alternative splicing of various pre-mRNAs. Previously, modularly assembled ligands targeting r(CUG)(exp) were designed using information in an RNA motif-ligand database. These studies showed that a bis-benzimidazole (H) binds the 5'CUG/3'GUC motif in r(CUG)(exp.) Therefore, we designed multivalent ligands to bind simultaneously multiple copies of this motif in r(CUG)(exp). Herein, we report that the designed compounds improve DM1-associated defects including improvement of translational and pre-mRNA splicing defects and the disruption of nuclear foci. These studies may establish a foundation to exploit other RNA targets in genomic sequence. PMID:22332923

  5. Rationally Designed Small Molecules Targeting the RNA That Causes Myotonic Dystrophy Type 1 Are Potently Bioactive

    PubMed Central

    Childs-Disney, Jessica L.; Hoskins, Jason; Rzuczek, Suzanne G.; Thornton, Charles A.; Disney, Matthew D.

    2012-01-01

    RNA is an important drug target, but it is difficult to design or discover small molecules that modulate RNA function. In the present study, we report that rationally designed, modularly assembled small molecules that bind the RNA that causes myotonic dystrophy type 1 (DM1) are potently bioactive in cell culture models. DM1 is caused when an expansion of r(CUG) repeats, or r(CUG)exp, is present in the 3′ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. r(CUG)exp folds into a hairpin with regularly repeating 5′CUG/3′GUC motifs and sequester muscleblind-like 1 protein (MBNL1). A variety of defects are associated with DM1 including: (i) formation of nuclear foci, (ii) decreased translation of DMPK mRNA due to its nuclear retention, and (iii) pre-mRNA splicing defects due to inactivation of MBNL1, which controls the alternative splicing of various pre-mRNAs. Previously, modularly assembled ligands targeting r(CUG)exp were designed using information in an RNA motif-ligand database. These studies showed that a bis-benzimidazole (H) binds the 5′CUG/3′GUC motif in r(CUG)exp. Therefore, we designed multivalent ligands to bind multiple copies of this motif simultaneously in r(CUG)exp. Herein, we report that the designed compounds improve DM1-associated defects including improvement of translational and pre-mRNA splicing defects and the disruption of nuclear foci. These studies may establish a foundation to exploit other RNA targets in genomic sequence. PMID:22332923

  6. Silencing stromal interaction molecule 1 by RNA interference inhibits the proliferation and migration of endothelial progenitor cells

    SciTech Connect

    Kuang, Chun-yan; Yu, Yang; Guo, Rui-wei; Qian, De-hui; Wang, Kui; Den, Meng-yang; Shi, Yan-kun; Huang, Lan

    2010-07-23

    Research highlights: {yields} STIM1 and TRPC1 are expressed in EPCs. {yields} Knockdown of STIM1 inhibits the proliferation, migration and SOCE of EPCs. {yields} TRPC1-SOC cooperates with STIM1 to mediate the SOCE of EPCs. -- Abstract: Knockdown of stromal interaction molecule 1 (STIM1) significantly suppresses neointima hyperplasia after vascular injury. Endothelial progenitor cells (EPCs) are the major source of cells that respond to endothelium repair and contribute to re-endothelialization by reducing neointima formation after vascular injury. We hypothesized that the effect of STIM1 on neointima hyperplasia inhibition is mediated through its effect on the biological properties of EPCs. In this study, we investigated the effects of STIM1 on the proliferation and migration of EPCs and examined the effect of STIM1 knockdown using cultured rat bone marrow-derived EPCs. STIM1 was expressed in EPCs, and knockdown of STIM1 by adenoviral delivery of small interfering RNA (siRNA) significantly suppressed the proliferation and migration of EPCs. Furthermore, STIM1 knockdown decreased store-operated channel entry 48 h after transfection. Replenishment with recombinant human STIM1 reversed the effects of STIM1 knockdown. Our data suggest that the store-operated transient receptor potential canonical 1 channel is involved in regulating the biological properties of EPCs through STIM1. STIM1 is a potent regulator of cell proliferation and migration in rat EPCs and may play an important role in the biological properties of EPCs.

  7. Pragmatic turn in biology: From biological molecules to genetic content operators.

    PubMed

    Witzany, Guenther

    2014-08-26

    Erwin Schrödinger's question "What is life?" received the answer for decades of "physics + chemistry". The concepts of Alain Turing and John von Neumann introduced a third term: "information". This led to the understanding of nucleic acid sequences as a natural code. Manfred Eigen adapted the concept of Hammings "sequence space". Similar to Hilbert space, in which every ontological entity could be defined by an unequivocal point in a mathematical axiomatic system, in the abstract "sequence space" concept each point represents a unique syntactic structure and the value of their separation represents their dissimilarity. In this concept molecular features of the genetic code evolve by means of self-organisation of matter. Biological selection determines the fittest types among varieties of replication errors of quasi-species. The quasi-species concept dominated evolution theory for many decades. In contrast to this, recent empirical data on the evolution of DNA and its forerunners, the RNA-world and viruses indicate cooperative agent-based interactions. Group behaviour of quasi-species consortia constitute de novo and arrange available genetic content for adaptational purposes within real-life contexts that determine epigenetic markings. This review focuses on some fundamental changes in biology, discarding its traditional status as a subdiscipline of physics and chemistry. PMID:25225596

  8. Pragmatic turn in biology: From biological molecules to genetic content operators

    PubMed Central

    Witzany, Guenther

    2014-01-01

    Erwin Schrödinger‘s question “What is life?” received the answer for decades of “physics + chemistry”. The concepts of Alain Turing and John von Neumann introduced a third term: “information”. This led to the understanding of nucleic acid sequences as a natural code. Manfred Eigen adapted the concept of Hammings “sequence space”. Similar to Hilbert space, in which every ontological entity could be defined by an unequivocal point in a mathematical axiomatic system, in the abstract ”sequence space” concept each point represents a unique syntactic structure and the value of their separation represents their dissimilarity. In this concept molecular features of the genetic code evolve by means of self-organisation of matter. Biological selection determines the fittest types among varieties of replication errors of quasi-species. The quasi-species concept dominated evolution theory for many decades. In contrast to this, recent empirical data on the evolution of DNA and its forerunners, the RNA-world and viruses indicate cooperative agent-based interactions. Group behaviour of quasi-species consortia constitute de novo and arrange available genetic content for adaptational purposes within real-life contexts that determine epigenetic markings. This review focuses on some fundamental changes in biology, discarding its traditional status as a subdiscipline of physics and chemistry. PMID:25225596

  9. Small Molecule Microarrays of RNA-Focused Peptoids Identifies Inhibitors of a Pathogenic Group I Intron RNA

    PubMed Central

    Labuda, Lucas P.; Pushechnikov, Alexei; Disney, Matthew D.

    2009-01-01

    Peptoids that inhibit the group I intron RNA from Candida albicans, an opportunistic pathogen that kills immunocompromised hosts, have been identified using microarrays. The arrayed peptoid library was constructed using submonomers with moieties similar to ones found in small molecules known to bind RNA. Library members that passed quality control analysis were spotted onto a microarray and screened for binding to the C. albicans group I intron ribozyme. Each ligand binder identified from microarray-based screening inhibited self-splicing in the presence of 1 mM nucleotide concentration of bulk yeast tRNA with IC50’s between 150 and 2200 µM. The binding signals and the corresponding IC50’s were used to identify features in the peptoids that predispose them for RNA binding. After statistical analysis of the peptoids’ structures that bind, a second generation of inhibitors was constructed using these important features; all second generation inhibitors have improved potencies with IC50’s <100 µM. The most potent inhibitor is composed of one phenylguanidine and three tryptamine submonomers and has an IC50 of 31 µM. This compound is 6-fold more potent than pentamidine, a clinically used drug that inhibits self-splicing. These results show that: 1.) modulators of RNA function can be identified by designing RNA-focused chemical libraries and screening them via microarray; 2.) statistical analysis of ligand binders can identify features in leads that predispose them for binding to their targets; and 3.) features can then be programmed into second generation inhibitors to design ligands with improved potencies. PMID:19278238

  10. Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases

    PubMed Central

    Krumm, Stefanie A.; Ndungu, J. Maina; Yoon, Jeong-Joong; Dochow, Melanie; Sun, Aiming; Natchus, Michael; Snyder, James P.; Plemper, Richard K.

    2011-01-01

    Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp) activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid adaptation to a pathogen

  11. Nanocarrier-based co-delivery of small molecules and siRNA/miRNA for treatment of cancer.

    PubMed

    Chitkara, Deepak; Singh, Saurabh; Mittal, Anupama

    2016-04-01

    Aberrant gene expression can trigger several vital molecular events that not only result in carcinogenesis but also cause chemoresistance, metastasis and relapse. Gene-based therapies using siRNA/miRNA have been suggested as new treatment method to improve the current regimen. Although these agents can restore the normal molecular cascade thereby resensitizing the cancer cells, delivering a standard regimen (either subsequently or simultaneously) is necessary to achieve the therapeutic benefit. However, co-delivery using a single carrier could give an additional advantage of similar biodistribution profile of the loaded agents. While much research has been carried out in this field in recent years, challenges involved in designing combination formulations including efficient coloading, stability, appropriate biodistribution and target specificity have hampered their clinical translation. This article highlights current aspects of nano-carriers used for co-delivery of small molecules and genes to treat cancer. PMID:27010986

  12. Prediction of RNA secondary structures: from theory to models and real molecules

    NASA Astrophysics Data System (ADS)

    Schuster, Peter

    2006-05-01

    RNA secondary structures are derived from RNA sequences, which are strings built form the natural four letter nucleotide alphabet, {AUGC}. These coarse-grained structures, in turn, are tantamount to constrained strings over a three letter alphabet. Hence, the secondary structures are discrete objects and the number of sequences always exceeds the number of structures. The sequences built from two letter alphabets form perfect structures when the nucleotides can form a base pair, as is the case with {GC} or {AU}, but the relation between the sequences and structures differs strongly from the four letter alphabet. A comprehensive theory of RNA structure is presented, which is based on the concepts of sequence space and shape space, being a space of structures. It sets the stage for modelling processes in ensembles of RNA molecules like evolutionary optimization or kinetic folding as dynamical phenomena guided by mappings between the two spaces. The number of minimum free energy (mfe) structures is always smaller than the number of sequences, even for two letter alphabets. Folding of RNA molecules into mfe energy structures constitutes a non-invertible mapping from sequence space onto shape space. The preimage of a structure in sequence space is defined as its neutral network. Similarly the set of suboptimal structures is the preimage of a sequence in shape space. This set represents the conformation space of a given sequence. The evolutionary optimization of structures in populations is a process taking place in sequence space, whereas kinetic folding occurs in molecular ensembles that optimize free energy in conformation space. Efficient folding algorithms based on dynamic programming are available for the prediction of secondary structures for given sequences. The inverse problem, the computation of sequences for predefined structures, is an important tool for the design of RNA molecules with tailored properties. Simultaneous folding or cofolding of two or more RNA

  13. Effect of Inducible Co-Stimulatory Molecule siRNA in Cerebral Infarction Rat Models

    PubMed Central

    Luo, Yingquan; Yang, Yu; Zhang, Hui; Zhang, Ting; Wang, Yina; Tan, Shengyu; Xu, Yan; Li, Dan; Ye, Ling; Chen, Ping

    2015-01-01

    Background T cell-induced inflammatory response and related cytokine secretion at the injury site may participate in the pathogenesis of cerebral infarction. Recent studies established inducible co-stimulatory molecule (ICOS) as a novel T cell-related factor for its activation and functions. We thus investigate the role of ICOS in cerebral infarction. Material/Methods The siRNA of ICOS was first used to suppress the gene expression in cultured lymphocytes. An in vivo study was then performed by intravenous application of ICOS siRNA in cerebral infarction rats. Survival rates, neurological scores, serum tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-17 levels were observed. Results The expression of ICOS in cultured lymphocytes was significantly suppressed by siRNA. In the in vivo study, the application of siRNA effectively lowered mortality rates of rats, in addition to the improvement of neurological behaviors and amelioration of cerebral tissue damage. Serum levels of TNF-α, IL-1 and IL-17 were all significantly suppressed after siRNA injection. Conclusions ICOS siRNA can protect brain tissues from ischemia injuries after cerebral infarction, improve limb movement and coordination, lower the mortality rate of rats, and inhibit T cell-induced cytokines. These results collectively suggest the potential treatment efficacy of ICOS siRNA against cerebral infarction. PMID:26436531

  14. Investigation of RNA Polymerase I Transcription under Force-Free Condition by Single Molecule Technique

    NASA Astrophysics Data System (ADS)

    Ucuncuoglu, Suleyman; Schneider, David A.; Dunlap, David; Finzi, Laura

    2014-03-01

    RNA Polymerase I (Pol I) conducts more than 60% of all the transcriptional activity in cells and also is responsible for synthesizing the RNA structure of the ribosome in eukaryotic cells. It is evident in many studies that Pol I transcription is affected by tumor suppressors and oncogenes which makes Pol I as a target for the anticancer therapeutics. The mechanistic pathways and kinetics of the Pol I transcription needs to be understood more precisely. Even though previous bulk studies measured the kinetics of the Pol I transcription, the results may hinder the intermediate states such as processivity and pausing during elongation. Here we used the single molecule approach to show that Pol I pauses more than Pol II during elongation step by using a novel single molecule instrument, multiplexed tethered particle motion microscopy (TPM). Our in-house developed TPM equipment is able to concurrently observe hundreds of single molecules. TPM technique has a major advantage to observe pausing under force-free condition unlike other single molecule techniques such as magnetic tweezers and optical tweezers. We also report that the processivity of Pol I is very low where only one out of fifteen transcription event reached the run-off site. We anticipate that our single molecule assays paved the way for observing more sophisticated aspects of Pol I transcription and it's relation with initiation and transcriptional factors.

  15. In Vivo Target Validation Using Biological Molecules in Drug Development.

    PubMed

    Sim, Derek S; Kauser, Katalin

    2016-01-01

    Drug development is a resource-intensive process requiring significant financial and time investment. Preclinical target validation studies and in vivo testing of the therapeutic molecules in clinically relevant disease models can accelerate and significantly de-risk later stage clinical development. In this chapter, we will focus on (1) in vivo animal models and (2) pharmacological tools for target validation. PMID:26552401

  16. Abiotic synthesis of RNA in water: a common goal of prebiotic chemistry and bottom-up synthetic biology.

    PubMed

    Cafferty, Brian J; Hud, Nicholas V

    2014-10-01

    For more than half a century chemists have searched for a plausible prebiotic synthesis of RNA. The initial advances of the 1960s and 1970s were followed by decades of measured progress and a growing pessimism about overcoming remaining challenges. Fortunately, the past few years have provided a number of important advances, including new abiotic routes for the synthesis of nucleobases, nucleosides, and nucleotides. Recent discoveries also provide additional support for the hypothesis that RNA is the product of evolution, being preceded by ancestral genetic polymers, or pre-RNAs, that are synthesized more easily than RNA. In some cases, parallel searches for plausible prebiotic routes to RNA and pre-RNAs have provided more than one experimentally verified synthesis of RNA substructures and possible predecessors. Just as the synthesis of a contemporary biological molecule cannot be understood without knowledge of cellular metabolism, it is likely that an integrated approach that takes into account both plausible prebiotic reactions and plausible prebiotic environments will ultimately provide the most satisfactory and unifying chemical scenarios for the origin of nucleic acids. In this context, recent advances towards the abiotic synthesis of RNA and candidates for pre-RNAs are beginning to suggest that some molecules (e.g., urea) were multi-faceted contributors to the origin of nucleic acids, and the origin of life. PMID:25438801

  17. Semiconductor Quantum Rods as Single Molecule FluorescentBiological Labels

    SciTech Connect

    Fu, Aihua; Gu, Weiwei; Boussert, Benjamine; Koski, Kristie; Gerion, Daniele; Manna, Liberato; Le Gros, Mark; Larabell, Carolyn; Alivisatos, A. Paul

    2006-05-29

    In recent years, semiconductor quantum dots have beenapplied with great advantage in a wide range of biological imagingapplications. The continuing developments in the synthesis of nanoscalematerials and specifically in the area of colloidal semiconductornanocrystals have created an opportunity to generate a next generation ofbiological labels with complementary or in some cases enhanced propertiescompared to colloidal quantum dots. In this paper, we report thedevelopment of rod shaped semiconductor nanocrystals (quantum rods) asnew fluorescent biological labels. We have engineered biocompatiblequantum rods by surface silanization and have applied them fornon-specific cell tracking as well as specific cellular targeting. Theproperties of quantum rods as demonstrated here are enhanced sensitivityand greater resistance for degradation as compared to quantum dots.Quantum rods have many potential applications as biological labels insituations where their properties offer advantages over quantumdots.

  18. A nickel complex cleaves uridine in folded RNA structures: application to E. coli tmRNA and related engineered molecules.

    PubMed

    Hickerson, R P; Watkins-Sims, C D; Burrows, C J; Atkins, J F; Gesteland, R F; Felden, B

    1998-06-12

    To gain more insight about Escherichia coli tmRNA structure, NiCR, a square planar macrocyclic nickel (II) complex, was used to probe guanine N7 exposure. On the basis of this additional structural information, a refined secondary structure of the molecule is proposed. In addition to its known specificity for guanine N7, we show here that the chemical probe can also cleave at specific uridine residues. In contrast to the alkaline-labile modification of guanine, the reactivity of NiCR at these uridine residues results in direct strand scission. To better characterize the uridine cleavage sites and assess the importance of the RNA structure for the reaction to occur, smaller RNA molecules derived from one pseudoknot (PK4) of E. coli tmRNA containing two uridine cleavage sites were engineered and probed. It is shown that this pseudoknot can fold by itself in solution and that the expected uridine residues are also cleaved by the nickel complex, suggesting that only a local sequence and/or structural context is required for cleavage. In E. coli tmRNA, the five uridine cleavage sites are located in double-stranded regions. These sites contain a G-U wobble base-pair and a downstream uridine which is cleaved. Using smaller RNAs derived from one stem of PK4, systematic changes in the proposed recognition motif indicate that the G-U pair is required for cleavage. Furthermore, there is no cleavage if the G-U pair is reversed. If the recognition motif is moved within the stem, the cleavage site moves accordingly. Additionally, if the recognition motif is changed such that the G-U pair is flanked by two uridine residues, the reactivity occurs only at the 3' uridine. Radical quenching studies have indicated that sulfate radical, as in the case of guanine oxidation, is involved in uridine oxidation. Although additional studies are required to better characterize the reaction, this paper reports a novel specificity for a chemical probe which may be useful for investigating

  19. Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules

    SciTech Connect

    Dugan, L

    2006-02-08

    The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.

  20. Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA

    PubMed Central

    Yang, Wang-Yong; Wilson, Henry D.; Velagapudi, Sai Pradeep

    2016-01-01

    One major class of disease-causing RNAs is expanded repeating transcripts. These RNAs cause diseases via multiple mechanisms, including: (i) gain-of-function, in which repeating RNAs bind and sequester proteins involved in RNA biogenesis and (ii) repeat associated non-ATG (RAN) translation, in which repeating transcripts are translated into toxic proteins without use of a canonical, AUG, start codon. Herein, we develop and study chemical probes that bind and react with an expanded r(CGG) repeat (r(CGG)exp) present in a 5′ untranslated region that causes fragile X-associated tremor/ataxia syndrome (FXTAS). Reactive compounds bind to r(CGG)exp in cellulo as shown with Chem-CLIP-Map, an approach to map small molecule binding sites within RNAs in cells. Compounds also potently improve FXTAS-associated pre-mRNA splicing and RAN translational defects, while not affecting translation of the downstream open reading frame. In contrast, oligonucleotides affect both RAN and canonical translation when they bind to r(CGG)exp, which is mechanistically traced to a decrease in polysome loading. Thus, designer small molecules that react with RNA targets can be used to profile the RNAs to which they bind in cells, including identification of binding sites, and can modulate several aspects of RNA-mediated disease pathology in a manner that may be more beneficial than oligonucleotides. PMID:25825793

  1. Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA.

    PubMed

    Yang, Wang-Yong; Wilson, Henry D; Velagapudi, Sai Pradeep; Disney, Matthew D

    2015-04-29

    One major class of disease-causing RNAs is expanded repeating transcripts. These RNAs cause diseases via multiple mechanisms, including: (i) gain-of-function, in which repeating RNAs bind and sequester proteins involved in RNA biogenesis and (ii) repeat associated non-ATG (RAN) translation, in which repeating transcripts are translated into toxic proteins without use of a canonical, AUG, start codon. Herein, we develop and study chemical probes that bind and react with an expanded r(CGG) repeat (r(CGG)(exp)) present in a 5' untranslated region that causes fragile X-associated tremor/ataxia syndrome (FXTAS). Reactive compounds bind to r(CGG)(exp) in cellulo as shown with Chem-CLIP-Map, an approach to map small molecule binding sites within RNAs in cells. Compounds also potently improve FXTAS-associated pre-mRNA splicing and RAN translational defects, while not affecting translation of the downstream open reading frame. In contrast, oligonucleotides affect both RAN and canonical translation when they bind to r(CGG)(exp), which is mechanistically traced to a decrease in polysome loading. Thus, designer small molecules that react with RNA targets can be used to profile the RNAs to which they bind in cells, including identification of binding sites, and can modulate several aspects of RNA-mediated disease pathology in a manner that may be more beneficial than oligonucleotides. PMID:25825793

  2. Identification of Small Molecule Inhibitors of Pre-mRNA Splicing*

    PubMed Central

    Pawellek, Andrea; McElroy, Stuart; Samatov, Timur; Mitchell, Lee; Woodland, Andrew; Ryder, Ursula; Gray, David; Lührmann, Reinhard; Lamond, Angus I.

    2014-01-01

    Eukaryotic pre-mRNA splicing is an essential step in gene expression for all genes that contain introns. In contrast to transcription and translation, few well characterized chemical inhibitors are available with which to dissect the splicing process, particularly in cells. Therefore, the identification of specific small molecules that either inhibit or modify pre-mRNA splicing would be valuable for research and potentially also for therapeutic applications. We have screened a highly curated library of 71,504 drug-like small molecules using a high throughput in vitro splicing assay. This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. One of these splicing modulators, DDD00107587 (termed “madrasin,” i.e. 2-((7methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one RNAsplicing inhibitor), was studied in more detail. Madrasin interferes with the early stages of spliceosome assembly and stalls spliceosome assembly at the A complex. Madrasin is cytotoxic at higher concentrations, although at lower concentrations it induces cell cycle arrest, promotes a specific reorganization of subnuclear protein localization, and modulates splicing of multiple pre-mRNAs in both HeLa and HEK293 cells. PMID:25281741

  3. 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.

  4. Legume genomics: understanding biology through DNA and RNA sequencing

    PubMed Central

    O'Rourke, Jamie A.; Bolon, Yung-Tsi; Bucciarelli, Bruna; Vance, Carroll P.

    2014-01-01

    Background The legume family (Leguminosae) consists of approx. 17 000 species. A few of these species, including, but not limited to, Phaseolus vulgaris, Cicer arietinum and Cajanus cajan, are important dietary components, providing protein for approx. 300 million people worldwide. Additional species, including soybean (Glycine max) and alfalfa (Medicago sativa), are important crops utilized mainly in animal feed. In addition, legumes are important contributors to biological nitrogen, forming symbiotic relationships with rhizobia to fix atmospheric N2 and providing up to 30 % of available nitrogen for the next season of crops. The application of high-throughput genomic technologies including genome sequencing projects, genome re-sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) by the legume research community has provided major insights into genome evolution, genomic architecture and domestication. Scope and Conclusions This review presents an overview of the current state of legume genomics and explores the role that next-generation sequencing technologies play in advancing legume genomics. The adoption of next-generation sequencing and implementation of associated bioinformatic tools has allowed researchers to turn each species of interest into their own model organism. To illustrate the power of next-generation sequencing, an in-depth overview of the transcriptomes of both soybean and white lupin (Lupinus albus) is provided. The soybean transcriptome focuses on analysing seed development in two near-isogenic lines, examining the role of transporters, oil biosynthesis and nitrogen utilization. The white lupin transcriptome analysis examines how phosphate deficiency alters gene expression patterns, inducing the formation of cluster roots. Such studies illustrate the power of next-generation sequencing and bioinformatic analyses in elucidating the gene networks underlying biological processes. PMID:24769535

  5. Detection of biological molecules using chemical amplification and optical sensors

    SciTech Connect

    Antwerp, W.P. van; Mastrototaro, J.J.

    2000-01-04

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  6. Detection of biological molecules using chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph

    2000-01-01

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  7. Detection of biological molecules using chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph

    2004-10-12

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  8. Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

    PubMed Central

    2011-01-01

    Background We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity. Results We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. Conclusions The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics. PMID:21356118

  9. Inhibition of pathologic immunoglobulin free light chain production by small interfering RNA molecules

    PubMed Central

    Phipps, Jonathan E.; Kestler, Daniel P.; Foster, James S.; Kennel, Stephen J.; Donnell, Robert; Weiss, Deborah T.; Solomon, Alan; Wall, Jonathan S.

    2010-01-01

    Objectives Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal Ig light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved anti-plasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference (RNAi). Materials and Methods SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the CMV promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the ATCC. Both were treated with small interfering RNA (siRNA) directed specifically to the V, J, or C portions of the molecules and then analyzed by ELISA, flow cytometry and real time PCR. Results Transfected cells were found to constitutively express detectable quantities of mRNA and protein Wil and, after exposure to siRNAs, an ~40% reduction in mRNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells. Conclusion Our results have shown that RNAi can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma. PMID:20637260

  10. Spatial Simulations in Systems Biology: From Molecules to Cells

    PubMed Central

    Klann, Michael; Koeppl, Heinz

    2012-01-01

    Cells are highly organized objects containing millions of molecules. Each biomolecule has a specific shape in order to interact with others in the complex machinery. Spatial dynamics emerge in this system on length and time scales which can not yet be modeled with full atomic detail. This review gives an overview of methods which can be used to simulate the complete cell at least with molecular detail, especially Brownian dynamics simulations. Such simulations require correct implementation of the diffusion-controlled reaction scheme occurring on this level. Implementations and applications of spatial simulations are presented, and finally it is discussed how the atomic level can be included for instance in multi-scale simulation methods. PMID:22837728

  11. Spatial simulations in systems biology: from molecules to cells.

    PubMed

    Klann, Michael; Koeppl, Heinz

    2012-01-01

    Cells are highly organized objects containing millions of molecules. Each biomolecule has a specific shape in order to interact with others in the complex machinery. Spatial dynamics emerge in this system on length and time scales which can not yet be modeled with full atomic detail. This review gives an overview of methods which can be used to simulate the complete cell at least with molecular detail, especially Brownian dynamics simulations. Such simulations require correct implementation of the diffusion-controlled reaction scheme occurring on this level. Implementations and applications of spatial simulations are presented, and finally it is discussed how the atomic level can be included for instance in multi-scale simulation methods. PMID:22837728

  12. Detection of biological molecules using chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph

    2001-01-01

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal. Specifically, the analyte transducer immobilized in a polymeric matrix can be a boronic acid moiety.

  13. 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

  14. Small Molecule Targeting of a MicroRNA Associated with Hepatocellular Carcinoma.

    PubMed

    Childs-Disney, Jessica L; Disney, Matthew D

    2016-02-19

    Development of precision therapeutics is of immense interest, particularly as applied to the treatment of cancer. By analyzing the preferred cellular RNA targets of small molecules, we discovered that 5"-azido neomycin B binds the Drosha processing site in the microRNA (miR)-525 precursor. MiR-525 confers invasive properties to hepatocellular carcinoma (HCC) cells. Although HCC is one of the most common cancers, treatment options are limited, making the disease often fatal. Herein, we find that addition of 5"-azido neomycin B and its FDA-approved precursor, neomycin B, to an HCC cell line selectively inhibits production of the mature miRNA, boosts a downstream protein, and inhibits invasion. Interestingly, neomycin B is a second-line agent for hepatic encephalopathy (HE) and bacterial infections due to cirrhosis. Our results provocatively suggest that neomycin B, or second-generation derivatives, may be dual functioning molecules to treat both HE and HCC. Collectively, these studies show that rational design approaches can be tailored to disease-associated RNAs to afford potential lead therapeutics. PMID:26551630

  15. From early lessons to new frontiers: the worm as a treasure trove of small RNA biology.

    PubMed

    Youngman, Elaine M; Claycomb, Julie M

    2014-01-01

    In the past 20 years, the tiny soil nematode Caenorhabditis elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNA interference pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology. PMID:25505902

  16. Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity

    PubMed Central

    Horn, Abigail E.; Kugel, Jennifer F.; Goodrich, James A.

    2016-01-01

    Transcription by RNA polymerase II (Pol II) is a complex process that requires general transcription factors and Pol II to assemble on DNA into preinitiation complexes that can begin RNA synthesis upon binding of NTPs (nucleoside triphosphate). The pathways by which preinitiation complexes form, and how this impacts transcriptional activity are not completely clear. To address these issues, we developed a single molecule system using TIRF (total internal reflection fluorescence) microscopy and purified human transcription factors, which allows us to visualize transcriptional activity at individual template molecules. We see that stable interactions between polymerase II (Pol II) and a heteroduplex DNA template do not depend on general transcription factors; however, transcriptional activity is highly dependent upon TATA-binding protein, TFIIB and TFIIF. We also found that subsets of general transcription factors and Pol II can form stable complexes that are precursors for functional transcription complexes upon addition of the remaining factors and DNA. Ultimately we found that Pol II, TATA-binding protein, TFIIB and TFIIF can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA. Single molecule studies can be used to learn how different modes of preinitiation complex assembly impact transcriptional activity. PMID:27112574

  17. Structure of the myotonic dystrophy type 2 RNA and designed small molecules that reduce toxicity.

    PubMed

    Childs-Disney, Jessica L; Yildirim, Ilyas; Park, HaJeung; Lohman, Jeremy R; Guan, Lirui; Tran, Tuan; Sarkar, Partha; Schatz, George C; Disney, Matthew D

    2014-02-21

    Myotonic dystrophy type 2 (DM2) is an incurable neuromuscular disorder caused by a r(CCUG) expansion (r(CCUG)(exp)) that folds into an extended hairpin with periodically repeating 2×2 nucleotide internal loops (5'CCUG/3'GUCC). We designed multivalent compounds that improve DM2-associated defects using information about RNA-small molecule interactions. We also report the first crystal structure of r(CCUG) repeats refined to 2.35 Å. Structural analysis of the three 5'CCUG/3'GUCC repeat internal loops (L) reveals that the CU pairs in L1 are each stabilized by one hydrogen bond and a water-mediated hydrogen bond, while CU pairs in L2 and L3 are stabilized by two hydrogen bonds. Molecular dynamics (MD) simulations reveal that the CU pairs are dynamic and stabilized by Na(+) and water molecules. MD simulations of the binding of the small molecule to r(CCUG) repeats reveal that the lowest free energy binding mode occurs via the major groove, in which one C residue is unstacked and the cross-strand nucleotides are displaced. Moreover, we modeled the binding of our dimeric compound to two 5'CCUG/3'GUCC motifs, which shows that the scaffold on which the RNA-binding modules are displayed provides an optimal distance to span two adjacent loops. PMID:24341895

  18. Structure of the Myotonic Dystrophy Type 2 RNA and Designed Small Molecules That Reduce Toxicity

    PubMed Central

    Park, HaJeung; Lohman, Jeremy R.; Guan, Lirui; Tran, Tuan; Sarkar, Partha; Schatz, George C.; Disney, Matthew D.

    2014-01-01

    Myotonic dystrophy type 2 (DM2) is an untreatable neuromuscular disorder caused by a r(CCUG) expansion (r(CCUG)exp) that folds into an extended hairpin with periodically repeating 2×2 nucleotide internal loops (5’CCUG/3’GUCC). We designed multivalent compounds that improve DM2-associated defects using information about RNA-small molecule interactions. We also report the first crystal structure of r(CCUG)exp refined to 2.35 Å. Structural analysis of the three 5’CCUG/3’GUCC repeat internal loops (L) reveals that the CU pairs in L1 are each stabilized by one hydrogen bond and a water-mediated hydrogen bond while CU pairs in L2 and L3 are stabilized by two hydrogen bonds. Molecular dynamics (MD) simulations reveal that the CU pairs are dynamic and stabilized by Na+ and water molecules. MD simulations of the binding of the small molecule to r(CCUG) repeats reveal that the lowest free energy binding mode occurs via the major groove, in which one C residue is unstacked and the cross-strand nucleotides are displaced. Moreover, we modeled the binding of our dimeric compound to two 5’CCUG/3’GUCC motifs, which shows that the scaffold on which the RNA-binding modules are displayed provides an optimal distance to span two adjacent loops. PMID:24341895

  19. Cellular and System Biology of Memory: Timing, Molecules, and Beyond.

    PubMed

    Korte, Martin; Schmitz, Dietmar

    2016-04-01

    The storage of information in the mammalian nervous systems is dependent on a delicate balance between change and stability of neuronal networks. The induction and maintenance of processes that lead to changes in synaptic strength to a multistep process which can lead to long-lasting changes, which starts and ends with a highly choreographed and perfectly timed dance of molecules in different cell types of the central nervous system. This is accompanied by synchronization of specific networks, resulting in the generation of characteristic "macroscopic" rhythmic electrical fields, whose characteristic frequencies correspond to certain activity and information-processing states of the brain. Molecular events and macroscopic fields influence each other reciprocally. We review here cellular processes of synaptic plasticity, particularly functional and structural changes, and focus on timing events that are important for the initial memory acquisition, as well as mechanisms of short- and long-term memory storage. Then, we cover the importance of epigenetic events on the long-time range. Furthermore, we consider how brain rhythms at the network level participate in processes of information storage and by what means they participating in it. Finally, we examine memory consolidation at the system level during processes of sleep. PMID:26960344

  20. Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity

    PubMed Central

    Martin, Robert M.; Rino, José; Carvalho, Célia; Kirchhausen, Tomas; Carmo-Fonseca, Maria

    2013-01-01

    SUMMARY Removal of introns from pre-mRNAs via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we have directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that beta-globin introns are transcribed and excised in 20-30 s. We further show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min-1, and that transcription can be rate limiting for splicing. These results have important implications for mechanistic understanding of co-transcriptional splicing regulation in the live-cell context. PMID:24035393

  1. Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

    PubMed Central

    Wu, Xiaoqing; Lan, Lan; Wilson, David Michael; Marquez, Rebecca T.; Tsao, Wei-chung; Gao, Philip; Roy, Anuradha; Turner, Benjamin Andrew; McDonald, Peter; Tunge, Jon A; Rogers, Steven A; Dixon, Dan A.; Aubé, Jeffrey; Xu, Liang

    2015-01-01

    HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3′-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR–ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ~6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR–ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers. PMID:25750985

  2. Manipulating lipid bilayer material properties using biologically active amphipathic molecules

    NASA Astrophysics Data System (ADS)

    Ashrafuzzaman, Md; Lampson, M. A.; Greathouse, D. V.; Koeppe, R. E., II; Andersen, O. S.

    2006-07-01

    Lipid bilayers are elastic bodies with properties that can be manipulated/controlled by the adsorption of amphipathic molecules. The resulting changes in bilayer elasticity have been shown to regulate integral membrane protein function. To further understand the amphiphile-induced modulation of bilayer material properties (thickness, intrinsic monolayer curvature and elastic moduli), we examined how an enantiomeric pair of viral anti-fusion peptides (AFPs)—Z-Gly-D-Phe and Z-Gly-Phe, where Z denotes a benzyloxycarbonyl group, as well as Z-Phe-Tyr and Z-D-Phe-Phe-Gly—alters the function of enantiomeric pairs of gramicidin channels of different lengths in planar bilayers. For both short and long channels, the channel lifetimes and appearance frequencies increase as linear functions of the aqueous AFP concentration, with no apparent effect on the single-channel conductance. These changes in channel function do not depend on the chirality of the channels or the AFPs. At pH 7.0, the relative changes in channel lifetimes do not vary when the channel length is varied, indicating that these compounds exert their effects primarily by causing a positive-going change in the intrinsic monolayer curvature. At pH 4.0, the AFPs are more potent than at pH 7.0 and have greater effects on the shorter channels, indicating that these compounds now change the bilayer elastic moduli. When AFPs of different anti-fusion potencies are compared, the rank order of the anti-fusion activity and the channel-modifying activity is similar, but the relative changes in anti-fusion potency are larger than the changes in channel-modifying activity. We conclude that gramicidin channels are useful as molecular force transducers to probe the influence of small amphiphiles upon lipid bilayer material properties.

  3. Surface functionalization of bioactive glasses with natural molecules of biological significance, Part I: Gallic acid as model molecule

    NASA Astrophysics Data System (ADS)

    Zhang, Xin; Ferraris, Sara; Prenesti, Enrico; Verné, Enrica

    2013-12-01

    Gallic acid (3,4,5-trihydroxybenzoic acid, GA) and its derivatives are a group of biomolecules (polyphenols) obtained from plants. They have effects which are potentially beneficial to heath, for example they are antioxidant, anticarcinogenic and antibacterial, as recently investigated in many fields such as medicine, food and plant sciences. The main drawbacks of these molecules are both low stability and bioavailability. In this research work the opportunity to graft GA to bioactive glasses is investigated, in order to deliver the undamaged biological molecule into the body, using the biomaterial surfaces as a localized carrier. GA was considered for functionalization since it is a good model molecule for polyphenols and presents several interesting biological activities, like antibacterial, antioxidant and anticarcinogenic properties. Two different silica based bioactive glasses (SCNA and CEL2), with different reactivity, were employed as substrates. UV photometry combined with the Folin&Ciocalteu reagent was adopted to test the concentration of GA in uptake solution after functionalization. This test verified how much GA consumption occurred with surface modification and it was also used on solid samples to test the presence of GA on functionalized glasses. XPS and SEM-EDS techniques were employed to characterize the modification of material surface properties and functional group composition before and after functionalization.

  4. DOCK 6: combining techniques to model RNA-small molecule complexes.

    PubMed

    Lang, P Therese; Brozell, Scott R; Mukherjee, Sudipto; Pettersen, Eric F; Meng, Elaine C; Thomas, Veena; Rizzo, Robert C; Case, David A; James, Thomas L; Kuntz, Irwin D

    2009-06-01

    With an increasing interest in RNA therapeutics and for targeting RNA to treat disease, there is a need for the tools used in protein-based drug design, particularly DOCKing algorithms, to be extended or adapted for nucleic acids. Here, we have compiled a test set of RNA-ligand complexes to validate the ability of the DOCK suite of programs to successfully recreate experimentally determined binding poses. With the optimized parameters and a minimal scoring function, 70% of the test set with less than seven rotatable ligand bonds and 26% of the test set with less than 13 rotatable bonds can be successfully recreated within 2 A heavy-atom RMSD. When DOCKed conformations are rescored with the implicit solvent models AMBER generalized Born with solvent-accessible surface area (GB/SA) and Poisson-Boltzmann with solvent-accessible surface area (PB/SA) in combination with explicit water molecules and sodium counterions, the success rate increases to 80% with PB/SA for less than seven rotatable bonds and 58% with AMBER GB/SA and 47% with PB/SA for less than 13 rotatable bonds. These results indicate that DOCK can indeed be useful for structure-based drug design aimed at RNA. Our studies also suggest that RNA-directed ligands often differ from typical protein-ligand complexes in their electrostatic properties, but these differences can be accommodated through the choice of potential function. In addition, in the course of the study, we explore a variety of newly added DOCK functions, demonstrating the ease with which new functions can be added to address new scientific questions. PMID:19369428

  5. Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase

    PubMed Central

    Xu, Hong-Tao; Colby-Germinario, Susan P.; Hassounah, Said; Quashie, Peter K.; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R.

    2015-01-01

    The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. PMID:26574011

  6. Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

    PubMed

    Xu, Hong-Tao; Colby-Germinario, Susan P; Hassounah, Said; Quashie, Peter K; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R; Wainberg, Mark A

    2016-01-01

    The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. PMID:26574011

  7. Determination of the Absolute Number of Cytokine mRNA Molecules within Individual Activated Human T Cells

    NASA Technical Reports Server (NTRS)

    Karr, Laurel J.; Marshall, Gwen; Hockett, Richard D.; Bucy, R. Pat; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    A primary function of activated T cells is the expression and subsequent secretion of cytokines, which orchestrate the differentiation of other lymphocytes, modulate antigen presenting cell activity, and alter vascular endothelium to mediate an immune response. Since many features of immune regulation probably result from modest alterations of endogenous rates of multiple interacting processes, quantitative analysis of the frequency and specific activity of individual T cells is critically important. Using a coordinated set of quantitative methods, the absolute number of molecules of several key cytokine mRNA species in individual T cells has been determined. The frequency of human blood T cells activated in vitro by mitogens and recall protein antigens was determined by intracellular cytokine protein staining, in situ hybridization for cytokine mRNA, and by limiting dilution analysis for cytokine mRNA+ cells. The absolute number of mRNA molecules was simultaneously determined in both homogenates of the entire population of cells and in individual cells obtained by limiting dilution, using a quantitative, competitive RT-PCR assay. The absolute numbers of mRNA molecules in a population of cells divided by the frequency of individual positive cells, yielded essentially the same number of mRNA molecules per cell as direct analysis of individual cells by limiting dilution analysis. Mean numbers of mRNA per positive cell from both mitogen and antigen activated T cells, using these stimulation conditions, were 6000 for IL-2, 6300 for IFN-gamma, and 1600 for IL-4.

  8. Experimental approaches for addressing fundamental biological questions in living, functioning cells with single molecule precision

    PubMed Central

    Lenn, Tchern; Leake, Mark C.

    2012-01-01

    In recent years, single molecule experimentation has allowed researchers to observe biological processes at the sensitivity level of single molecules in actual functioning, living cells, thereby allowing us to observe the molecular basis of the key mechanistic processes in question in a very direct way, rather than inferring these from ensemble average data gained from traditional molecular and biochemical techniques. In this short review, we demonstrate the impact that the application of single molecule bioscience experimentation has had on our understanding of various cellular systems and processes, and the potential that this approach has for the future to really address very challenging and fundamental questions in the life sciences. PMID:22773951

  9. Small molecule BMH-compounds that inhibit RNA polymerase I and cause nucleolar stress.

    PubMed

    Peltonen, Karita; Colis, Laureen; Liu, Hester; Jäämaa, Sari; Zhang, Zhewei; Af Hällström, Taija; Moore, Henna M; Sirajuddin, Paul; Laiho, Marikki

    2014-11-01

    Activation of the p53 pathway has been considered a therapeutic strategy to target cancers. We have previously identified several p53-activating small molecules in a cell-based screen. Two of the compounds activated p53 by causing DNA damage, but this modality was absent in the other four. We recently showed that one of these, BMH-21, inhibits RNA polymerase I (Pol I) transcription, causes the degradation of Pol I catalytic subunit RPA194, and has potent anticancer activity. We show here that three remaining compounds in this screen, BMH-9, BMH-22, and BMH-23, cause reorganization of nucleolar marker proteins consistent with segregation of the nucleolus, a hallmark of Pol I transcription stress. Further, the compounds destabilize RPA194 in a proteasome-dependent manner and inhibit nascent rRNA synthesis and expression of the 45S rRNA precursor. BMH-9- and BMH-22-mediated nucleolar stress was detected in ex vivo-cultured human prostate tissues indicating good tissue bioactivity. Testing of closely related analogues showed that their activities were chemically constrained. Viability screen for BMH-9, BMH-22, and BMH-23 in the NCI60 cancer cell lines showed potent anticancer activity across many tumor types. Finally, we show that the Pol I transcription stress by BMH-9, BMH-22, and BMH-23 is independent of p53 function. These results highlight the dominant impact of Pol I transcription stress on p53 pathway activation and bring forward chemically novel lead molecules for Pol I inhibition, and, potentially, cancer targeting. PMID:25277384

  10. Imaging of single mRNA molecules moving within a living cell nucleus

    SciTech Connect

    Tadakuma, Hisashi; Ishihama, Yo; Shibuya, Toshiharu; Tani, Tokio; Funatsu, Takashi . E-mail: funatsu@mail.ecc.u-tokyo.ac.jp

    2006-06-09

    In eukaryotic cells, pre-mRNAs are transcribed in the nucleus, processed by 5' capping, 3'-polyadenylation, and splicing, and exported to the cytoplasm for translation. To examine the nuclear mRNA transport mechanism, intron-deficient mRNAs of truncated {beta}-globin and EGFP were synthesized, fluorescently labeled in vitro, and injected into the nucleus of living Xenopus A6 cells. The trajectories of single mRNA molecules in the nucleus were visualized using video-rate confocal microscopy. Approximately half the mRNAs moved by Brownian motion in the nucleoplasm, except the nucleoli, with an apparent diffusion coefficient of 0.2 {mu}m{sup 2}/s, about 1/150 of that in water. The slow diffusion could not be explained by simple diffusion obeying the Stokes-Einstein equation, suggesting interactions of the mRNAs with nuclear components. The remaining mRNAs were stationary with an average residence time of about 30 s, comparable to the time required for mRNA diffusion from the site of synthesis to nuclear pores.

  11. A renaissance in RNA synthetic biology: new mechanisms, applications and tools for the future.

    PubMed

    Chappell, James; Watters, Kyle E; Takahashi, Melissa K; Lucks, Julius B

    2015-10-01

    Since our ability to engineer biological systems is directly related to our ability to control gene expression, a central focus of synthetic biology has been to develop programmable genetic regulatory systems. Researchers are increasingly turning to RNA regulators for this task because of their versatility, and the emergence of new powerful RNA design principles. Here we review advances that are transforming the way we use RNAs to engineer biological systems. First, we examine new designable RNA mechanisms that are enabling large libraries of regulators with protein-like dynamic ranges. Next, we review emerging applications, from RNA genetic circuits to molecular diagnostics. Finally, we describe new experimental and computational tools that promise to accelerate our understanding of RNA folding, function and design. PMID:26093826

  12. Targeting Th17 Cells with Small Molecules and Small Interference RNA

    PubMed Central

    Lin, Hui; Song, Pingfang; Zhao, Yi; Xue, Li-Jia; Liu, Yi; Chu, Cong-Qiu

    2015-01-01

    T helper 17 (Th17) cells play a central role in inflammatory and autoimmune diseases via the production of proinflammatory cytokines interleukin- (IL-) 17, IL-17F, and IL-22. Anti-IL-17 monoclonal antibodies show potent efficacy in psoriasis but poor effect in rheumatoid arthritis (RA) and Crohn's disease. Alternative agents targeting Th17 cells may be a better way to inhibit the development and function of Th17 cells than antibodies of blocking a single effector cytokine. Retinoic acid-related orphan receptor gamma t (RORγt) which acts as the master transcription factor of Th17 differentiation has been an attractive pharmacologic target for the treatment of Th17-mediated autoimmune disease. Recent progress in technology of chemical screen and engineering nucleic acid enable two new classes of therapeutics targeting RORγt. Chemical screen technology identified several small molecule specific inhibitors of RORγt from a small molecule library. Systematic evolution of ligands by exponential enrichment (SELEX) technology enabled target specific aptamers to be isolated from a random sequence oligonucleotide library. In this review, we highlight the development and therapeutic potential of small molecules inhibiting Th17 cells by targeting RORγt and aptamer mediated CD4+ T cell specific delivery of small interference RNA against RORγt gene expression to inhibit pathogenic effector functions of Th17 lineage. PMID:26792955

  13. Unbinding forces and energies between a siRNA molecule and a dendrimer measured by force spectroscopy.

    PubMed

    Dumitru, Andra C; Herruzo, Elena T; Rausell, Estrella; Ceña, Valentin; Garcia, Ricardo

    2015-12-21

    We have measured the intermolecular forces between small interference RNA (siRNA) and polyamidoamine dendrimers at the single molecular level. A single molecule force spectroscopy approach has been developed to measure the unbinding forces and energies between a siRNA molecule and polyamidoamine dendrimers deposited on a mica surface in a buffer solution. We report three types of unbinding events which are characterized by forces and free unbinding energies, respectively, of 28 pN, 0.709 eV; 38 pN, 0.722 eV; and 50 pN, 0.724 eV. These events reflect different possible electrostatic interactions between the positive charges of one or two dendrimers and the negatively charged phosphate groups of a single siRNA. We have evidence of a high binding affinity of siRNA towards polyamidoamine dendrimers that leads to a 45% probability of measuring specific unbinding events. PMID:26580848

  14. Unbinding forces and energies between a siRNA molecule and a dendrimer measured by force spectroscopy

    NASA Astrophysics Data System (ADS)

    Dumitru, Andra C.; Herruzo, Elena T.; Rausell, Estrella; Ceña, Valentin; Garcia, Ricardo

    2015-11-01

    We have measured the intermolecular forces between small interference RNA (siRNA) and polyamidoamine dendrimers at the single molecular level. A single molecule force spectroscopy approach has been developed to measure the unbinding forces and energies between a siRNA molecule and polyamidoamine dendrimers deposited on a mica surface in a buffer solution. We report three types of unbinding events which are characterized by forces and free unbinding energies, respectively, of 28 pN, 0.709 eV; 38 pN, 0.722 eV; and 50 pN, 0.724 eV. These events reflect different possible electrostatic interactions between the positive charges of one or two dendrimers and the negatively charged phosphate groups of a single siRNA. We have evidence of a high binding affinity of siRNA towards polyamidoamine dendrimers that leads to a 45% probability of measuring specific unbinding events.

  15. Lights, camera, action! Capturing the spliceosome and pre-mRNA splicing with single-molecule fluorescence microscopy.

    PubMed

    DeHaven, Alexander C; Norden, Ian S; Hoskins, Aaron A

    2016-09-01

    The process of removing intronic sequences from a precursor to messenger RNA (pre-mRNA) to yield a mature mRNA transcript via splicing is an integral step in eukaryotic gene expression. Splicing is carried out by a cellular nanomachine called the spliceosome that is composed of RNA components and dozens of proteins. Despite decades of study, many fundamentals of spliceosome function have remained elusive. Recent developments in single-molecule fluorescence microscopy have afforded new tools to better probe the spliceosome and the complex, dynamic process of splicing by direct observation of single molecules. These cutting-edge technologies enable investigators to monitor the dynamics of specific splicing components, whole spliceosomes, and even cotranscriptional splicing within living cells. WIREs RNA 2016, 7:683-701. doi: 10.1002/wrna.1358 For further resources related to this article, please visit the WIREs website. PMID:27198613

  16. Ribosomal protein S1 induces a conformational change of tmRNA; more than one protein S1 per molecule of tmRNA.

    PubMed

    Bordeau, Valérie; Felden, Brice

    2002-08-01

    tmRNA (10Sa RNA, ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. Ribosomal protein S1 is required for tmRNA binding to isolated and poly U-programmed ribosomes. Mobility assays on native gels indicate that the binding curves of both recombinant and purified proteins S1 from E. coli is biphasic with apparent binding constants of approximately 90 and approximately 300 nM, respectively, suggesting that more than one protein interacts with tmRNA. Structural probing of native tmRNA in the presence and absence of the purified protein suggest that when S1 binds, tmRNA undergoes a significant conformational change. In the presence of the protein, nucleotides from tmRNA with enhanced (H2, H3, PK1, PK2, PK4, in and around the first triplet to be translated), or decreased (H5 and PK2), reactivity towards a probe specific for RNA single-strands are scattered throughout the molecule, with the exception of the tRNA-like domain that may be dispensable for the interaction. Converging experimental evidence suggests that ribosomal protein S1 binds to pseudoknot PK2. Previous structural studies of tmRNA in solution have revealed several discrepancies between the probing data and the phylogeny, and most of these are reconciled when analyzing tmRNA structure in complex with the protein(s). Ribosomal protein(s) S1 is proposed to set tmRNA in the mRNA mode, relieving strains that may develop when translating a looped mRNA. PMID:12457560

  17. Models of single-molecule experiments with periodic perturbations reveal hidden dynamics in RNA folding.

    PubMed

    Li, Ying; Qu, Xiaohui; Ma, Ao; Smith, Glenna J; Scherer, Norbert F; Dinner, Aaron R

    2009-05-28

    Traditionally, microscopic fluctuations of molecules have been probed by measuring responses of an ensemble to perturbations. Now, single-molecule experiments are capable of following fluctuations without introducing perturbations. However, dynamics not readily sampled at equilibrium should be accessible to nonequilibrium single-molecule measurements. In a recent study [Qu, X. et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6602-6607], the efficiency of fluorescence resonance energy transfer (FRET) between probes on the L18 loop and 3' terminus of the 260 nucleotide RNase P RNA from Bacillus stearothermophilus was found to exhibit complex kinetics that depended on the (periodically alternating) concentration of magnesium ions ([Mg2+]) in solution. Specifically, this time series was found to exhibit a quasi-periodic response to a square-wave pattern of [Mg2+] changes. Because these experiments directly probe only one of the many degrees of freedom in the macromolecule, models are needed to interpret these data. We find that Hidden Markov Models are inadequate for describing the nonequilibrium dynamics, but they serve as starting points for the construction of models in which a discrete observable degree of freedom is coupled to a continuously evolving (hidden) variable. Consideration of several models of this general form indicates that the quasi-periodic response in the nonequilibrium experiments results from the switching (back and forth) in positions of the minima of the effective potential for the hidden variable. This switching drives oscillation of that variable and synchronizes the population to the changing [Mg2+]. We set the models in the context of earlier theoretical and experimental studies and conclude that single-molecule experiments with periodic peturbations can indeed yield qualitatively new information beyond that obtained at equilibrium. PMID:19415919

  18. Experimental and Computational Characterization of Biological Liquid Crystals: A Review of Single-Molecule Bioassays

    PubMed Central

    Eom, Kilho; Yang, Jaemoon; Park, Jinsung; Yoon, Gwonchan; Soo Sohn, Young; Park, Shinsuk; Yoon, Dae Sung; Na, Sungsoo; Kwon, Taeyun

    2009-01-01

    Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM) have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins. PMID:19865530

  19. Unintended consequences? Water molecules at biological and crystallographic protein-protein interfaces.

    PubMed

    Ahmed, Mostafa H; Habtemariam, Mesay; Safo, Martin K; Scarsdale, J Neel; Spyrakis, Francesca; Cozzini, Pietro; Mozzarelli, Andrea; Kellogg, Glen E

    2013-12-01

    The importance of protein-protein interactions (PPIs) is becoming increasingly appreciated, as these interactions lie at the core of virtually every biological process. Small molecule modulators that target PPIs are under exploration as new therapies. One of the greatest obstacles faced in crystallographically determining the 3D structures of proteins is coaxing the proteins to form "artificial" PPIs that lead to uniform crystals suitable for X-ray diffraction. This work compares interactions formed naturally, i.e., "biological", with those artificially formed under crystallization conditions or "non-biological". In particular, a detailed analysis of water molecules at the interfaces of high-resolution (≤2.30 Å) X-ray crystal structures of protein-protein complexes, where 140 are biological protein-protein complex structures and 112 include non-biological protein-protein interfaces, was carried out using modeling tools based on the HINT forcefield. Surprisingly few and relatively subtle differences were observed between the two types of interfaces: (i) non-biological interfaces are more polar than biological interfaces, yet there is better organized hydrogen bonding at the latter; (ii) biological associations rely more on water-mediated interactions with backbone atoms compared to non-biological associations; (iii) aromatic/planar residues play a larger role in biological associations with respect to water, and (iv) Lys has a particularly large role at non-biological interfaces. A support vector machines (SVMs) classifier using descriptors from this study was devised that was able to correctly classify 84% of the two interface types. PMID:24076743

  20. Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging

    PubMed Central

    Gahlmann, Andreas; Moerner, W. E.

    2014-01-01

    The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field. PMID:24336182

  1. Sensors and Biosensors for the Determination of Small Molecule Biological Toxins

    PubMed Central

    Wang, Xiang-Hong; Wang, Shuo

    2008-01-01

    The following review of sensors and biosensors focuses on the determination of commonly studied small molecule biological toxins, including mycotoxins and small molecule neurotoxins. Because of the high toxicity of small molecule toxins, an effective analysis technique for determining their toxicity is indispensable. Sensors and biosensors have emerged as sensitive and rapid techniques for toxicity analysis in the past decade. Several different sensors for the determination of mycotoxins and other small molecule neurotoxins have been reported in the literature, and many of these sensors such as tissue biosensors, enzyme sensors, optical immunosensors, electrochemical sensors, quartz crystal sensors, and surface plasmon resonance biosensors are reviewed in this paper. Sensors are a practical and convenient monitoring tool in the area of routine analysis, and their specificity, sensitivity, reproducibility and analysis stability should all be improved in future work. In addition, accuracy field portable sensing devices and multiplexing analysis devices will be important requirement for the future.

  2. Single-molecule tracking of the transcription cycle by sub-second RNA detection

    PubMed Central

    Zhang, Zhengjian; Revyakin, Andrey; Grimm, Jonathan B; Lavis, Luke D; Tjian, Robert

    2014-01-01

    Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (∼10e7 M−1s−1), and allows deterministic detection of single nascent transcripts. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. DOI: http://dx.doi.org/10.7554/eLife.01775.001 PMID:24473079

  3. Dual-Channel Single-Molecule Fluorescence Resonance Energy Transfer to Establish Distance Parameters for RNA Nanoparticles

    PubMed Central

    2010-01-01

    The increasing interest in RNA nanotechnology and the demonstrated feasibility of using RNA nanoparticles as therapeutics have prompted the need for imaging systems with nanometer-scale resolution for RNA studies. Phi29 dimeric pRNAs can serve as building blocks in assembly into the hexameric ring of the nanomotors, as modules of RNA nanoparciles, and as vehicles for specific delivery of therapeutics to cancers or viral infected cells. The understanding of the 3D structure of this novel RNA dimeric particle is fundamentally and practically important. Although a 3D model of pRNA dimer has been proposed based on biochemical analysis, no distance measurements or X-ray diffraction data have been reported. Here we evaluated the application of our customized single-molecule dual-viewing system for distance measurement within pRNA dimers using single-molecule Fluorescence Resonance Energy Transfer (smFRET). Ten pRNA monomers labeled with single donor or acceptor fluorophores at various locations were constructed and eight dimers were assembled. smFRET signals were detected for six dimers. The tethered arm sizes of the fluorophores were estimated empirically from dual-labeled RNA/DNA standards. The distances between donor and acceptor were calculated and used as distance parameters to assess and refine the previously reported 3D model of the pRNA dimer. Distances between nucleotides in pRNA dimers were found to be different from those of the dimers bound to procapsid, suggesting a conformational change of the pRNA dimer upon binding to the procapsid. PMID:20954698

  4. Current advances in Phi29 pRNA biology and its application in drug delivery.

    PubMed

    Ye, Xin; Hemida, Maged; Zhang, Huifang M; Hanson, Paul; Ye, Qiu; Yang, Decheng

    2012-01-01

    Bacteriophage 29 (Phi29) packaging RNA (pRNA) is one of the key components in the viral DNA-packaging motor. It contains two functional domains facilitating the translocation of DNA into the viral capsid by interacting with other elements in the motor and promoting adenosine triphosphates hydrolysis. Through the connection between interlocking loops in adjacent pRNA monomers, pRNA functions in the form of multimer ring in the motor. Previous studies have addressed the unique structure and conformation of pRNA. However, there are different DNA-packaging models proposed for the viral genome transportation mechanism. The DNA-packaging ability and the unique features of pRNA have been attracting efforts to study its potential applications in nanotechnology. The pRNA has been proved to be a promising tool for delivering nucleic acid-based therapeutic molecules by covalent linkage with ribozymes, small interfering RNAs, aptamers, and artificial microRNAs. The flexibility in constructing dimers, trimers, and hexamers enables the assembly of polyvalent nanoparticles to carry drug molecules for therapeutic purposes, cell ligands for target delivery, image detector for drug entry monitoring, and endosome disrupter for drug release. Besides these fascinating pharmacological advantages, pRNA-based drug delivery has also been demonstrated to prolong the drug half life with minimal induction of immune response and toxicity. PMID:22362726

  5. Suppression and enhancement of non-native molecules within biological systems

    NASA Astrophysics Data System (ADS)

    Jones, E. A.; Lockyer, N. P.; Vickerman, J. C.

    2006-07-01

    With the aim of evaluating the potential of SIMS to provide molecular information from small molecules within biological systems, here we investigate the effect of different biological compounds as they act as matrices. The results highlight the fact that the chemical environment of a molecule can have a significant effect on its limit of detection. This has implications for the imaging of drugs and xenobiotics in tissue sections and other biological matrices. A 1:1 mixture of the organic acid 2,4,6-trihydroxyacetophenone and the dipeptide valine-valine demonstrates that almost complete suppression of the [M + H] + ion of one compound can be caused by the presence of a compound of higher proton affinity. The significance of this is highlighted when two similar drug molecules, atropine (a neutral molecule) and ipratropium bromide (a quaternary nitrogen containing salt) are mixed with brain homogenate. The atropine [M + H] + ion shows significant suppression whilst the [M - Br] + of ipratopium bromide is detected at an intensity that can be rationalised by its decreased surface concentration. By investigating the effect of two abundant tissue lipids, cholesterol and dipalmitoylphosphatidyl choline (DPPC), on the atropine [M + H] + signal detected in mixtures with these lipids we see that the DPPC has a strong suppressing effect, which may be attributed to gas phase proton transfer.

  6. Non-coding RNA: a new frontier in regulatory biology

    PubMed Central

    Fu, Xiang-Dong

    2015-01-01

    A striking finding in the past decade is the production of numerous non-coding RNAs (ncRNAs) from mammalian genomes. While it is entirely possible that many of those ncRNAs are transcription noises or by-products of RNA processing, increasing evidence suggests that a large fraction of them are functional and provide various regulatory activities in the cell. Thus, functional genomics and proteomics are incomplete without understanding functional ribonomics. As has been long suggested by the ‘RNA world’ hypothesis, many ncRNAs have the capacity to act like proteins in diverse biochemical processes. The enormous amount of information residing in the primary sequences and secondary structures of ncRNAs makes them particularly suited to function as scaffolds for molecular interactions. In addition, their functions appear to be stringently controlled by default via abundant nucleases when not engaged in specific interactions. This review focuses on the functional properties of regulatory ncRNAs in comparison with proteins and emphasizes both the opportunities and challenges in future ncRNA research. PMID:25821635

  7. Multicomponent redox catalysts for reduction of large biological molecules using molecular hydrogen as the reductant

    SciTech Connect

    Chao, S.; Simon, R.A.; Mallouk, T.E.; Wrighton, M.S.

    1988-03-30

    One-electron reduction of the large biological molecules horse heart cytochrome c, sperm whale myoglobin, and horseradish peroxidase using H/sub 2/ as the reductant can be catalyzed by two-component, high surface area heterogeneous catalysts. The catalysts can be prepared by first functionalizing high surface area SiO/sub 2/ with a polycationic polymer into which is dispersed MCl/sub 4//sup 2 -/ (M = Pd, Pt). Reduction with H/sub 2/ yields elemental Pd or Pt dispersed in the polymer. The particles are finally functionalized with a redox polymer derived from hydrolysis of Si(OR)/sub 3/ groups of an N,N'-dialkyl-4,4'-bipyridinium- or from a cobalticenium-based monomer. The two components of the heterogeneous catalysts are the buried noble metal capable of activating the H/sub 2/ and the redox polymer, which can equilibrate both with the noble metal and with the large biological molecule. Reduction of the large biological molecules in aqueous solution can be effected at room temperature and 1 atm H/sub 2/ using the catalysts under conditions where the biological materials would not be reducible with H/sub 2/ alone or when the noble metal alone would be used as the catalyst.

  8. Electronic and Transport Properties of Quasi-1D Wires of Biological Molecules

    NASA Astrophysics Data System (ADS)

    Oetzel, Björn; Matthes, Lars; Tandetzky, Falk; Ortmann, Frank; Bechstedt, Friedhelm; Hannewald, Karsten

    2010-03-01

    In the search for organic materials with good charge-transport properties, artificial stacks of biological molecules are considered attractive candidates [1,2]. In this spirit, we present ab-initio DFT calculations of the structural, electronic, and quantum-transport properties of quasi-1D wires based on guanine and eumelanin molecules [3]. Hereby, a special focus is put on the results for the electronic bandwidths and the consequences for potential applications. [4pt] [1] R. di Felice et al., Phys. Rev. B 65, 045104 (2001) [0pt] [2] P. Meredith et al., Pigment Cell Res. 19, 572 (2006) [0pt] [3] B. Oetzel et al. (unpublished)

  9. Modulation of Host Biology by Pseudomonas aeruginosa Quorum Sensing Signal Molecules: Messengers or Traitors

    PubMed Central

    Liu, Yi-Chia; Chan, Kok-Gan; Chang, Chien-Yi

    2015-01-01

    Bacterial cells sense their population density and respond accordingly by producing various signal molecules to the surrounding environments thereby trigger a plethora of gene expression. This regulatory pathway is termed quorum sensing (QS). Plenty of bacterial virulence factors are controlled by QS or QS-mediated regulatory systems and QS signal molecules (QSSMs) play crucial roles in bacterial signaling transduction. Moreover, bacterial QSSMs were shown to interfere with host cell signaling and modulate host immune responses. QSSMs not only regulate the expression of bacterial virulence factors but themselves act in the modulation of host biology that can be potential therapeutic targets. PMID:26617576

  10. Ambient pollutants, polymorphisms associated with microRNA processing and adhesion molecules: the Normative Aging Study

    PubMed Central

    2011-01-01

    Background Particulate air pollution has been associated with cardiovascular morbidity and mortality, but it remains unclear which time windows and pollutant sources are most critical. MicroRNA (miRNA) is thought to be involved in cardiovascular regulation. However, little is known about whether polymorphisms in genes that process microRNAs influence response to pollutant exposure. We hypothesized that averaging times longer than routinely measured one or two day moving averages are associated with higher soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) levels, and that stationary and mobile sources contribute differently to these effects. We also investigated whether single nucleotide polymorphisms (SNPs) in miRNA-processing genes modify these associations. Methods sICAM-1 and sVCAM-1 were measured from 1999-2008 and matched to air pollution monitoring for fine particulate matter (PM2.5) black carbon, and sulfates (SO42-). We selected 17 SNPs in five miRNA-processing genes. Mixed-effects models were used to assess effects of pollutants, SNPs, and interactions under recessive inheritance models using repeated measures. Results 723 participants with 1652 observations and 1-5 visits were included in our analyses for black carbon and PM2.5. Sulfate data was available for 672 participants with 1390 observations. An interquartile range change in seven day moving average of PM2.5 (4.27 μg/m3) was associated with 3.1% (95%CI: 1.6, 4.6) and 2.5% (95%CI: 0.6, 4.5) higher sICAM-1 and sVCAM-1. Interquartile range changes in sulfates (1.39 μg/m3) were associated with 1.4% higher (95%CI: 0.04, 2.7) and 1.6% (95%CI: -0.4, 3.7) higher sICAM-1 and sVCAM-1 respectively. No significant associations were observed for black carbon. In interaction models with PM2.5, both sICAM-1 and sVCAM-1 levels were lower in rs1062923 homozygous carriers. These interactions remained significant after multiple comparisons adjustment. Conclusions PM

  11. Mass amplifying probe for sensitive fluorescence anisotropy detection of small molecules in complex biological samples.

    PubMed

    Cui, Liang; Zou, Yuan; Lin, Ninghang; Zhu, Zhi; Jenkins, Gareth; Yang, Chaoyong James

    2012-07-01

    detection of small molecules by means of FA in complex biological samples. PMID:22686244

  12. siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

    PubMed Central

    Bett, John S.; Ibrahim, Adel F. M.; Garg, Amit K.; Rocha, Sonia; Hay, Ronald T.

    2014-01-01

    Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.  To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA “ubiquitome” library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.  Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.  An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening.  The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.  Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments. PMID:24893647

  13. Site-specific fluorescent probing of RNA molecules by unnatural base-pair transcription for local structural conformation analysis.

    PubMed

    Hikida, Yasushi; Kimoto, Michiko; Yokoyama, Shigeyuki; Hirao, Ichiro

    2010-07-01

    Methods for fluorescent probing at a defined position of RNA provide powerful tools for analyzing the local structural conformation of functional RNA molecules by tracking fluorescence changes. In this article, we describe the site-specific fluorescent probing of RNA by transcription with an expanded genetic alphabet, using an extra, unnatural base pair between 2-amino-6-(2-thienyl)purine (s) and pyrrole-2-carbaldehyde (Pa). The protocol comprises template DNA preparation containing Pa, transcription involving fluorescent s incorporation and structural analysis of transcripts. The s base is strongly fluorescent, and its nucleoside 5'-triphosphate is site-specifically incorporated into RNA transcripts, opposite Pa in DNA templates, by conventional T7 transcription. The fluorescent intensity of s changes depending on its environment around the probe site, providing clues about the local structural features of RNA molecules. This is the first protocol for RNA transcript preparation with fluorescent labeling at a desired position. The procedure for s-containing RNA preparation takes about 2-3 d. PMID:20595959

  14. MicroRNA Biomarkers of Toxicity in Biological Matrices.

    PubMed

    Harrill, Alison H; McCullough, Shaun D; Wood, Charles E; Kahle, Juliette J; Chorley, Brian N

    2016-08-01

    Biomarker measurements that reliably correlate with tissue injury and that can be measured within accessible biofluids offer benefits in terms of cost, time, and convenience when assessing chemical and drug-induced toxicity in model systems or human cohorts. MicroRNAs (miRNAs) have emerged in recent years as a promising new class of biomarker for monitoring toxicity. Recent enthusiasm for miRNA biomarker research has been fueled by evidence that certain miRNAs are cell-type specific and are released during injury, thus raising the possibility of using biofluid-based miRNAs as a "liquid biopsy" that may be obtained by sampling extracellular fluids. As biomarkers, miRNAs demonstrate improved stability as compared with many protein markers and sequences are largely conserved across species, simplifying analytical techniques. Recent efforts have sought to identify miRNAs that are released into accessible biofluids following xenobiotic exposure, using compounds that target specific organs. Whereas still early in the discovery phase, miRNA biomarkers will have an increasingly important role in the assessment of adverse effects of both environmental chemicals and pharmaceutical drugs. Here, we review the current findings of biofluid-based miRNAs, as well as highlight technical challenges in assessing toxicologic pathology using these biomarkers. PMID:27462126

  15. Directly measuring single molecule heterogeneity in proteins and RNA using force spectroscopy

    NASA Astrophysics Data System (ADS)

    Hinczewski, Michael; Hyeon, Changbong; Thirumalai, Devarajan

    One of the most intriguing results of single molecule experiments on proteins and nucleic acids is the discovery of functional heterogeneity: the observation that complex cellular machines exhibit multiple, biologically active conformations. The structural differences between these conformations may be subtle, but each distinct state can be remarkably long-lived, with stochastic interconversions occurring only at macroscopic timescales, fractions of a second or longer. Though we now have proof of functional heterogeneity in a handful of systems--enzymes, motors, adhesion complexes--identifying and measuring it remains a formidable challenge. We show that evidence of this phenomenon is more widespread than previously known, encoded in data collected from some of the most well-established single molecule techniques: AFM or optical tweezer pulling experiments. We present a theoretical procedure for analyzing distributions of rupture/unfolding forces recorded at different pulling speeds. This results in a single parameter, quantifying the degree of heterogeneity, and also leads to bounds on the equilibration and conformational interconversion timescales. Our work suggests experimental approaches for estimating the timescales of these fluctuations with unprecedented accuracy.

  16. Singlet oxygen production from the reactions of ozone with biological molecules

    SciTech Connect

    Kanofsky, J.R.; Sima, P. )

    1991-05-15

    The reaction of ozone with a number of biological molecules was found to produce singlet oxygen in high yield. At pH 7.0, the reaction of ozone with an equimolar amount of biological molecule produced the following singlet oxygen yields (mole of singlet oxygen/mole of ozone): cysteine, 0.49 +/- 0.02; methionine, 1.13 +/- 0.11; reduced glutathione, 0.33 +/- 0.02; albumin, 1.00 +/- 0.05; uric acid, 0.64 +/- 0.09; ascorbic acid, 0.96 +/- 0.007; NADPH, 1.07 +/- 0.07; NADH, 0.95 +/- 0.01. Thus, singlet oxygen may be an important intermediate in the biochemical damage caused by ozone.

  17. Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

    PubMed

    Guzman-Villanueva, Diana; El-Sherbiny, Ibrahim M; Vlassov, Alexander V; Herrera-Ruiz, Dea; Smyth, Hugh D C

    2014-10-01

    The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases. However, the siRNA therapy has been limited by its low stability and rapid degradation in presence of nucleases, low cellular uptake, and immune response activation. In order to overcome these drawbacks, we propose the synthesis and characterization of non-viral delivery systems using chitosan derivatives to obtain siRNA complexes (polyplexes). The non-viral delivery systems synthesized included PEG-g-OCs (oligochitosan) and PEG-g-Cs (chitosan medium molecular weight). Both systems allowed the formation of siRNA polyplexes, increased the stability of siRNA in the presence of nucleases, enhanced cellular internalization, and showed low toxicity in the A549 cell line. Finally, the complexes obtained with the PEG-g-OCs system showed silencing activity in a GFP model in the cell line A549 in comparison with naked siRNA. PMID:25063077

  18. The corrosion inhibition of iron and aluminum by various naturally occurring biological molecules

    SciTech Connect

    McCafferty, E.; Hansen, D.C.

    1995-12-31

    Biological polymers that exhibit a strong affinity for metal surfaces are increasingly becoming the focus of research toward the development of environmentally friendly corrosion inhibitors. This paper deals with the use of various naturally occurring organic molecules as corrosion inhibitors for iron or aluminum. Among the organic molecules considered are catecholate and hydroxamate siderophores isolated from bacteria, the adhesive protein from the blue mussel Mytilus edulis L, and caffeic acid and chlorogenic acid. FTIR analysis, anodic polarization curves, and AC impedance measurements were used to determine the adsorption and effectiveness of the various organic molecules as corrosion inhibitors. Parabactin, a catecholate siderophore, was effective in inhibiting both the corrosion of iron in hydrochloric acid and the pitting of aluminum in 0.1 M sodium chloride. The adhesive protein from the blue mussel was also effective in inhibiting the pitting of aluminum.

  19. Structure-property relationship of quinuclidinium surfactants--Towards multifunctional biologically active molecules.

    PubMed

    Skočibušić, Mirjana; Odžak, Renata; Štefanić, Zoran; Križić, Ivana; Krišto, Lucija; Jović, Ozren; Hrenar, Tomica; Primožič, Ines; Jurašin, Darija

    2016-04-01

    Motivated by diverse biological and pharmacological activity of quinuclidine and oxime compounds we have synthesized and characterized novel class of surfactants, 3-hydroxyimino quinuclidinium bromides with different alkyl chains lengths (CnQNOH; n=12, 14 and 16). The incorporation of non conventional hydroxyimino quinuclidinium headgroup and variation in alkyl chain length affects hydrophilic-hydrophobic balance of surfactant molecule and thereby physicochemical properties important for its application. Therefore, newly synthesized surfactants were characterized by the combination of different experimental techniques: X-ray analysis, potentiometry, electrical conductivity, surface tension and dynamic light scattering measurements, as well as antimicrobial susceptibility tests. Comprehensive investigation of CnQNOH surfactants enabled insight into structure-property relationship i.e., way in which the arrangement of surfactant molecules in the crystal phase correlates with their solution behavior and biologically activity. The synthesized CnQNOH surfactants exhibited high adsorption efficiency and relatively low critical micelle concentrations. In addition, all investigated compounds showed very potent and promising activity against Gram-positive and clinically relevant Gram-negative bacterial strains compared to conventional antimicrobial agents: tetracycline and gentamicin. The overall results indicate that bicyclic headgroup with oxime moiety, which affects both hydrophilicity and hydrophobicity of CnQNOH molecule in addition to enabling hydrogen bonding, has dominant effect on crystal packing and physicochemical properties. The unique structural features of cationic surfactants with hydroxyimino quinuclidine headgroup along with diverse biological activity have made them promising structures in novel drug discovery. Obtained fundamental understanding how combination of different functionalities in a single surfactant molecule affects its physicochemical

  20. Graphene as a transparent conductive support for studying biological molecules by transmission electron microscopy

    SciTech Connect

    Nair, R. R.; Anissimova, S.; Novoselov, K. S.; Blake, P.; Blake, J. R.; Geim, A. K.; Zan, R.; Bangert, U.; Golovanov, A. P.; Morozov, S. V.; Latychevskaia, T.

    2010-10-11

    We demonstrate the application of graphene as a support for imaging individual biological molecules in transmission electron microscope (TEM). A simple procedure to produce free-standing graphene membranes has been designed. Such membranes are extremely robust and can support practically any submicrometer object. Tobacco mosaic virus has been deposited on graphene samples and observed in a TEM. High contrast has been achieved even though no staining has been applied.

  1. Small molecule activators of pre-mRNA 3′ cleavage

    PubMed Central

    Ryan, Kevin; Khleborodova, Asya; Pan, Jingyi; Ryan, Xiaozhou P.

    2009-01-01

    3′ Cleavage and polyadenylation are obligatory steps in the biogenesis of most mammalian pre-mRNAs. In vitro reconstitution of the 3′ cleavage reaction from human cleavage factors requires high concentrations of creatine phosphate (CP), though how CP activates cleavage is not known. Previously, we proposed that CP might work by competitively inhibiting a cleavage-suppressing serine/threonine (S/T) phosphatase. Here we show that fluoride/EDTA, a general S/T phosphatase inhibitor, activates in vitro cleavage in place of CP. Subsequent testing of inhibitors specific for different S/T phosphatases showed that inhibitors of the PPM family of S/T phosphatases, which includes PP2C, but not the PPP family, which includes PP1, PP2A, and PP2B, activated 3′ cleavage in vitro. In particular, NCI 83633, an inhibitor of PP2C, activated extensive 3′ cleavage at a concentration 50-fold below that required by fluoride or CP. The testing of structural analogs led to the identification of a more potent compound that activated 3′ cleavage at 200 μM. While testing CP analogs to understand the origin of its cleavage activation effect, we found phosphocholine to be a more effective activator than CP. The minimal structural determinants of 3′ cleavage activation by phosphocholine were identified. Our results describe a much improved small molecule activator of in vitro pre-mRNA cleavage, identify the molecular determinants of cleavage activation by phosphoamines such as phosphocholine, and suggest that a PPM family phosphatase is involved in the negative regulation of mammalian pre-mRNA 3′ cleavage. PMID:19155323

  2. Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency.

    PubMed

    Maddox, J F; Aherne, K M; Reddy, C C; Sordillo, L M

    1999-05-01

    Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation. Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome. Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH-Px) and plays an important role in the maintenance of the redox state of a cell. Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland. However, the mechanisms by which Se modulates neutrophil migration have not been elucidated. Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC. The effect of Se on functional activities was examined by measuring neutrophil adherence to Se-deficient and Se-supplemented EC. Se-deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor alpha (TNF-alpha) for 4 or 24 h, interleukin-1 for 12 h, or H2O2 for 20 min (P < 0.05). To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM-1, E-selectin, and P-selectin were examined at the molecular level by a competitive reverse transcription-polymerase chain reaction. Results revealed higher mRNA expression for E-selectin and ICAM-1 in Se-deficient EC stimulated with TNF-alpha for 3 and 6 h, and greater expression of P-selectin mRNA in Se-supplemented EC with 3-h TNF-alpha stimulation. These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium. PMID:10331495

  3. Potential siRNA Molecules for Nucleoprotein and M2/L Overlapping Region of Respiratory Syncytial Virus: In Silico Design

    PubMed Central

    Shatizadeh Malekshahi, Somayeh; Arefian, Ehsan; Salimi, Vahid; Mokhtari Azad, Talat; Yavarian, Jila

    2016-01-01

    Background Human respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in the pediatric population, elderly and in immunosuppressed individuals. Respiratory syncytial virus is also responsible for bronchiolitis, pneumonia, and chronic obstructive pulmonary infections in all age groups. With this high disease burden and the lack of an effective RSV treatment and vaccine, there is a clear need for discovery and development of novel, effective and safe drugs to prevent and treat RSV disease. The most innovative approach is the use of small interfering RNAs (siRNAs) which represent a revolutionary new concept in human therapeutics. The nucleoprotein gene of RSV which is known as the most conserved gene and the M2/L mRNA, which encompass sixty-eight overlapping nucleotides, were selected as suitable targets for siRNA design. Objectives The present study is aimed to design potential siRNAs for silencing nucleoprotein and an overlapping region of M2-L coding mRNAs by computational analysis. Materials and Methods Various computational methods (target alignment, similarity search, secondary structure prediction, and RNA interaction calculation) have been used for siRNA designing against different strains of RSV. Results In this study, seven siRNA molecules were rationally designed against the nucleoprotein gene and validated using various computational methods for silencing different strains of RSV. Additionally, three effective siRNA molecules targeting the overlapping region of M2/L mRNA were designed. Conclusions This approach provides insight and a validated strategy for chemical synthesis of an antiviral RNA molecule which meets many sequence features for efficient silencing and treatment at the genomic level. PMID:27303618

  4. 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. PMID:27378726

  5. Photoelectron spectra of some important biological molecules: symmetry-adapted-cluster configuration interaction study.

    PubMed

    Farrokhpour, Hossein; Ghandehari, Maryam

    2013-05-23

    In this work, the valence vertical ionization energies (up to 5) of some important biologically active molecules including 2,4-dinitrophenol, 2,4-dinitroanisole, nicotinic acid, nicotinic acid methyl ester, nicotinamide, N,N-diethylnicotinamide, barbituric acid, uric acid, cytosine, β-carotene, and menadione were calculated in the gas phase and compared with the experimental data reported in the literature. The symmetry-adapted-cluster configuration interaction (SAC-CI) general-R method was used to calculate the ionization energies. The intensity of each ionization band was evaluated using the monopole approximation. Comparison of the calculated photoelectron spectrum of each molecule with its corresponding experimental spectra allowed for assigning the photoelectron bands by natural bonding orbital (NBO) calculations even though some of the associated bands were significantly overlapped for some molecules. Among the considered molecules, there was no agreement between the experimental and calculated photoelectron spectrum of β-carotene. The reason for this disagreement was theoretically investigated and attributed to the degradation and decomposition of β-carotene. The calculated first ionization energies of the considered molecules were correlated with their Hückel k-index to obtain Coulomb (α) and resonance (β) integrals of the Hückel molecular orbital theory for the biomolecules considered in this study. A linear correlation was found between the first ionization energy and the Hückel k-index. PMID:23659524

  6. Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS

    NASA Astrophysics Data System (ADS)

    Nagy, Julia; Grohmann, Dina; Cheung, Alan C. M.; Schulz, Sarah; Smollett, Katherine; Werner, Finn; Michaelis, Jens

    2015-01-01

    The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Förster resonance energy transfer and the Bayesian parameter estimation-based Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH.

  7. Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS.

    PubMed

    Nagy, Julia; Grohmann, Dina; Cheung, Alan C M; Schulz, Sarah; Smollett, Katherine; Werner, Finn; Michaelis, Jens

    2015-01-01

    The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Förster resonance energy transfer and the Bayesian parameter estimation-based Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH. PMID:25635909

  8. Fluorescence imaging for bacterial cell biology: from localization to dynamics, from ensembles to single molecules.

    PubMed

    Yao, Zhizhong; Carballido-López, Rut

    2014-01-01

    Fluorescent proteins and developments in superresolution (nanoscopy) and single-molecule techniques bring high sensitivity, speed, and one order of magnitude gain in spatial resolution to live-cell imaging. These technologies have only recently been applied to prokaryotic cell biology, revealing the exquisite subcellular organization of bacterial cells. Here, we review the parallel evolution of fluorescence microscopy methods and their application to bacteria, mainly drawing examples from visualizing actin-like MreB proteins in the model bacterium Bacillus subtilis. We describe the basic principles of nanoscopy and conventional techniques and their advantages and limitations to help microbiologists choose the most suitable technique for their biological question. Looking ahead, multidimensional live-cell nanoscopy combined with computational image analysis tools, systems biology approaches, and mathematical modeling will provide movie-like, mechanistic, and quantitative description of molecular events in bacterial cells. PMID:25002084

  9. Efficient read-through of Tn9 and IS1 by RNA polymerase molecules that initiate at rRNA promoters.

    PubMed Central

    Siehnel, R J; Morgan, E A

    1983-01-01

    Transcription and translation are coupled in most Escherichia coli operons. As a consequence, ribosomes must be present on an mRNA molecule while transcription of the mRNA is in progress or else premature termination of transcription may result. This requirement is most clearly manifested when premature nonsense codons result in polarity in multicistronic operons. Polarity can also result from insertions of transposons and insertion sequences. However, since rRNA operons are not translated, some property of these operons must allow transcription to be uncoupled from translation. In this paper we demonstrate that transposon Tn9 and insertion sequence IS1 are nonpolar or incompletely polar in rRNA operons during normal growth. We also show that essentially all expression of rrn sequences distal to IS1 and Tn9 results from transcripts that originate at rRNA promoters. These results suggest either that rRNA operons possess some mechanism which reduces or prevents termination within rRNA operons or that Tn9 and IS1 can be very inefficient at blocking normal transcription. Insertions of Tn10 in rRNA operons are substantially but incompletely polar. We could not determine whether the residual downstream transcription observed results from promoters within Tn10 or from read-through of Tn10. We discuss the meaning of read-through of Tn9 and IS1 and the residual expression of genes downstream from Tn10 with regard to rRNA operon structure and previous experiments in which polarity of transposons or insertion sequences was observed in protein-encoding operons. Images PMID:6185465

  10. Single-molecule detection and tracking of RNA transcripts in living cells using phosphorothioate-optimized 2'-O-methyl RNA molecular beacons.

    PubMed

    Zhao, Dan; Yang, Yantao; Qu, Na; Chen, Mingming; Ma, Zhao; Krueger, Christopher J; Behlke, Mark A; Chen, Antony K

    2016-09-01

    Molecular Beacons (MBs) composed of 2'-O-methyl RNA (2Me) and phosphorothioate (PS) linkages throughout the backbone (2Me/PSFULL MBs) have enabled long-term imaging of RNA in living cells, but excess PS modification can induce nonspecific binding, causing false-positive signals. In this study, we evaluate the intracellular stability of MBs composed of 2Me with various PS modifications, and found that false-positive signals could be reduced to marginal levels when the MBs possess a fully PS-modified loop domain and a phosphodiester stem (2Me/PSLOOP MB). Additionally, 2Me/PSLOOP MBs exhibited uncompromised hybridization kinetics, prolonged functionality and >88% detection accuracy for single RNA transcripts, and could do so without interfering with gene expression or cell growth. Finally, 2Me/PSLOOP MBs could image the dynamics of single mRNA transcripts in the nucleus and the cytoplasm simultaneously, regardless of whether the MBs targeted the 5'- or the 3'-UTR. Together, these findings demonstrate the effectiveness of loop-domain PS modification in reducing nonspecific signals and the potential for sensitive and accurate imaging of individual RNAs at the single-molecule level. With the growing interest in the role of RNA localization and dynamics in health and disease, 2Me/PSLOOP MBs could enable new discoveries in RNA research. PMID:27261815

  11. A human haploid gene trap collection to study lncRNAs with unusual RNA biology

    PubMed Central

    Kornienko, Aleksandra E.; Vlatkovic, Irena; Neesen, Jürgen; Barlow, Denise P; Pauler, Florian M.

    2016-01-01

    ABSTRACT Many thousand long non-coding (lnc) RNAs are mapped in the human genome. Time consuming studies using reverse genetic approaches by post-transcriptional knock-down or genetic modification of the locus demonstrated diverse biological functions for a few of these transcripts. The Human Gene Trap Mutant Collection in haploid KBM7 cells is a ready-to-use tool for studying protein-coding gene function. As lncRNAs show remarkable differences in RNA biology compared to protein-coding genes, it is unclear if this gene trap collection is useful for functional analysis of lncRNAs. Here we use the uncharacterized LOC100288798 lncRNA as a model to answer this question. Using public RNA-seq data we show that LOC100288798 is ubiquitously expressed, but inefficiently spliced. The minor spliced LOC100288798 isoforms are exported to the cytoplasm, whereas the major unspliced isoform is nuclear localized. This shows that LOC100288798 RNA biology differs markedly from typical mRNAs. De novo assembly from RNA-seq data suggests that LOC100288798 extends 289kb beyond its annotated 3' end and overlaps the downstream SLC38A4 gene. Three cell lines with independent gene trap insertions in LOC100288798 were available from the KBM7 gene trap collection. RT-qPCR and RNA-seq confirmed successful lncRNA truncation and its extended length. Expression analysis from RNA-seq data shows significant deregulation of 41 protein-coding genes upon LOC100288798 truncation. Our data shows that gene trap collections in human haploid cell lines are useful tools to study lncRNAs, and identifies the previously uncharacterized LOC100288798 as a potential gene regulator. PMID:26670263

  12. The Juxtaposition of Ribose Hydroxyl Groups: The Root of Biological Catalysis and the RNA World?

    NASA Astrophysics Data System (ADS)

    Bernhardt, Harold S.

    2015-06-01

    We normally think of enzymes as being proteins; however, the RNA world hypothesis suggests that the earliest biological catalysts may have been composed of RNA. One of the oldest surviving RNA enzymes we are aware of is the peptidyl transferase centre (PTC) of the large ribosomal RNA, which joins amino acids together to form proteins. Recent evidence indicates that the enzymatic activity of the PTC is principally due to ribose 2 '-OHs. Many other reactions catalyzed by RNA and/or in which RNA is a substrate similarly utilize ribose 2 '-OHs, including phosphoryl transfer reactions that involve the cleavage and/or ligation of the ribose-phosphate backbone. It has recently been proposed by Yakhnin (2013) that phosphoryl transfer reactions were important in the prebiotic chemical evolution of RNA, by enabling macromolecules composed of polyols joined by phosphodiester linkages to undergo recombination reactions, with the reaction energy supplied by the phosphodiester bond itself. The almost unique juxtaposition of the ribose 2'-hydroxyl and 3'-oxygen in ribose-containing polymers such as RNA, which gives ribose the ability to catalyze such reactions, may have been an important factor in the selection of ribose as a component of the first biopolymer. In addition, the juxtaposition of hydroxyl groups in free ribose: (i) allows coordination of borate ions, which could have provided significant and preferential stabilization of ribose in a prebiotic environment; and (ii) enhances the rate of permeation by ribose into a variety of lipid membrane systems, possibly favouring its incorporation into early metabolic pathways and an ancestral ribose-phosphate polymer. Somewhat more speculatively, hydrogen bonds formed by juxtaposed ribose hydroxyl groups may have stabilized an ancestral ribose-phosphate polymer against degradation (Bernhardt and Sandwick 2014). I propose that the almost unique juxtaposition of ribose hydroxyl groups constitutes the root of both biological

  13. Screening for Small-Molecule Modulators of Long Noncoding RNA-Protein Interactions Using AlphaScreen

    PubMed Central

    Pedram Fatemi, Roya; Salah-Uddin, Sultan; Modarresi, Farzaneh; Khoury, Nathalie; Wahlestedt, Claes

    2015-01-01

    Long non–protein coding RNAs (lncRNAs) are an important class of molecules that help orchestrate key cellular events. Although their functional roles in cells are not well understood, thousands of lncRNAs and a number of possible mechanisms by which they act have been reported. LncRNAs can exert their regulatory function in cells by interacting with epigenetic enzymes. In this study, we developed a tool to study lncRNA-protein interactions for high-throughput screening of small-molecule modulators using AlphaScreen technology. We tested the interaction of two lncRNAs: brain-derived neurotrophic factor antisense (BDNF-AS) and Hox transcript antisense RNA (HOTAIR), with Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase against a phytochemical library, to look for small-molecule inhibitors that can alter the expression of downstream target genes. We identified ellipticine, a compound that up-regulates BDNF transcription. Our study shows the feasibility of using high-throughput screening to identify modulators of lncRNA-protein interactions and paves the road for targeting lncRNAs that are dysregulated in human disorders using small-molecule therapies. PMID:26173710

  14. Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules.

    PubMed

    Childs-Disney, Jessica L; Stepniak-Konieczna, Ewa; Tran, Tuan; Yildirim, Ilyas; Park, HaJeung; Chen, Catherine Z; Hoskins, Jason; Southall, Noel; Marugan, Juan J; Patnaik, Samarjit; Zheng, Wei; Austin, Chris P; Schatz, George C; Sobczak, Krzysztof; Thornton, Charles A; Disney, Matthew D

    2013-01-01

    The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)(exp)). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. A thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)(exp) and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)(exp). This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)(exp) and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of myotonic dystrophy type 1. PMID:23806903

  15. Induction and Reversal of Myotonic Dystrophy Type 1 Pre-mRNA Splicing Defects by Small Molecules

    PubMed Central

    Childs-Disney, Jessica L.; Stepniak-Konieczna, Ewa; Tran, Tuan; Yildirim, Ilyas; Park, HaJeung; Chen, Catherine Z.; Hoskins, Jason; Southall, Noel; Marugan, Juan J.; Patnaik, Samarjit; Zheng, Wei; Austin, Chris P.; Schatz, George C.; Sobczak, Krzysztof; Thornton, Charles A.; Disney, Matthew D.

    2013-01-01

    The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 (DM1) is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)exp). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. The thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)exp and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)exp. This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)exp and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of DM1. PMID:23806903

  16. Engineering Bacteria to Search for Specific Concentrations of Molecules by a Systematic Synthetic Biology Design Method

    PubMed Central

    Chen, Bor-Sen

    2016-01-01

    Bacteria navigate environments full of various chemicals to seek favorable places for survival by controlling the flagella’s rotation using a complicated signal transduction pathway. By influencing the pathway, bacteria can be engineered to search for specific molecules, which has great potential for application to biomedicine and bioremediation. In this study, genetic circuits were constructed to make bacteria search for a specific molecule at particular concentrations in their environment through a synthetic biology method. In addition, by replacing the “brake component” in the synthetic circuit with some specific sensitivities, the bacteria can be engineered to locate areas containing specific concentrations of the molecule. Measured by the swarm assay qualitatively and microfluidic techniques quantitatively, the characteristics of each “brake component” were identified and represented by a mathematical model. Furthermore, we established another mathematical model to anticipate the characteristics of the “brake component”. Based on this model, an abundant component library can be established to provide adequate component selection for different searching conditions without identifying all components individually. Finally, a systematic design procedure was proposed. Following this systematic procedure, one can design a genetic circuit for bacteria to rapidly search for and locate different concentrations of particular molecules by selecting the most adequate “brake component” in the library. Moreover, following simple procedures, one can also establish an exclusive component library suitable for other cultivated environments, promoter systems, or bacterial strains. PMID:27096615

  17. Core-shell nanoparticles as scavengers for hydrophobic molecules in biological systems

    NASA Astrophysics Data System (ADS)

    Wong, Jeffrey

    2005-11-01

    Core-shell nanoparticles containing hydrophobic cores and hydrophilic shells were synthesized based on a tertiary microemulsion system. Due to their hydrophobic interior, this system is proposed to work as a scavenger of non polar species, like pesticides or drugs present in concentrations higher than what the body can tolerate. The ability to control the core and shell sizes using different molecules to fill the core was studied with different techniques. Hydrophobic fluorescent dyes were used as model non polar molecules to study the uptake abilities of the system synthesized. Derivatization of the surface of the core-shell particles with different groups, including biotin and poly(ethylene glycol) was performed to improve the biocompatibility of the system. Binding to streptavidin via the biotin units on the surface was performed to study the biocompatibility of the derivatized nanocapsules in biological relevant systems.

  18. Single-molecule Spectroscopy: Exploring Heterogeneity in Chemical and Biological Systems.

    PubMed

    Ghosh, Shirsendu; Bhattacharyya, Kankan

    2016-04-01

    Many chemical and biological systems are heterogeneous in the molecular length scale (∼ 1 nm). Heterogeneity in many chemical systems and organized assemblies may be monitored using single-molecule spectroscopy (SMS). In SMS, the size of the focal spot (i.e., the smallest region to be probed) is nearly half of the excitation wavelength (λ/2, i.e., 200-375 nm) for visible light (400-750 nm). We discuss how one can get spatial resolutions better than 200 nm using molecules as nanometric probes. We show that polymer hydrogels, lipid vesicles, room temperature ionic liquids (RTILs), and binary liquid mixtures exhibit such heterogeneity. Another important observation is solute-dependent friction in RTILs. In an RTIL, diffusion of an ionic solute is slower than that of a neutral solute. PMID:26814020

  19. Ab Initio Calculations of the Electronic Structures and Biological Functions of Protein Molecules

    NASA Astrophysics Data System (ADS)

    Zheng, Haoping

    The self-consistent cluster-embedding (SCCE) calculation method reduces the computational effort from M3 to about M1 (M is the number of atoms in the system) with precise calculations. Thus the ab initio, all-electron calculation of the electronic structure and biological function of protein molecule has become a reality, which will promote new proteomics considerably. The calculated results of two real protein molecules, the trypsin inhibitor from the seeds of squash Cucurbita maxima (CMTI-I, 436 atoms) and the ascaris trypsin inhibitor (912 atoms, two three-dimensional structures), will be presented in this paper. The reactive sites of the inhibitors are determined and explained. The accuracy of structure determination of the inhibitors are tested theoretically.

  20. Ab Initio Calculations of the Electronic Structures and Biological Functions of Protein Molecules

    NASA Astrophysics Data System (ADS)

    Zheng, Haoping

    2003-04-01

    The self-consistent cluster-embedding (SCCE) calculation method reduces the computational effort from M3 to about M1 (M is the number of atoms in the system) with unchanged calculation precision. So the ab initio, all-electron calculation of the electronic structure and biological function of protein molecule becomes a reality, which will promote new proteomics considerably. The calculated results of two real protein molecules, the trypsin inhibitor from the seeds of squash Cucurbita maxima (CMTI-I, 436 atoms) and the Ascaris trypsin inhibitor (912 atoms, two three-dimensional structures), are presented. The reactive sites of the inhibitors are determined and explained. The precision of structure determination of inhibitors are tested theoretically.

  1. RNA around the clock – regulation at the RNA level in biological timing

    PubMed Central

    Nolte, Christine; Staiger, Dorothee

    2015-01-01

    The circadian timing system in plants synchronizes their physiological functions with the environment. This is achieved by a global control of gene expression programs with a considerable part of the transcriptome undergoing 24-h oscillations in steady-state abundance. These circadian oscillations are driven by a set of core clock proteins that generate their own 24-h rhythm through periodic feedback on their own transcription. Additionally, post-transcriptional events are instrumental for oscillations of core clock genes and genes in clock output. Here we provide an update on molecular events at the RNA level that contribute to the 24-h rhythm of the core clock proteins and shape the circadian transcriptome. We focus on the circadian system of the model plant Arabidopsis thaliana but also discuss selected regulatory principles in other organisms. PMID:25999975

  2. Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle.

    PubMed

    Skinner, Gary M; Baumann, Christoph G; Quinn, Diana M; Molloy, Justin E; Hoggett, James G

    2004-01-30

    A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape. PMID:14597619

  3. New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins

    PubMed Central

    Mannion, Niamh; Arieti, Fabiana; Gallo, Angela; Keegan, Liam P.; O’Connell, Mary A.

    2015-01-01

    The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Inosine can have a profound effect on the RNAs that are edited, not only changing the base-pairing properties, but can also result in recoding, as inosine behaves as if it were guanosine. In mammals there are three ADAR proteins and two ADAR-related proteins (ADAD) expressed. All have a very similar modular structure; however, both their expression and biological function differ significantly. Only two of the ADAR proteins have enzymatic activity. However, both ADAR and ADAD proteins possess the ability to bind double-strand RNA. Mutations in ADARs have been associated with many diseases ranging from cancer, innate immunity to neurological disorders. Here, we will discuss in detail the domain structure of mammalian ADARs, the effects of RNA editing, and the role of ADARs in human diseases. PMID:26437436

  4. Pulse requirements for x-ray diffraction imaging of single biological molecules

    SciTech Connect

    Hau-Riege, S; London, R; Huldt, G; Chapman, H

    2005-01-05

    It has been suggested that x-ray free electron lasers will enable single-particle diffraction imaging of biological molecules. In this paper we present a model to estimate the required pulse parameters based on a trade-off between minimizing image degradation due to damage and maximizing the image signal-to-noise ratio. We discuss several means to alleviate the photon requirements, and compare the requirements with existing or planned x-ray sources such as short-pulse x-ray free-electron lasers.

  5. Long noncoding RNAs: Novel molecules in cardiovascular biology, disease and regeneration.

    PubMed

    Hou, Jingying; Zhou, Changqing; Long, Huibao; Zheng, Shaoxin; Guo, Tianzhu; Wu, Quanhua; Wu, Hao; Zhong, Tingting; Wang, Tong

    2016-06-01

    Remarkable breakthroughs made in genomic technologies have facilitated the discovery of thousands of novel transcripts that do not template protein synthesis. Numerous RNAs termed as long noncoding RNAs (lncRNAs) generated from this pervasive transcription function vividly in gene regulatory networks and a variety of biological and cellular processes. Here, we make a brief description of the known and putative functions of lncRNAs in cardiovascular biology and disease. The association between lncRNAs and stem cells mediated cardiomyocytes differentiation and neovascularization is discussed then. It will provide a new clue for further studies on these novel molecules in cardiovascular disease and bring bright prospects for their future applications in cardiac regenerative medicine. PMID:27180105

  6. Small Molecule Binding, Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

    SciTech Connect

    Hames, Mary C; McFeeters, Hana; Holloway, W Blake; Stanley, Christopher B; Urban, Volker S; McFeeters, Robert L

    2013-01-01

    Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contributing to understanding how Pth1 interacts with its substrate, advancing the current model for cleavage, and demonstrating feasibility of small molecule inhibition.

  7. The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules.

    PubMed Central

    van Noort, J M; Duisterwinkel, F J; Jonák, J; Sedlácek, J; Kraal, B; Bosch, L

    1982-01-01

    The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6765192

  8. The Non-Coding RNA Ontology (NCRO): a comprehensive resource for the unification of non-coding RNA biology.

    PubMed

    Huang, Jingshan; Eilbeck, Karen; Smith, Barry; Blake, Judith A; Dou, Dejing; Huang, Weili; Natale, Darren A; Ruttenberg, Alan; Huan, Jun; Zimmermann, Michael T; Jiang, Guoqian; Lin, Yu; Wu, Bin; Strachan, Harrison J; He, Yongqun; Zhang, Shaojie; Wang, Xiaowei; Liu, Zixing; Borchert, Glen M; Tan, Ming

    2016-01-01

    In recent years, sequencing technologies have enabled the identification of a wide range of non-coding RNAs (ncRNAs). Unfortunately, annotation and integration of ncRNA data has lagged behind their identification. Given the large quantity of information being obtained in this area, there emerges an urgent need to integrate what is being discovered by a broad range of relevant communities. To this end, the Non-Coding RNA Ontology (NCRO) is being developed to provide a systematically structured and precisely defined controlled vocabulary for the domain of ncRNAs, thereby facilitating the discovery, curation, analysis, exchange, and reasoning of data about structures of ncRNAs, their molecular and cellular functions, and their impacts upon phenotypes. The goal of NCRO is to serve as a common resource for annotations of diverse research in a way that will significantly enhance integrative and comparative analysis of the myriad resources currently housed in disparate sources. It is our belief that the NCRO ontology can perform an important role in the comprehensive unification of ncRNA biology and, indeed, fill a critical gap in both the Open Biological and Biomedical Ontologies (OBO) Library and the National Center for Biomedical Ontology (NCBO) BioPortal. Our initial focus is on the ontological representation of small regulatory ncRNAs, which we see as the first step in providing a resource for the annotation of data about all forms of ncRNAs. The NCRO ontology is free and open to all users, accessible at: http://purl.obolibrary.org/obo/ncro.owl. PMID:27152146

  9. Probing Solvation Dynamics around Aromatic and Biological Molecules at the Single-Molecular Level.

    PubMed

    Dopfer, Otto; Fujii, Masaaki

    2016-05-11

    Solvation processes play a crucial role in chemical reactions and biomolecular recognition phenomena. Although solvation dynamics of interfacial or biological water has been studied extensively in aqueous solution, the results are generally averaged over several solvation layers and the motion of individual solvent molecules is difficult to capture. This review describes the development and application of a new experimental approach, namely, picosecond time-resolved pump-probe infrared spectroscopy of size- and isomer-selected aromatic clusters, in which for the first time the dynamics of a single individual solvent molecule can be followed in real time. The intermolecular isomerization reaction is triggered by resonant photoionization (pump), and infrared photodissociation (probe) at variable delay generates the spectroscopic signature of salient properties of the reaction, including rates, yields, pathways, branching ratios of competing reactions, existence of reaction intermediates, occurrence of back reactions, and time scales of energy relaxation processes. It is shown that this relevant information can reliably be decoded from the experimental spectra by sophisticated molecular dynamics simulations. This review covers a description of the experimental strategies and spectroscopic methods along with all applications to date, which range from aromatic clusters with nonpolar solvent molecules to aromatic monohydrated biomolecules. PMID:27054835

  10. Molecules in interstellar clouds. [physical and chemical conditions of star formation and biological evolution

    NASA Technical Reports Server (NTRS)

    Irvine, W. M.; Hjalmarson, A.; Rydbeck, O. E. H.

    1981-01-01

    The physical conditions and chemical compositions of the gas in interstellar clouds are reviewed in light of the importance of interstellar clouds for star formation and the origin of life. The Orion A region is discussed as an example of a giant molecular cloud where massive stars are being formed, and it is pointed out that conditions in the core of the cloud, with a kinetic temperature of about 75 K and a density of 100,000-1,000,000 molecules/cu cm, may support gas phase ion-molecule chemistry. The Taurus Molecular Clouds are then considered as examples of cold, dark, relatively dense interstellar clouds which may be the birthplaces of solar-type stars and which have been found to contain the heaviest interstellar molecules yet discovered. The molecular species identified in each of these regions are tabulated, including such building blocks of biological monomers as H2O, NH3, H2CO, CO, H2S, CH3CN and H2, and more complex species such as HCOOCH3 and CH3CH2CN.

  11. Application of Fourier transform infrared ellipsometry to assess the concentration of biological molecules.

    PubMed

    Garcia-Caurel, Enric; Drévillon, Bernard; De Martino, Antonello; Schwartz, Laurent

    2002-12-01

    Spectroscopic ellipsometry is a noninvasive optical characterization technique mainly used in the semiconductor field to characterize bare substrates and thin films. In particular, it allows the gathering of information concerning the physical structure of the sample, such as roughness and film thickness, as well as its optical response. In the mid-infrared (IR) range each molecule exhibits a characteristic absorption fingerprint, which makes this technique chemically selective. Phase-modulated IR ellipsometry does not require a baseline correction procedure or suppression of atmospheric CO2 and water-vapor absorption bands, thus greatly reducing the subjectivity in data analysis. We have found that ellipsometric measurements of thin films, such as the solid residuals left on a plane surface after evaporation of a liquid drop containing a given compound in solution, are particularly favorable for dosing purposes because the intensity of IR absorptions shows a linear behavior along a wide range of solution concentrations of the given compound. Our aim is to illustrate with a concrete example and to justify theoretically the linearity experimentally found between radiation absorption and molecule concentration. For the example, we prepared aqueous solutions of glycogen, a molecule of huge biological importance currently tested in biochemical analyses, at concentrations ranging from 1 mg/l to 1 g/l which correspond to those found in physiological conditions. The results of this example are promising for the application of ellipsometry for dosing purposes in biochemistry and biomedicine. PMID:12477127

  12. Next generation techniques in the high resolution spectroscopy of biologically relevant molecules.

    PubMed

    Neill, Justin L; Douglass, Kevin O; Pate, Brooks H; Pratt, David W

    2011-04-28

    Recent advances in the technology of test and measurement equipment driven by the computer and telecommunications industries have made possible the development of a new broadband, Fourier-transform microwave spectrometer that operates on principles similar to FTNMR. This technique uses a high sample-rate arbitrary waveform generator to construct a phase-locked chirped microwave pulse that gives a linear frequency sweep over a wide frequency range in 1 μs. The chirped pulse efficiently polarizes the molecular sample at all frequencies lying within this band. The subsequent free induction decay of this polarization is measured with a high-speed digitizer and then fast Fourier-transformed to yield a broadband, frequency-resolved rotational spectrum, spanning up to 11.5 GHz and containing lines that are as narrow as 100 kHz. This new technique is called chirped-pulse Fourier transform microwave (CP-FTMW) spectroscopy. The technique offers the potential to determine the structural and dynamical properties of very large molecules solely from fully resolved pure rotational spectra. FTMW double resonance techniques employing a low-resolution UV laser facilitate an easy assignment of overlapping spectra produced by different conformers in the sample. Of particular interest are the energy landscapes of conformationally flexible molecules of biological importance, including studies of their interaction with solvent and/or other weakly bound molecules. An example is provided from the authors' work on p-methoxyphenethylamine, a neurotransmitter, and its complexes with water. PMID:21394332

  13. Single-molecule RNA observation in vivo reveals dynamics of co-transcriptional splicing

    NASA Astrophysics Data System (ADS)

    Ferguson, M. L.; Coulon, A.; de Turris, V.; Palangat, M.; Chow, C. C.; Singer, R. H.; Larson, D. R.

    2013-03-01

    The synthesis of pre-mRNA and the splicing of that pre-mRNA to form completed transcripts requires coordination between two large multi-subunit complexes (the transcription elongation complex and the spliceosome). How this coordination occurs in vivo is unknown. Here we report the first experimental observation of transcription and splicing occurring at the same gene in living cells. By utilizing the PP7/MS2 fluorescent RNA reporter system, we can directly observe two distinct regions of the nascent RNA, allowing us to measure the rise and fall time of the intron and exon of a reporter gene stably integrated into a human cell line. The reporter gene consists of a beta globin gene where we have inserted a 24 RNA hairpin cassette into the intron/exon. Upon synthesis, the RNA hairpins are tightly bound by fluorescently-labeled PP7/MS2 bacteriophage coat proteins. After gene induction, a single locus of active transcription in the nucleus shows fluorescence intensity changes characteristic of the synthesis and excision of the intron/exon. Using fluctuation analysis, we determine the elongation rate to be 1.5 kb/min. From the temporal cross correlation function, we determine that splicing of this gene must be co-transcriptional with a splicing time of ~100 seconds before termination and a ~200 second pause at termination. We propose that dual-color RNA imaging may be extended to investigate other mechanisms of transcription, gene regulation, and RNA processing.

  14. The T box mechanism: tRNA as a regulatory molecule

    PubMed Central

    Green, Nicholas J.; Grundy, Frank J.; Henkin, Tina M.

    2009-01-01

    The T box mechanism is widely used in Gram-positive bacteria to regulate expression of aminoacyl-tRNA synthetase genes and genes involved in amino acid biosynthesis and uptake. Binding of a specific uncharged tRNA to a riboswitch element in the nascent transcript causes a structural change in the transcript that promotes expression of the downstream coding sequence. In most cases, this occurs by stabilization of an antiterminator element that competes with formation of a terminator helix. Specific tRNA recognition by the nascent transcript results in increased expression of genes important for tRNA aminoacylation in response to decreased pools of charged tRNA. PMID:19932103

  15. Of social molecules: The interactive assembly of ASH1 mRNA-transport complexes in yeast

    PubMed Central

    Niedner, Annika; Edelmann, Franziska T; Niessing, Dierk

    2014-01-01

    Asymmetric, motor-protein dependent transport of mRNAs and subsequent localized translation is an important mechanism of gene regulation. Due to the high complexity of such motile particles, our mechanistic understanding of mRNA localization is limited. Over the last two decades, ASH1 mRNA localization in budding yeast has served as comparably simple and accessible model system. Recent advances have helped to draw an increasingly clear picture on the molecular mechanisms governing ASH1 mRNA localization from its co-transcriptional birth to its delivery at the site of destination. These new insights help to better understand the requirement of initial nuclear mRNPs, the molecular basis of specific mRNA-cargo recognition via cis-acting RNA elements, the different stages of RNP biogenesis and reorganization, as well as activation of the motile activity upon cargo binding. We discuss these aspects in context of published findings from other model organisms. PMID:25482892

  16. High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

    PubMed Central

    Chen, Jin; Dalal, Ravindra V.; Petrov, Alexey N.; Tsai, Albert; O’Leary, Seán E.; Chapin, Karen; Cheng, Janice; Ewan, Mark; Hsiung, Pei-Lin; Lundquist, Paul; Turner, Stephen W.; Hsu, David R.; Puglisi, Joseph D.

    2014-01-01

    Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community. PMID:24379388

  17. Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA.

    PubMed

    Weitzmann, C J; Cunningham, P R; Ofengand, J

    1990-06-25

    The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed. PMID:2194163

  18. Single-Molecule Observation of the Induction of k-Turn RNA Structure on Binding L7Ae Protein

    PubMed Central

    Wang, Jia; Fessl, Tomáš; Schroeder, Kersten T.; Ouellet, Jonathan; Liu, Yijin; Freeman, Alasdair D.J.; Lilley, David M.J.

    2012-01-01

    The k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection of the kinked structure, or a more active process in which the protein manipulates the RNA structure. We have devised a single-molecule experiment whereby immobilized L7Ae protein binds Cy3-Cy5-labeled RNA from free solution. We find that all bound RNA is in the kinked geometry, with no evidence for transitions to an extended form at the millisecond timescale of the camera. Furthermore, real-time binding experiments provide no evidence for a more extended intermediate even at the earliest times, at a time resolution of 16 ms. The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form. PMID:23260056

  19. Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact.

    PubMed

    Karousis, Evangelos D; Nasif, Sofia; Mühlemann, Oliver

    2016-09-01

    Nonsense-mediated mRNA decay (NMD) was originally coined to define a quality control mechanism that targets mRNAs with truncated open reading frames due to the presence of a premature termination codon. Meanwhile, it became clear that NMD has a much broader impact on gene expression and additional biological functions beyond quality control are continuously being discovered. We review here the current views regarding the molecular mechanisms of NMD, according to which NMD ensues on mRNAs that fail to terminate translation properly, and point out the gaps in our understanding. We further summarize the recent literature on an ever-rising spectrum of biological processes in which NMD appears to be involved, including homeostatic control of gene expression, development and differentiation, as well as viral defense. WIREs RNA 2016, 7:661-682. doi: 10.1002/wrna.1357 For further resources related to this article, please visit the WIREs website. PMID:27173476

  20. Single Molecule Detection in Living Biological Cells using Carbon Nanotube Optical Probes

    NASA Astrophysics Data System (ADS)

    Strano, Michael

    2009-03-01

    Nanoscale sensing elements offer promise for single molecule analyte detection in physically or biologically constrained environments. Molecular adsorption can be amplified via modulation of sharp singularities in the electronic density of states that arise from 1D quantum confinement [1]. Single-walled carbon nanotubes (SWNT), as single molecule optical sensors [2-3], offer unique advantages such as photostable near-infrared (n-IR) emission for prolonged detection through biological media, single-molecule sensitivity and, nearly orthogonal optical modes for signal transduction that can be used to identify distinct classes of analytes. Selective binding to the SWNT surface is difficult to engineer [4]. In this lecture, we will briefly review the immerging field of fluorescent diagnostics using band gap emission from SWNT. In recent work, we demonstrate that even a single pair of SWNT provides at least four optical modes that can be modulated to uniquely fingerprint chemical agents by the degree to which they alter either the emission band intensity or wavelength. We validate this identification method in vitro by demonstrating detection and identification of six genotoxic analytes, including chemotherapeutic drugs and reactive oxygen species (ROS), which are spectroscopically differentiated into four distinct classes. We also demonstrate single-molecule sensitivity in detecting hydrogen peroxide, one of the most common genotoxins and an important cellular signal. Finally, we employ our sensing and fingerprinting method of these analytes in real time within live 3T3 cells, demonstrating the first multiplexed optical detection from a nanoscale biosensor and the first label-free tool to optically discriminate between genotoxins. We will also discuss our recent efforts to fabricate biomedical sensors for real time detection of glucose and other important physiologically relevant analytes in-vivo. The response of embedded SWNT in a swellable hydrogel construct to

  1. Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash

    2008-12-01

    Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2+) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells

  2. Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.

    PubMed

    Rodriguez-Manzano, Jesus; Karymov, Mikhail A; Begolo, Stefano; Selck, David A; Zhukov, Dmitriy V; Jue, Erik; Ismagilov, Rustem F

    2016-03-22

    Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests. PMID:26900709

  3. RNA. Introduction.

    PubMed

    Bao, Marie Z; Kruger, Robert P; Rivas, Fabiola; Smith, Orla; Szewczak, Lara

    2009-02-20

    Two scientists walk into a bar. After a pint and an exchange of pleasantries, one says to the other, "Where do you come from? Scientifically, I mean." The queried scientist responds, "Out of the RNA world." "Don't we all," the asker responds chuckling. Fifteen years ago, the joke would have been made with a nod to the notion that life arose from an RNA-based precursor, the so-called "RNA world." Yet had this conversation happened last week, the scientists would also be grinning in appreciation of the extent to which contemporary cellular biology is steeped in all things RNA. Ours is truly an RNA world.In this year's special review issue, the Cell editorial team has brought together articles focused on RNA in the modern world, providing perspectives on classical and emerging areas of inquiry. We extend our thanks to the many distinguished experts who contributed their time and effort as authors and reviewers to make the issue informative, thought-provoking, and timely. We hope that this collection of articles, written as we stand on the verge of a new wave of RNA biology, edifies and inspires by revealing the inner workings of these versatile molecules and by highlighting the next key questions that need to be addressed as we strive to understand the full functional scope of RNA in cells. PMID:19263588

  4. RNAiFold 2.0: a web server and software to design custom and Rfam-based RNA molecules

    PubMed Central

    Garcia-Martin, Juan Antonio; Dotu, Ivan; Clote, Peter

    2015-01-01

    Several algorithms for RNA inverse folding have been used to design synthetic riboswitches, ribozymes and thermoswitches, whose activity has been experimentally validated. The RNAiFold software is unique among approaches for inverse folding in that (exhaustive) constraint programming is used instead of heuristic methods. For that reason, RNAiFold can generate all sequences that fold into the target structure or determine that there is no solution. RNAiFold 2.0 is a complete overhaul of RNAiFold 1.0, rewritten from the now defunct COMET language to C++. The new code properly extends the capabilities of its predecessor by providing a user-friendly pipeline to design synthetic constructs having the functionality of given Rfam families. In addition, the new software supports amino acid constraints, even for proteins translated in different reading frames from overlapping coding sequences; moreover, structure compatibility/incompatibility constraints have been expanded. With these features, RNAiFold 2.0 allows the user to design single RNA molecules as well as hybridization complexes of two RNA molecules. Availability: the web server, source code and linux binaries are publicly accessible at http://bioinformatics.bc.edu/clotelab/RNAiFold2.0. PMID:26019176

  5. Surface-enhanced Raman spectroscopy at single-molecule scale and its implications in biology.

    PubMed

    Wang, Yuling; Irudayaraj, Joseph

    2013-02-01

    Single-molecule (SM) spectroscopy has been an exciting area of research offering significant promise and hope in the field of sensor development to detect targets at ultra-low levels down to SM resolution. To the experts and developers in the field of surface-enhanced Raman spectroscopy (SERS), this has often been a challenge and a significant opportunity for exploration. Needless to say, the opportunities and excitement of this multidisciplinary area impacts span the fields of physics, chemistry and engineering, along with a significant thrust in applications constituting areas in medicine, biology, environment and agriculture among others. In this review, we will attempt to provide a quick snapshot of the basics of SM-SERS, nanostructures and devices that can enable SM Raman measurement. We will conclude with a discussion on SERS implications in biomedical sciences. PMID:23267180

  6. Accelerating the Discovery of Biologically Active Small Molecules Using a High-Throughput Yeast Halo Assay#

    PubMed Central

    Gassner, Nadine C.; Tamble, Craig M.; Bock, Jonathan E.; Cotton, Naomi; White, Kimberly N.; Tenney, Karen; St. Onge, Robert P.; Proctor, Michael J.; Giaever, Guri; Davis, Ronald W.; Crews, Phillip; Holman, Theodore R.; Lokey, R. Scott

    2008-01-01

    The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3,104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation resulting in the identification of crambescidin 800 as a potent antifungal agent. PMID:17291044

  7. Developing novel organocatalyzed aldol reactions for the enantioselective synthesis of biologically active molecules

    PubMed Central

    Bhanushali, Mayur; Zhao, Cong-Gui

    2011-01-01

    Aldol reaction is one of the most important methods for the formation of carbon-carbon bonds. Because of its significance and usefulness, asymmetric versions of this reaction have been realized with different approaches in the past. Over the last decade, the area of organocatalysis has made significant progresses. As one of most studied reactions in organocatalyses, organocatalyzed aldol reaction has emerged as a powerful tool for the synthesis of a large number of useful products in optically enriched forms. In this review, we summarize our efforts on the development of novel organocatalyzed aldol reactions for the enantioselective synthesis of biological active molecules. Literatures closely related to our studies are also covered. PMID:21918584

  8. Surface-enhanced Raman spectroscopy at single-molecule scale and its implications in biology

    PubMed Central

    Wang, Yuling; Irudayaraj, Joseph

    2013-01-01

    Single-molecule (SM) spectroscopy has been an exciting area of research offering significant promise and hope in the field of sensor development to detect targets at ultra-low levels down to SM resolution. To the experts and developers in the field of surface-enhanced Raman spectroscopy (SERS), this has often been a challenge and a significant opportunity for exploration. Needless to say, the opportunities and excitement of this multidisciplinary area impacts span the fields of physics, chemistry and engineering, along with a significant thrust in applications constituting areas in medicine, biology, environment and agriculture among others. In this review, we will attempt to provide a quick snapshot of the basics of SM-SERS, nanostructures and devices that can enable SM Raman measurement. We will conclude with a discussion on SERS implications in biomedical sciences. PMID:23267180

  9. CD22ΔE12 as a molecular target for corrective repair using a RNA trans-splicing strategy: Anti-leukemic activity of a rationally designed RNA trans-splicing molecule

    PubMed Central

    Uckun, Fatih M.; Qazi, Sanjive; Ma, Hong; Reaman, Gregory H.; Mitchell, Lloyd G.

    2015-01-01

    Our recent studies have demonstrated that the CD22 exon 12 deletion (CD22ΔE12) is a characteristic genetic defect of therapy-refractory clones in pediatric B-precursor acute lymphoblastic leukemia (BPL) and implicated the CD22ΔE12 genetic defect in the aggressive biology of relapsed or therapy-refractory pediatric BPL. The purpose of the present study was to further evaluate the biologic significance of the CD22ΔE12 molecular lesion and determine if it could serve as a molecular target for corrective repair using RNA trans-splicing therapy. We show that both pediatric and adult B-lineage lymphoid malignancies are characterized by a very high incidence of the CD22ΔE12 genetic defect. We provide experimental evidence that the correction of the CD22ΔE12 genetic defect in human CD22ΔE12+ BPL cells using a rationally designed CD22 RNA trans-splicing molecule (RTM) caused a pronounced reduction of their clonogenicity. The RTM-mediated correction replaced the downstream mutation-rich segment of Intron 12 and remaining segments of the mutant CD22 pre-mRNA with wildtype CD22 Exons 10-14, thereby preventing the generation of the cis-spliced aberrant CD22ΔE12 product. The anti-leukemic activity of this RTM against BPL xenograft clones derived from CD22ΔE12+ leukemia patients provides the preclinical proof-of-concept that correcting the CD22ΔE12 defect with rationally designed CD22 RTMs may provide the foundation for therapeutic innovations that are needed for successful treatment of high-risk and relapsed BPL patients. PMID:25567759

  10. Statistical detection of differentially expressed genes based on RNA-seq: from biological to phylogenetic replicates.

    PubMed

    Gu, Xun

    2016-03-01

    RNA-seq has been an increasingly popular high-throughput platform to identify differentially expressed (DE) genes, which is much more reproducible and accurate than the previous microarray technology. Yet, a number of statistical issues remain to be resolved in data analysis, largely due to the high-throughput data volume and over-dispersion of read counts. These problems become more challenging for those biologists who use RNA-seq to measure genome-wide expression profiles in different combinations of sampling resources (species or genotypes) or treatments. In this paper, the author first reviews the statistical methods available for detecting DE genes, which have implemented negative binomial (NB) models and/or quasi-likelihood (QL) approaches to account for the over-dispersion problem in RNA-seq samples. The author then studies how to carry out the DE test in the context of phylogeny, i.e., RNA-seq samples are from a range of species as phylogenetic replicates. The author proposes a computational framework to solve this phylo-DE problem: While an NB model is used to account for data over-dispersion within biological replicates, over-dispersion among phylogenetic replicates is taken into account by QL, plus some special treatments for phylogenetic bias. This work helps to design cost-effective RNA-seq experiments in the field of biodiversity or phenotype plasticity that may involve hundreds of species under a phylogenetic framework. PMID:26108230

  11. The small regulatory RNA molecule MicA is involved in Salmonella enterica serovar Typhimurium biofilm formation

    PubMed Central

    2010-01-01

    Background LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules. Results Analysis of additional S. Typhimurium luxS mutants indicated that the LuxS enzyme itself is not a prerequisite for a wild type mature biofilm. However, in close proximity of the luxS coding sequence, a small RNA molecule, MicA, is encoded on the opposite DNA strand. Interference with the MicA expression level showed that a balanced MicA level is essential for mature Salmonella biofilm formation. Several MicA targets known to date have previously been reported to be implicated in biofilm formation in Salmonella or in other bacterial species. Additionally, we showed by RT-qPCR analysis that MicA levels are indeed altered in some luxS mutants, corresponding to their biofilm formation phenotype. Conclusions We show that the S. Typhimurium biofilm formation phenotype of a luxS mutant in which the complete coding region is deleted, is dependent on the sRNA molecule MicA, encoded in the luxS adjacent genomic region, rather than on LuxS itself. Future studies are required to fully elucidate the role of MicA in Salmonella biofilm formation. PMID:21044338

  12. Incorporating small molecules or biologics into nanofibers for optimized drug release: A review.

    PubMed

    Sebe, István; Szabó, Péter; Kállai-Szabó, Barnabás; Zelkó, Romána

    2015-10-15

    Over the past several decades, the formulation of novel nanofiber-based drug delivery systems focusing on specific delivery purposes has been investigated worldwide with a continuous level of interest. The unique structure and properties of nanoscale fibrous systems, such as their high specific-area-to-volume ratio and high porosity and the possibility of controlling their crystalline-amorphous phase transitions, make them a desirable formulation pathway to satisfy the needs of recent pharmaceutical development. Fibrous delivery systems can facilitate the accelerated dissolution and increased solubility of small molecules and can also be useful in controlling drug delivery over time (for local or systemic drug administration). In the latter case, the release periods can be tuned over a wide range (from hours to weeks), e.g., by adjusting the fiber diameter and selecting the appropriate polymers. The solubility of the polymer, the fiber diameter and the fiber structure are the primary parameters affecting drug release. In addition to immediate and sustained release, other release profiles, such as biphasic release, can also be achieved. Chemical conjugation and surface functionalization offer further possibilities for the control of drug release. In the case of small molecules, developments focus mostly on overcoming the unfavorable physicochemical nature of the active agents. By contrast, in the preparation of macromolecule-loaded nanofibers, maximizing the biological activity of the macromolecules presents the greatest challenge. The authors' intent is to provide a comprehensive overview of the key parameters of advanced drug delivery systems of this type. PMID:26307263

  13. UCSF Small Molecule Discovery Center: innovation, collaboration and chemical biology in the Bay Area.

    PubMed

    Arkin, Michelle R; Ang, Kenny K H; Chen, Steven; Davies, Julia; Merron, Connie; Tang, Yinyan; Wilson, Christopher G M; Renslo, Adam R

    2014-05-01

    The Small Molecule Discovery Center (SMDC) at the University of California, San Francisco, works collaboratively with the scientific community to solve challenging problems in chemical biology and drug discovery. The SMDC includes a high throughput screening facility, medicinal chemistry, and research labs focused on fundamental problems in biochemistry and targeted drug delivery. Here, we outline our HTS program and provide examples of chemical tools developed through SMDC collaborations. We have an active research program in developing quantitative cell-based screens for primary cells and whole organisms; here, we describe whole-organism screens to find drugs against parasites that cause neglected tropical diseases. We are also very interested in target-based approaches for so-called "undruggable", protein classes and fragment-based lead discovery. This expertise has led to several pharmaceutical collaborations; additionally, the SMDC works with start-up companies to enable their early-stage research. The SMDC, located in the biotech-focused Mission Bay neighborhood in San Francisco, is a hub for innovative small-molecule discovery research at UCSF. PMID:24661212

  14. Self-Optimized Biological Channels in Facilitating the Transmembrane Movement of Charged Molecules

    PubMed Central

    Huyen, V. T. N.; Lap, Vu Cong; Nguyen, V. Lien

    2016-01-01

    We consider an anisotropically two-dimensional diffusion of a charged molecule (particle) through a large biological channel under an external voltage. The channel is modeled as a cylinder of three structure parameters: radius, length, and surface density of negative charges located at the channel interior-lining. These charges induce inside the channel a potential that plays a key role in controlling the particle current through the channel. It was shown that to facilitate the transmembrane particle movement the channel should be reasonably self-optimized so that its potential coincides with the resonant one, resulting in a large particle current across the channel. Observed facilitation appears to be an intrinsic property of biological channels, regardless of the external voltage or the particle concentration gradient. This facilitation is very selective in the sense that a channel of definite structure parameters can facilitate the transmembrane movement of only particles of proper valence at corresponding temperatures. Calculations also show that the modeled channel is nonohmic with the ion conductance which exhibits a resonance at the same channel potential as that identified in the current. PMID:27022394

  15. Force per cross-sectional area from molecules to muscles: a general property of biological motors

    PubMed Central

    Meyer-Vernet, Nicole

    2016-01-01

    We propose to formally extend the notion of specific tension, i.e. force per cross-sectional area—classically used for muscles, to quantify forces in molecular motors exerting various biological functions. In doing so, we review and compare the maximum tensions exerted by about 265 biological motors operated by about 150 species of different taxonomic groups. The motors considered range from single molecules and motile appendages of microorganisms to whole muscles of large animals. We show that specific tensions exerted by molecular and non-molecular motors follow similar statistical distributions, with in particular, similar medians and (logarithmic) means. Over the 1019 mass (M) range of the cell or body from which the motors are extracted, their specific tensions vary as Mα with α not significantly different from zero. The typical specific tension found in most motors is about 200 kPa, which generalizes to individual molecular motors and microorganisms a classical property of macroscopic muscles. We propose a basic order-of-magnitude interpretation of this result. PMID:27493785

  16. Self-Optimized Biological Channels in Facilitating the Transmembrane Movement of Charged Molecules.

    PubMed

    Huyen, V T N; Ho, Le Bin; Lap, Vu Cong; Nguyen, V Lien

    2016-01-01

    We consider an anisotropically two-dimensional diffusion of a charged molecule (particle) through a large biological channel under an external voltage. The channel is modeled as a cylinder of three structure parameters: radius, length, and surface density of negative charges located at the channel interior-lining. These charges induce inside the channel a potential that plays a key role in controlling the particle current through the channel. It was shown that to facilitate the transmembrane particle movement the channel should be reasonably self-optimized so that its potential coincides with the resonant one, resulting in a large particle current across the channel. Observed facilitation appears to be an intrinsic property of biological channels, regardless of the external voltage or the particle concentration gradient. This facilitation is very selective in the sense that a channel of definite structure parameters can facilitate the transmembrane movement of only particles of proper valence at corresponding temperatures. Calculations also show that the modeled channel is nonohmic with the ion conductance which exhibits a resonance at the same channel potential as that identified in the current. PMID:27022394

  17. MicroRNA-221 controls expression of intercellular adhesion molecule-1 in epithelial cells in response to Cryptosporidium parvum infection

    PubMed Central

    Gong, Ai-Yu; Hu, Guoku; Zhou, Rui; Liu, Jun; Feng, Yaoyu; Soukup, Garrett A.; Chen, Xian-Ming

    2011-01-01

    Cryptosporidium parvum is a protozoan parasite that infects gastrointestinal epithelial cells and causes diarrheal disease in humans and animals globally. Pathological changes following C. parvum infection include crypt hyperplasia, a modest inflammatory reaction with increased infiltration of lymphocytes into intestinal mucosa. Expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), on infected epithelial cell surfaces may facilitate adhesion and recognition of lymphocytes at infection sites. MicroRNAs (miRNAs) are small RNA molecules of 23 nucleotides that negatively regulate protein-coding gene expression via translational suppression or mRNA degradation. We recently reported that microRNA-221 (miR-221) regulates ICAM-1 translation through targeting the ICAM-1 3′-untranslated region (UTR). In this study, we tested the role of miR-221 in regulating ICAM-1 expression in epithelial cells in response to C. parvum infection using an in vitro model of human biliary cryptosporidiosis. Up-regulation of ICAM-1 at both message and protein levels was detected in epithelial cells following C. parvum infection. Inhibition of ICAM-1 transcription with actinomycin D could only partially block C. parvum-induced ICAM-1 expression at the protein level. Cryptosporidium parvum infection decreased miR-221 expression in infected epithelial cells. When cells were transfected with a luciferase reporter construct covering the miR-221 binding site in the ICAM-1 3′-UTR and then exposed to C. parvum, an enhanced luciferase activity was detected. Transfection of miR-221 precursor abolished C. parvum-stimulated ICAM-1 protein expression. In addition, expression of ICAM-1 on infected epithelial cells facilitated epithelial adherence of co-cultured Jurkat cells. These results indicate that miR-221-mediated translational suppression controls ICAM-1 expression in epithelial cells in response to C. parvum infection. PMID:21236259

  18. RNA Profiling for the Identification of the Tissue Origin of Dried Stains in Forensic Biology.

    PubMed

    Hanson, E K; Ballantyne, J

    2010-07-01

    Examination of crime scene items for biological evidence typically begins with a preliminary screening for the presence of biological fluids in order to identify possible sources of DNA. Conventional biochemical and immunological assays employed for this screening require multiple tests to be performed in a serial manner, can consume a significant amount of valuable evidentiary material, and can require a significant amount of time and labor for completion. Moreover, the presence of several biological fluids, such as saliva, vaginal secretions, and menstrual blood, cannot be conclusively identified using current methods. Due to the disadvantages of conventional body fluid testing, some operational crime laboratories have chosen to bypass the body fluid identification process and proceed directly to DNA analysis. However, while reducing the time spent on each case, this "shortcut" could result in a failure to provide important probative information regarding the nature of the crime as well as result in increased cost to crime laboratories if unnecessary DNA testing is performed. In the past several years, a number of forensic researchers have attempted to develop molecular-based approaches to body fluid identification that would provide operational crime laboratories with significantly improved specificity. This has resulted in an increased interest in the use of RNA profiling strategies for the identification of forensically relevant biological fluids. This review provides an overview of studies carried out on the use of both messenger RNA and small (micro) RNA profiling. The results of these studies are encouraging and presage the routine identification the tissue source(s) of forensic evidence using molecular-based approaches. PMID:26242593

  19. The landscape of fusion transcripts in spitzoid melanoma and biologically indeterminate spitzoid tumors by RNA sequencing

    PubMed Central

    Wu, Gang; Barnhill, Raymond L.; Lee, Seungjae; Li, Yongjin; Shao, Ying; Easton, John; Dalton, James; Zhang, Jinghui; Pappo, Alberto; Bahrami, Armita

    2016-01-01

    Kinase activation by chromosomal translocations is a common mechanism that drives tumorigenesis in spitzoid neoplasms. To explore the landscape of fusion transcripts in these tumors, we performed whole-transcriptome sequencing using formalin-fixed paraffin-embedded tissues in malignant or biologically indeterminate spitzoid tumors from 7 patients (age 2–14 years). RNA sequence libraries enriched for coding regions were prepared and the sequencing was analyzed by a novel assembly-based algorithm designed for detecting complex fusions. In addition, tumor samples were screened for hotspot TERT promoter mutations, and telomerase expression was assessed by TERT mRNA in situ hybridization (ISH). Two patients had widespread metastasis and subsequently died of disease, and 5 patients had a benign clinical course on limited follow-up (mean: 30 months). RNA sequencing and TERT mRNA ISH were successful in 6 tumors and unsuccessful in 1 disseminating tumor due to low RNA quality. RNA sequencing identified a kinase fusion in 5 of the 6 sequenced tumors: TPM3–NTRK1 (2 tumors), complex rearrangements involving TPM3, ALK, and IL6R (1 tumor), BAIAP2L1–BRAF (1 tumor), and EML4–BRAF (1 disseminating tumor). All predicted chimeric transcripts were expressed at high levels and contained the intact kinase domain. In addition, 2 tumors each contained a second fusion gene, ARID1B-SNX9 or PTPRZ1-NFAM1. The detected chimeric genes were validated by home-brew break-apart or fusion fluorescence in situ hybridization. The 2 disseminating tumors each harbored the TERT promoter −124C>T (Chr 5:1,295,228 hg19 coordinate) mutation whereas the remaining 5 tumors retained the wild-type gene. The presence of the −124C>T mutation correlated with telomerase expression by TERT mRNA ISH. In summary, we demonstrated complex fusion transcripts and novel partner genes for BRAF by RNA sequencing of FFPE samples. The diversity of gene fusions demonstrated by RNA sequencing defines the molecular

  20. 2012 SINGLE MOLECULE APPROACHES TO BIOLOGY GORDON RESEARCH CONFERENCE (JULY 15-20, 2012 - MOUNT SNOW RESORT, WEST DOVER VT)

    SciTech Connect

    Fernandez, Julio

    2012-04-20

    Single molecule techniques are rapidly occupying a central role in biological research at all levels. This transition was made possible by the availability and dissemination of robust techniques that use fluorescence and force probes to track the conformation of molecules one at a time, in vitro as well as in live cells. Single-molecule approaches have changed the way many biological problems are studied. These novel techniques provide previously unobtainable data on fundamental biochemical processes that are essential for all forms of life. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of the molecular systems that underpin the functioning of living cells. Hence, our conference seeks to disseminate the implementation and use of single molecule techniques in the pursuit of new biological knowledge. Topics covered include: Molecular Motors on the Move; Origin And Fate Of Proteins; Physical Principles Of Life; Molecules and Super-resolution Microscopy; Nanoswitches In Action; Active Motion Or Random Diffusion?; Building Blocks Of Living Cells; From Molecular Mechanics To Physiology; Tug-of-war: Force Spectroscopy Of Single Proteins.

  1. Novel hypovirulence-associated RNA mycovirus in the plant pathogenic fungus botrytis cinerea: molecular and biological characterization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botrytis cinerea is a pathogenic fungus causing gray mold disease on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain ...

  2. Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter.

    PubMed

    Tomescu, Alexandra I; Robb, Nicole C; Hengrung, Narin; Fodor, Ervin; Kapanidis, Achillefs N

    2014-08-12

    The influenza virus is a major human and animal pathogen responsible for seasonal epidemics and occasional pandemics. The genome of the influenza A virus comprises eight segments of single-stranded, negative-sense RNA with highly conserved 5' and 3' termini. These termini interact to form a double-stranded promoter structure that is recognized and bound by the viral RNA-dependent RNA polymerase (RNAP); however, no 3D structural information for the influenza polymerase-bound promoter exists. Functional studies have led to the proposal of several 2D models for the secondary structure of the bound promoter, including a corkscrew model in which the 5' and 3' termini form short hairpins. We have taken advantage of an insect-cell system to prepare large amounts of active recombinant influenza virus RNAP, and used this to develop a highly sensitive single-molecule FRET assay to measure distances between fluorescent dyes located on the promoter and map its structure both with and without the polymerase bound. These advances enabled the direct analysis of the influenza promoter structure in complex with the viral RNAP, and provided 3D structural information that is in agreement with the corkscrew model for the influenza virus promoter RNA. Our data provide insights into the mechanisms of promoter binding by the influenza RNAP and have implications for the understanding of the regulatory mechanisms involved in the transcription of viral genes and replication of the viral RNA genome. In addition, the simplicity of this system should translate readily to the study of any virus polymerase-promoter interaction. PMID:25071209

  3. Visualising single molecules of HIV-1 and miRNA nucleic acids

    PubMed Central

    2013-01-01

    Background The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19–26 nucleotides in length. Results We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1–2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. Conclusions Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1–2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence. PMID:23590669

  4. MicroRNA-1 properties in cancer regulatory networks and tumor biology.

    PubMed

    Weiss, Martin; Brandenburg, Lars-Ove; Burchardt, Martin; Stope, Matthias B

    2016-08-01

    Short non-coding microRNAs have been identified to orchestrate crucial mechanisms in cancer progression and treatment resistance. MicroRNAs are involved in posttranscriptional modulation of gene expression and therefore represent promising targets for anticancer therapy. As mircoRNA-1 (miR-1) exerted to be predominantly downregulated in the majority of examined tumors, miR-1 is classified to be a tumor suppressor with high potential to diminish tumor development and therapy resistance. Here we review the complex functionality of miR-1 in tumor biology. PMID:27286699

  5. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  6. Hairpins under tension: RNA versus DNA

    PubMed Central

    Bercy, Mathilde; Bockelmann, Ulrich

    2015-01-01

    We use optical tweezers to control the folding and unfolding of individual DNA and RNA hairpins by force. Four hairpin molecules are studied in comparison: two DNA and two RNA ones. We observe that the conformational dynamics is slower for the RNA hairpins than for their DNA counterparts. Our results indicate that structures made of RNA are dynamically more stable. This difference might contribute to the fact that DNA and RNA play fundamentally different biological roles in spite of chemical similarity. PMID:26323319

  7. Vascular activation of adhesion molecule mRNA and cell surface expression by ionizing radiation.

    PubMed

    Heckmann, M; Douwes, K; Peter, R; Degitz, K

    1998-01-10

    During cutaneous inflammatory reactions the recruitment of circulating leukocytes into the tissue critically depends on the regulated expression of endothelial cell adhesion molecules (CAMs). Various proinflammatory stimuli upregulate endothelial CAMs, including cytokines and UV irradiation. We have investigated the effects of ionizing radiation (IR) on endothelial CAM expression. Organ cultures of normal human skin as well as cultured human dermal microvascular endothelial cells (HDMEC) were exposed to IR. Expression of three major endothelial CAMs was studied in skin organ cultures by immunohistochemistry and in cell culture by Northern blot analysis and flow cytometry. In skin organ cultures vascular immunoreactivity for ICAM-1, E-selectin, and VCAM-1 was strongly induced 24 h after exposure to 5 or 10 Gy of IR, while immunoreactivity for CD31/PECAM-1, a constitutively expressed endothelial cell adhesion molecule, remained unchanged. In cultured HDMEC IR upregulated ICAM-1, VCAM-1, and E-selectin mRNAs and cell surface expression in a time- and dose-dependent fashion. Cellular morphology and viability remained unaltered by IR up to 24 h postirradiation. This study characterizes microvascular activation of adhesion molecule expression in response to ionizing radiation in a clinically relevant IR dose range. The findings also underscore the ability of endothelial cells to integrate environmental electromagnetic stimuli. PMID:9457067

  8. Immunological and biological properties of iodoinsulin labeled with one or less atoms of iodine per molecule

    PubMed Central

    Arquilla, Edward R.; Ooms, Henri; Mercola, Karen

    1968-01-01

    Experiments were designed to compare the distribution of free and antibody-bound unlabeled insulin to the distribution of free and antibody-bound insulin-125I. The insulin antibody was incorporated in a specific immune precipitate similar to the one used by Hales and Randle for the radioimmune assay of insulin. Insulin which was not bound by the specific immune precipitate was measured by the immune hemolysis inhibition assay. This report contains evidence that the addition of the unlabeled insulin in the radioimmune assay results in relatively more insulin-125I which remains free and less bound by antibodies than is the case with the unlabeled insulin. Methods are described for the separation of an electrophoretically homogeneous iodoinsulin from samples of crude iodoinsulin with average incorporations of less than 0.2 atoms iodine per molecule. These purified iodoinsulin fractions have a markedly attenuated biological activity. Evidence is presented which supports the postulate that only a portion of the antibodies in guinea pig insulin antiserum are capable of effectively binding with purified iodoinsulin. Images PMID:5637137

  9. RNA targeting by small molecule alkaloids: Studies on the binding of berberine and palmatine to polyribonucleotides and comparison to ethidium

    NASA Astrophysics Data System (ADS)

    Islam, Md. Maidul; Suresh Kumar, Gopinatha

    2008-03-01

    The binding affinity, energetics and conformational aspects of the interaction of isoquinoline alkaloids berberine and palmatine to four single stranded polyribonucleotides polyguanylic acid [poly(G)], polyinosinic acid [poly(I)], polycytidylic acid [poly(C)] and polyuridylic acid [poly(U)] were studied by absorption, fluorescence, isothermal titration calorimetry and circular dichroism spectroscopy and compared with ethidium. Berberine, palmatine and ethidium binds strongly with poly(G) and poly(I) with affinity in the order 10 5 M -1 while their binding to poly(C) and poly(U) were very weak or practically nil. The same conclusions have also emerged from isothermal titration calorimetric studies. The binding of all the three compounds to poly(C) and poly(I) was exothermic and favored by both negative enthalpy change and positive entropy change. Conformational change in the polymer associated with the binding was observed in poly(I) with all the three molecules and poly(U) with ethidium but not in poly(G) and poly(C) revealing differences in the orientation of the bound molecules in the hitherto different helical organization of these polymers. These fundamental results may be useful and serve as database for the development of futuristic RNA based small molecule therapeutics.

  10. Solid-supported cross-metathesis and a formal alkane metathesis for the generation of biologically relevant molecules.

    PubMed

    Méndez, Luciana; Mata, Ernesto G

    2015-02-01

    Solid-phase synthetic strategies toward the generation of libraries of biologically relevant molecules were developed using olefin cross-metathesis as a key step. It is remarkably the formal alkane metathesis based on a one-pot, microwave-assisted, ruthenium-catalyzed cross-metathesis and reduction to obtain Csp3-Csp3 linkages. PMID:25569690

  11. Introducing Bond-Line Organic Structures in High School Biology: An Activity that Incorporates Pleasant-Smelling Molecules

    ERIC Educational Resources Information Center

    Rios, Andro C.; French, Gerald

    2011-01-01

    Chemical education occurs in settings other than just the chemistry classroom. High school biology courses are frequently where students are introduced to organic molecules and their importance to cellular chemistry. However, structural representations are often intimidating because students have not been introduced to the language. As part of a…

  12. A model of compensatory molecular evolution involving multiple sites in RNA molecules.

    PubMed

    Kusumi, Junko; Ichinose, Motoshi; Takefu, Masasuke; Piskol, Robert; Stephan, Wolfgang; Iizuka, Masaru

    2016-01-01

    Consider two sites under compensatory fitness interaction, such as a Watson-Crick base pair in an RNA helix or two interacting residues in a protein. A mutation at any one of these two sites may reduce the fitness of an individual. However, fitness may be restored by the occurrence of a second mutation at the other site. Kimura modeled this process using a two-locus haploid fitness scheme with two alleles at each locus. He predicted that compensatory evolution following this model is very rare unless selection against the deleterious single mutations is weak and linkage between the interacting sites is tight. Here we investigate the question whether the rate of compensatory evolution increases if we take the context of the two directly interacting sites into account. By "context", we mean the effect of neighboring sites in an RNA helix. Interaction between the focal pair of sites under consideration and the context may lead to so-called indirect compensation. Thus, extending Kimura's classical model of compensatory evolution, we study the effects of both direct and indirect compensation on the rate of compensatory evolution. It is shown that the effects of indirect compensation are very strong. We find that recombination does not slow down the rate of compensatory evolution as predicted by the classical model. Instead, compensatory substitutions may be relatively frequent, even if linkage between the focal interacting sites is loose, selection against deleterious mutations is strong, and mutation rate is low. We compare our theoretical results with data on RNA secondary structures from vertebrate introns. PMID:26506471

  13. Molecular-scale quantitative charge density measurement of biological molecule by frequency modulation atomic force microscopy in aqueous solutions

    NASA Astrophysics Data System (ADS)

    Umeda, Kenichi; Kobayashi, Kei; Oyabu, Noriaki; Matsushige, Kazumi; Yamada, Hirofumi

    2015-07-01

    Surface charge distributions on biological molecules in aqueous solutions are essential for the interactions between biomolecules, such as DNA condensation, antibody-antigen interactions, and enzyme reactions. There has been a significant demand for a molecular-scale charge density measurement technique for better understanding such interactions. In this paper, we present the local electric double layer (EDL) force measurements on DNA molecules in aqueous solutions using frequency modulation atomic force microscopy (FM-AFM) with a three-dimensional force mapping technique. The EDL forces measured in a 100 mM KCl solution well agreed with the theoretical EDL forces calculated using reasonable parameters, suggesting that FM-AFM can be used for molecular-scale quantitative charge density measurements on biological molecules especially in a highly concentrated electrolyte.

  14. FRET Enabled Real Time Detection of RNA-Small Molecule Binding

    PubMed Central

    Xie, Yun; Dix, Andrew V.; Tor, Yitzhak

    2011-01-01

    A robust analysis and discovery platform for antibiotics targeting the bacterial rRNA A-site has been developed by incorporating a new emissive U surrogate into the RNA and labeling the aminoglycosides with an appropriate fluorescence acceptor. Specifically, a 5-methoxyquinazoline-2,4(1H,3H)-dione-based emissive uracil analogue was identified to be an ideal donor for 7-diethylaminocoumarin-3-carboxylic acid. This donor/acceptor pair displays a critical Förster radius (R0) of 27 Å, a value suitable for an A-site-aminoglycoside assembly. Titrating the coumarin labeled aminoglycosides into the emissive A-site construct, labeled at position U1406, shows a decrease in donor emission (at 395 nm) and concurrent increase of the acceptor emission (at 473 nm). Titration curves, obtained by fitting the donor’s emission quenching or the augmentation of the acceptor’s sensitized emission, faithfully generate EC50 values. Titration of unlabeled ligands into the preformed FRET complex showed a continuous increase of the donor emission, with a concurrent decrease of the acceptor emission, yielding valuable data regarding competitive displacement of aminoglycosides by A-site binders. Detection of antibiotic binding is therefore not dependent on changes in the environment of a single fluorophore, but rather on the responsive interaction between two chromophores acting as a FRET pair, facilitating the determination of direct binding and competitive displacement events with FRET accuracy. PMID:19908830

  15. Peptide nucleic acids rather than RNA may have been the first genetic molecule

    NASA Technical Reports Server (NTRS)

    Nelson, K. E.; Levy, M.; Miller, S. L.

    2000-01-01

    Numerous problems exist with the current thinking of RNA as the first genetic material. No plausible prebiotic processes have yet been demonstrated to produce the nucleosides or nucleotides or for efficient two-way nonenzymatic replication. Peptide nucleic acid (PNA) is a promising precursor to RNA, consisting of N-(2-aminoethyl)glycine (AEG) and the adenine, uracil, guanine, and cytosine-N-acetic acids. However, PNA has not yet been demonstrated to be prebiotic. We show here that AEG is produced directly in electric discharge reactions from CH(4), N(2), NH(3), and H(2)O. Electric discharges also produce ethylenediamine, as do NH(4)CN polymerizations. AEG is produced from the robust Strecker synthesis with ethylenediamine. The NH(4)CN polymerization in the presence of glycine leads to the adenine and guanine-N(9)-acetic acids, and the cytosine and uracil-N(1)-acetic acids are produced in high yield from the reaction of cyanoacetaldehyde with hydantoic acid, rather than urea. Preliminary experiments suggest that AEG may polymerize rapidly at 100 degrees C to give the polypeptide backbone of PNA. The ease of synthesis of the components of PNA and possibility of polymerization of AEG reinforce the possibility that PNA may have been the first genetic material.

  16. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  17. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  18. Max Delbruck Biological Physics Prize Talk: The Biophysics of Gene Regulation, Studied One Molecule at a Time

    NASA Astrophysics Data System (ADS)

    Block, Steven

    2008-03-01

    Advances have led to a new field, dubbed single molecule biophysics. Prominent among the new technologies is the optical trap, or `optical tweezers.' Sensitive systems for measuring force and displacement in optical traps permit the nanomechanical properties of individual macromolecules to be explored with unprecedented precision, revealing behaviors heretofore obscured by ensemble-based approaches. This talk will focus on some of our current work with single-molecule systems, including transcription by RNA polymerase and structural transitions in nucleic acids. We developed high-resolution instrumentation that has broken the nanometer barrier and is thereby able to detect displacements down to the atomic level, in aqueous buffer at room temperature. Consequently, we can monitor the motions of RNA polymerase molecules in real time as these step from base to base along DNA. On the practical side, base-pair resolution makes it possible to sequence DNA in a new way, based on enzyme motions, and points to new directions in nanoscience. The improved stability afforded by the current generation of optical trapping apparatus has allowed us to reconstruct the complete energy landscapes for folding transitions in nucleic-acid hairpins. Recently, we have turned our attention to the problem of co-transcriptional folding, aptamers, and riboswitches formed in nascent mRNAs, and to the DNA or RNA sequence elements that regulate expression.

  19. Short non-coding RNA biology and neurodegenerative disorders: novel disease targets and therapeutics

    PubMed Central

    Weinberg, Marc S.; Wood, Matthew J.A.

    2009-01-01

    Genomic studies in model organisms and in humans have shown that complexity in biological systems arises not from the absolute number of genes, but from the differential use of combinations of genetic programmes and the myriad ways in which these are regulated spatially and temporally during development, senescence and in disease. Nowhere is this lesson in biological complexity likely to be more apparent than in the human nervous system. Increasingly, the role of genomic non-protein coding small regulatory RNAs, in particular the microRNAs (miRNAs), in regulating cellular pathways controlling fundamental functions in the nervous system and in neurodegenerative disease is being appreciated. Not only might dysregulated expression of miRNAs serve as potential disease biomarkers but increasingly such short regulatory RNAs are being implicated directly in the pathogenesis of complex, sporadic neurodegenerative disease. Moreover, the targeting and exploitation of short RNA silencing pathways, commonly known as RNA interference, and the development of related tools, offers novel therapeutic approaches to target upstream disease components with the promise of providing future disease modifying therapies for neurodegenerative disorders. PMID:19297399

  20. RNA topology

    PubMed Central

    2013-01-01

    A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases. PMID:23603781

  1. Measuring fate and rate of single-molecule competition of amplification and restriction digestion, and its use for rapid genotyping tested with hepatitis C viral RNA.

    PubMed

    Sun, Bing; Rodriguez-Manzano, Jesus; Selck, David A; Khorosheva, Eugenia; Karymov, Mikhail A; Ismagilov, Rustem F

    2014-07-28

    We experimentally monitored, at the single-molecule level, the competition among reverse transcription, exponential amplification (RT-LAMP), and linear degradation (restriction enzymes) starting with hepatitis C viral RNA molecules. We found significant heterogeneity in the rate of single-molecule amplification; introduction of the restriction enzymes affected both the rate and the "fate" (the binary outcome) of single-molecule amplification. While end-point digital measurements were primarily sensitive to changes in fate, the bulk real-time kinetic measurements were dominated by the rate of amplification of the earliest molecules, and were not sensitive to fate of the rest of the molecules. We show how this competition of reactions can be used for rapid HCV genotyping with either digital or bulk readout. This work advances our understanding of single-molecule dynamics in reaction networks and may help bring genotyping capabilities out of clinical labs and into limited-resource settings. PMID:24889060

  2. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking.

    PubMed

    Dolgosheina, Elena V; Jeng, Sunny C Y; Panchapakesan, Shanker Shyam S; Cojocaru, Razvan; Chen, Patrick S K; Wilson, Peter D; Hawkins, Nancy; Wiggins, Paul A; Unrau, Peter J

    2014-10-17

    Because RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem and to allow the purification of fluorescently tagged RNA complexes, we have selected a high affinity RNA aptamer called RNA Mango. This aptamer binds a series of thiazole orange (fluorophore) derivatives with nanomolar affinity, while increasing fluorophore fluorescence by up to 1,100-fold. Visualization of RNA Mango by single-molecule fluorescence microscopy, together with injection and imaging of RNA Mango/fluorophore complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. By inserting RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating the fluorophore, we demonstrate that the aptamer can be used to simultaneously fluorescently label and purify biologically important RNAs. The high affinity and fluorescent properties of RNA Mango are therefore expected to simplify the study of RNA complexes. PMID:25101481

  3. IL-2 or IL-4 mRNA as a potential flow cytometric marker molecule for selective collection of living T helper 1 or T helper 2 lymphocytes.

    PubMed

    Ishibashi, Kaname; Tsuji, Akihiko

    2003-06-01

    Flow cytometry has been widely used to analyze and sort out particular types of living cells that have specific marker molecules. In many cases, marker proteins are present on the cell surface and are detected by monoclonal antibodies against them. However, there are some cases in which cells do not have specific marker molecules on their surface. In this situation, it would be useful if mRNA that is expressed specifically in the particular cell could be used as a marker molecule. We previously reported that mRNA can be detected in living cells by hybridizing a pair of fluoreophore (donor or acceptor)-labeled oligonucleotides to adjacent locations on the target mRNA in the cytoplasm of cells (Tsuji, A.; Koshimoto, H.; Sato, Y.; Hirano, M.; Sei-Iida, Y.; Kondo, S.; Ishibashi, K. Biophys. J. 2000, 78, 3260-3274). On the formed hybrid of the two fluorescent oligonucleotides with the target mRNA, the distance between the two fluorophores becomes very close, which results in fluorescence resonance energy transfer (FRET). Combining this fluorescent labeling method for mRNA with flow cytometry, we have examined the isolation of living CD4+ T helper lymphocytes expressing IL-2 mRNA (Th1) or IL-4 mRNA (Th2). A pair of fluorescent oligonucleotides for hybridizing to IL-2 or IL-4 mRNA were introduced into activated CD4+ T lymphocytes by electroporation. The cells were applied to FACS and analyzed by FRET signals. Th1 or Th2 lymphocytes were exclusively sorted from their mixed populations in activated CD4+ T cells. Our results demonstrate that it is possible to use mRNA as marker molecules to analyze and isolate living cells in flow cytometry. PMID:12948141

  4. Laser desorption/ionization mass spectrometry for direct profiling and imaging of small molecules from raw biological materials

    SciTech Connect

    Cha, Sangwon

    2008-01-01

    Matrix-assisted laser desorption/ionization(MALDI) mass spectrometry(MS) has been widely used for analysis of biological molecules, especially macromolecules such as proteins. However, MALDI MS has a problem in small molecule (less than 1 kDa) analysis because of the signal saturation by organic matrixes in the low mass region. In imaging MS (IMS), inhomogeneous surface formation due to the co-crystallization process by organic MALDI matrixes limits the spatial resolution of the mass spectral image. Therefore, to make laser desorption/ionization (LDI) MS more suitable for mass spectral profiling and imaging of small molecules directly from raw biological tissues, LDI MS protocols with various alternative assisting materials were developed and applied to many biological systems of interest. Colloidal graphite was used as a matrix for IMS of small molecules for the first time and methodologies for analyses of small metabolites in rat brain tissues, fruits, and plant tissues were developed. With rat brain tissues, the signal enhancement for cerebroside species by colloidal graphite was observed and images of cerebrosides were successfully generated by IMS. In addition, separation of isobaric lipid ions was performed by imaging tandem MS. Directly from Arabidopsis flowers, flavonoids were successfully profiled and heterogeneous distribution of flavonoids in petals was observed for the first time by graphite-assisted LDI(GALDI) IMS.

  5. Codelivery of small molecule hedgehog inhibitor and miRNA for treating pancreatic cancer.

    PubMed

    Kumar, Virender; Mondal, Goutam; Slavik, Paige; Rachagani, Satyanarayna; Batra, Surinder K; Mahato, Ram I

    2015-04-01

    Successful treatment of pancreatic ductal adenocarcinoma (PDAC) remains a challenge due to the desmoplastic microenvironment that promotes both tumor growth and metastasis and forms a barrier to chemotherapy. Hedgehog (Hh) signaling is implicated in initiation and progression of PDAC and also contributes to desmoplasia. While Hh levels are increased in pancreatic cancer cells, levels of tumor suppressor miR-let7b, which targets several genes involved in PDAC pathogenesis, is downregulated. Therefore, our overall objective was to inhibit Hh pathway and restore miR-let7b simultaneously for synergistically treating PDAC. miR-let7b and Hh inhibitor GDC-0449 could inhibit the proliferation of human pancreatic cancer cells (Capan-1, HPAF-II, T3M4, and MIA PaCa-2), and there was synergistic effect when miR-let7b and GDC-0449 were coformulated into micelles using methoxy poly(ethylene glycol)-block-poly(2-methyl- 2-carboxyl-propylenecarbonate-graft-dodecanol-graft-tetraethylene-pentamine) (mPEG-b-PCC-g-DC-g-TEPA). This copolymer self-assembled into micelles of <100 nm and encapsulated hydrophobic GDC-0449 into its core with 5% w/w drug loading and allowed complex formation between miR-let7b and its cationic pendant chains. Complete polyplex formation with miRNA was observed at the N/P ratio of 16/1. Almost 80% of GDC-0449 was released from the polyplex in a sustained manner in 2 days. miRNA in the micelle formulation was stable for up to 24 h in the presence of serum and high uptake efficiency was achieved with low cytotoxicity. This combination therapy effectively inhibited tumor growth when injected to athymic nude mice bearing ectopic tumor generated using MIA PaCa-2 cells compared to micelles carrying GDC-0449 or miR-let7b alone. Immunohistochemical analysis revealed decreased tumor cell proliferation with increased apoptosis in the animals treated with miR-let7b and GDC-0449 combination. PMID:25679326

  6. A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting.

    PubMed

    Tantale, Katjana; Mueller, Florian; Kozulic-Pirher, Alja; Lesne, Annick; Victor, Jean-Marc; Robert, Marie-Cécile; Capozi, Serena; Chouaib, Racha; Bäcker, Volker; Mateos-Langerak, Julio; Darzacq, Xavier; Zimmer, Christophe; Basyuk, Eugenia; Bertrand, Edouard

    2016-01-01

    Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. We additionally observe that the HIV-1 promoter displays stochastic fluctuations on two time scales, which we refer to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. We propose that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells. PMID:27461529

  7. A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting

    PubMed Central

    Tantale, Katjana; Mueller, Florian; Kozulic-Pirher, Alja; Lesne, Annick; Victor, Jean-Marc; Robert, Marie-Cécile; Capozi, Serena; Chouaib, Racha; Bäcker, Volker; Mateos-Langerak, Julio; Darzacq, Xavier; Zimmer, Christophe; Basyuk, Eugenia; Bertrand, Edouard

    2016-01-01

    Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. We additionally observe that the HIV-1 promoter displays stochastic fluctuations on two time scales, which we refer to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. We propose that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells. PMID:27461529

  8. Complex RNA Folding Kinetics Revealed by Single-Molecule FRET and Hidden Markov Models

    PubMed Central

    2014-01-01

    We have developed a hidden Markov model and optimization procedure for photon-based single-molecule FRET data, which takes into account the trace-dependent background intensities. This analysis technique reveals an unprecedented amount of detail in the folding kinetics of the Diels–Alderase ribozyme. We find a multitude of extended (low-FRET) and compact (high-FRET) states. Five states were consistently and independently identified in two FRET constructs and at three Mg2+ concentrations. Structures generally tend to become more compact upon addition of Mg2+. Some compact structures are observed to significantly depend on Mg2+ concentration, suggesting a tertiary fold stabilized by Mg2+ ions. One compact structure was observed to be Mg2+-independent, consistent with stabilization by tertiary Watson–Crick base pairing found in the folded Diels–Alderase structure. A hierarchy of time scales was discovered, including dynamics of 10 ms or faster, likely due to tertiary structure fluctuations, and slow dynamics on the seconds time scale, presumably associated with significant changes in secondary structure. The folding pathways proceed through a series of intermediate secondary structures. There exist both compact pathways and more complex ones, which display tertiary unfolding, then secondary refolding, and, subsequently, again tertiary refolding. PMID:24568646

  9. miRNA biogenesis: biological impact in the development of cancer.

    PubMed

    Romero-Cordoba, Sandra L; Salido-Guadarrama, Ivan; Rodriguez-Dorantes, Mauricio; Hidalgo-Miranda, Alfredo

    2014-01-01

    microRNAs (miRNAs) are non coding RNAs with different biological functions and pathological implications. Given their role as post-transcriptional gene expression regulators, they are involved in several important physiological processes like development, cell differentiation and cell signaling. miRNAs act as modulators of gene expression programs in different diseases, particularly in cancer, where they act through the repression of genes which are critical for carcinogenesis. The expression level of mature miRNAs is the result of a fine mechanism of biogenesis, carried out by different enzymatic complexes that exert their function at transcriptional and post-transcriptional levels. In this review, we will focus our discussion on the alterations in the miRNA biogenesis machinery, and its impact on the establishment and development of cancer programs. PMID:25482951

  10. Non-canonical roles of tRNAs and tRNA mimics in bacterial cell biology.

    PubMed

    Katz, Assaf; Elgamal, Sara; Rajkovic, Andrei; Ibba, Michael

    2016-08-01

    Transfer RNAs (tRNAs) are the macromolecules that transfer activated amino acids from aminoacyl-tRNA synthetases to the ribosome, where they are used for the mRNA guided synthesis of proteins. Transfer RNAs are ancient molecules, perhaps even predating the existence of the translation machinery. Albeit old, these molecules are tremendously conserved, a characteristic that is well illustrated by the fact that some bacterial tRNAs are efficient and specific substrates of eukaryotic aminoacyl-tRNA synthetases and ribosomes. Considering their ancient origin and high structural conservation, it is not surprising that tRNAs have been hijacked during evolution for functions outside of translation. These roles beyond translation include synthetic, regulatory and information functions within the cell. Here we provide an overview of the non-canonical roles of tRNAs and their mimics in bacteria, and discuss some of the common themes that arise when comparing these different functions. PMID:27169680

  11. Structural and Energetic Impact of Non-Natural 7-Deaza-8-Azaadenine and Its 7-Substituted Derivatives on H-Bonding Potential with Uracil in RNA Molecules.

    PubMed

    Chawla, Mohit; Credendino, Raffaele; Oliva, Romina; Cavallo, Luigi

    2015-10-15

    Non-natural (synthetic) nucleobases, including 7-ethynyl- and 7-triazolyl-8-aza-7-deazaadenine, have been introduced in RNA molecules for targeted applications, and have been characterized experimentally. However, no theoretical characterization of the impact of these modifications on the structure and energetics of the corresponding H-bonded base pair is available. To fill this gap, we performed quantum mechanics calculations, starting with the analysis of the impact of the 8-aza-7-deaza modification of the adenine skeleton, and we moved then to analyze the impact of the specific substituents on the modified 8-aza-7-deazaadenine. Our analysis indicates that, despite of these severe structural modifications, the H-bonding properties of the modified base pair gratifyingly replicate those of the unmodified base pair. Similar behavior is predicted when the same skeleton modifications are applied to guanine when paired to cytosine. To stress further the H-bonding pairing in the modified adenine-uracil base pair, we explored the impact of strong electron donor and electron withdrawing substituents on the C7 position. Also in this case we found minimal impact on the base pair geometry and energy, confirming the validity of this modification strategy to functionalize RNAs without perturbing its stability and biological functionality. PMID:26389789

  12. Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology

    PubMed Central

    2011-01-01

    Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution. PMID:21338175

  13. The Molecule Microscope: A New Instrument for Biological and Biomedical Research

    PubMed Central

    Weaver, James C.; King, John G.

    1973-01-01

    We describe a new instrument, the molecule microscope, which reveals directly spatial variations in the rate of evaporation of molecules from surfaces by using neutral molecules instead of light or charged particles used in existing kinds of microscopes. The surface composition of the sample determines the binding energy of the evaporating molecules and, hence, the rate of evaporation, which also depends on permeability of the sample when the molecules come either from within or from the other side. We show first results obtained with our apparatus, discuss the design of an instrument now under construction with ≈1-μm resolution, and describe briefly some more advanced versions under consideration with ≈100-Å resolution. Images PMID:4542778

  14. RNA-Mediated Silencing in Algae: Biological Roles and Tools for Analysis of Gene Function ▿

    PubMed Central

    Cerutti, Heriberto; Ma, Xinrong; Msanne, Joseph; Repas, Timothy

    2011-01-01

    Algae are a large group of aquatic, typically photosynthetic, eukaryotes that include species from very diverse phylogenetic lineages, from those similar to land plants to those related to protist parasites. The recent sequencing of several algal genomes has provided insights into the great complexity of these organisms. Genomic information has also emphasized our lack of knowledge of the functions of many predicted genes, as well as the gene regulatory mechanisms in algae. Core components of the machinery for RNA-mediated silencing show widespread distribution among algal lineages, but they also seem to have been lost entirely from several species with relatively small nuclear genomes. Complex sets of endogenous small RNAs, including candidate microRNAs and small interfering RNAs, have now been identified by high-throughput sequencing in green, red, and brown algae. However, the natural roles of RNA-mediated silencing in algal biology remain poorly understood. Limited evidence suggests that small RNAs may function, in different algae, in defense mechanisms against transposon mobilization, in responses to nutrient deprivation and, possibly, in the regulation of recently evolved developmental processes. From a practical perspective, RNA interference (RNAi) is becoming a promising tool for assessing gene function by sequence-specific knockdown. Transient gene silencing, triggered with exogenously synthesized nucleic acids, and/or stable gene repression, involving genome-integrated transgenes, have been achieved in green algae, diatoms, yellow-green algae, and euglenoids. The development of RNAi technology in conjunction with system level “omics” approaches may provide the tools needed to advance our understanding of algal physiological and metabolic processes. PMID:21803865

  15. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    PubMed Central

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-01-01

    Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing. PMID:26404236

  16. Conformational, spectroscopic and nonlinear optical properties of biologically active N,N-dimethyltryptamine molecule: a theoretical study.

    PubMed

    Öner, Nazmiye; Tamer, Ömer; Avcı, Davut; Atalay, Yusuf

    2014-12-10

    The effective psychoactive properties of N,N-dimethyltryptamine (DMT) known as the near-death molecule have encouraged the imagination of many research disciplines for several decades. Although there is no theoretical study, a number of paper composed by experimental techniques have been reported for DMT molecule. In this study, the molecular modeling of DMT was carried out using B3LYP and HSEh1PBE levels of density functional theory (DFT). Our calculations showed that the energy gap between HOMO and LUMO is low, demonstrating that DMT is a biologically active molecule. Large hyperconjugation interaction energies imply that molecular charge transfer occurs in DMT. Moreover, NLO analysis indicates that DMT can be used an effective NLO material. PMID:24983923

  17. Conformational, spectroscopic and nonlinear optical properties of biologically active N,N-dimethyltryptamine molecule: A theoretical study

    NASA Astrophysics Data System (ADS)

    Öner, Nazmiye; Tamer, Ömer; Avcı, Davut; Atalay, Yusuf

    2014-12-01

    The effective psychoactive properties of N,N-dimethyltryptamine (DMT) known as the near-death molecule have encouraged the imagination of many research disciplines for several decades. Although there is no theoretical study, a number of paper composed by experimental techniques have been reported for DMT molecule. In this study, the molecular modeling of DMT was carried out using B3LYP and HSEh1PBE levels of density functional theory (DFT). Our calculations showed that the energy gap between HOMO and LUMO is low, demonstrating that DMT is a biologically active molecule. Large hyperconjugation interaction energies imply that molecular charge transfer occurs in DMT. Moreover, NLO analysis indicates that DMT can be used an effective NLO material.

  18. Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells

    PubMed Central

    Santos-Carballal, B.; Aaldering, L. J.; Ritzefeld, M.; Pereira, S.; Sewald, N.; Moerschbacher, B. M.; Götte, M.; Goycoolea, F. M.

    2015-01-01

    Cancer gene therapy requires the design of non-viral vectors that carry genetic material and selectively deliver it with minimal toxicity. Non-viral vectors based on cationic natural polymers can form electrostatic complexes with negatively-charged polynucleotides such as microRNAs (miRNAs). Here we investigated the physicochemical/biophysical properties of chitosan–hsa-miRNA-145 (CS–miRNA) nanocomplexes and the biological responses of MCF-7 breast cancer cells cultured in vitro. Self-assembled CS–miRNA nanocomplexes were produced with a range of (+/−) charge ratios (from 0.6 to 8) using chitosans with various degrees of acetylation and molecular weight. The Z-average particle diameter of the complexes was <200 nm. The surface charge increased with increasing amount of chitosan. We observed that chitosan induces the base-stacking of miRNA in a concentration dependent manner. Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight. We found no evidence that these complexes were cytotoxic towards MCF-7 cells. Furthermore, CS–miRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells. Our data, therefore, shows that CS–miRNA complexes offer a promising non-viral platform for breast cancer gene therapy. PMID:26324407

  19. Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells.

    PubMed

    Santos-Carballal, B; Aaldering, L J; Ritzefeld, M; Pereira, S; Sewald, N; Moerschbacher, B M; Götte, M; Goycoolea, F M

    2015-01-01

    Cancer gene therapy requires the design of non-viral vectors that carry genetic material and selectively deliver it with minimal toxicity. Non-viral vectors based on cationic natural polymers can form electrostatic complexes with negatively-charged polynucleotides such as microRNAs (miRNAs). Here we investigated the physicochemical/biophysical properties of chitosan-hsa-miRNA-145 (CS-miRNA) nanocomplexes and the biological responses of MCF-7 breast cancer cells cultured in vitro. Self-assembled CS-miRNA nanocomplexes were produced with a range of (+/-) charge ratios (from 0.6 to 8) using chitosans with various degrees of acetylation and molecular weight. The Z-average particle diameter of the complexes was <200 nm. The surface charge increased with increasing amount of chitosan. We observed that chitosan induces the base-stacking of miRNA in a concentration dependent manner. Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight. We found no evidence that these complexes were cytotoxic towards MCF-7 cells. Furthermore, CS-miRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells. Our data, therefore, shows that CS-miRNA complexes offer a promising non-viral platform for breast cancer gene therapy. PMID:26324407

  20. Case studies in quantitative biology: Biochemistry on a leash and a single-molecule Hershey-Chase experiment

    NASA Astrophysics Data System (ADS)

    Van Valen, David

    2011-12-01

    The last 50 years of biological research has seen a marked increase in the amount of quantitative data that describes living systems. This wealth of data provides a unique opportunity to recast the pictorial level descriptions of biological processes in the language of mathematics, with the hope that such an undertaking will lead to deeper insights into the behavior of living systems. To achieve this end, we have undertaken three case studies in physical biology. In the first case study, we used statistical mechanics and polymer physics to construct a simple model that describes how flexible chains of amino acids, referred to as tethers, influence the information processing properties of signaling proteins. In the second case study, we studied the DNA ejection process of phage lambda in vitro. In particular, we used bulk and single-molecule methods to study the control parameters that govern the force and kinematics of the ejection process in vitro. In the last case study, we studied the DNA ejection process of phage lambda in vivo. We developed an assay that allows real-time monitoring of DNA ejection in vivo at the single-molecule level. We also developed a parallel system that allows the simultaneous visualization of both phage capsids and phage DNA at the single-cell level, constituting a true single-molecule Hershey-Chase experiment. The work described in this thesis outlines new tools, both in theory and experiment, that can be used to study biological systems as well as a paradigm that can be employed to mathematicize the cartoons of biology.

  1. In situ single molecule imaging of cell membranes: linking basic nanotechniques to cell biology, immunology and medicine

    NASA Astrophysics Data System (ADS)

    Pi, Jiang; Jin, Hua; Yang, Fen; Chen, Zheng W.; Cai, Jiye

    2014-10-01

    The cell membrane, which consists of a viscous phospholipid bilayer, different kinds of proteins and various nano/micrometer-sized domains, plays a very important role in ensuring the stability of the intracellular environment and the order of cellular signal transductions. Exploring the precise cell membrane structure and detailed functions of the biomolecules in a cell membrane would be helpful to understand the underlying mechanisms involved in cell membrane signal transductions, which could further benefit research into cell biology, immunology and medicine. The detection of membrane biomolecules at the single molecule level can provide some subtle information about the molecular structure and the functions of the cell membrane. In particular, information obtained about the molecular mechanisms and other information at the single molecule level are significantly different from that detected from a large amount of biomolecules at the large-scale through traditional techniques, and can thus provide a novel perspective for the study of cell membrane structures and functions. However, the precise investigations of membrane biomolecules prompts researchers to explore cell membranes at the single molecule level by the use of in situ imaging methods, as the exact conformation and functions of biomolecules are highly controlled by the native cellular environment. Recently, the in situ single molecule imaging of cell membranes has attracted increasing attention from cell biologists and immunologists. The size of biomolecules and their clusters on the cell surface are set at the nanoscale, which makes it mandatory to use high- and super-resolution imaging techniques to realize the in situ single molecule imaging of cell membranes. In the past few decades, some amazing imaging techniques and instruments with super resolution have been widely developed for molecule imaging, which can also be further employed for the in situ single molecule imaging of cell membranes. In

  2. Inferring Biological Structures from Super-Resolution Single Molecule Images Using Generative Models

    PubMed Central

    Maji, Suvrajit; Bruchez, Marcel P.

    2012-01-01

    Localization-based super resolution imaging is presently limited by sampling requirements for dynamic measurements of biological structures. Generating an image requires serial acquisition of individual molecular positions at sufficient density to define a biological structure, increasing the acquisition time. Efficient analysis of biological structures from sparse localization data could substantially improve the dynamic imaging capabilities of these methods. Using a feature extraction technique called the Hough Transform simple biological structures are identified from both simulated and real localization data. We demonstrate that these generative models can efficiently infer biological structures in the data from far fewer localizations than are required for complete spatial sampling. Analysis at partial data densities revealed efficient recovery of clathrin vesicle size distributions and microtubule orientation angles with as little as 10% of the localization data. This approach significantly increases the temporal resolution for dynamic imaging and provides quantitatively useful biological information. PMID:22629348

  3. Super-resolution imaging with stochastic single-molecule localization: concepts, technical developments, and biological applications.

    PubMed

    Oddone, Anna; Vilanova, Ione Verdeny; Tam, Johnny; Lakadamyali, Melike

    2014-07-01

    Light microscopy has undergone a revolution with the advent of super-resolution microscopy methods that can surpass the diffraction limit. These methods have generated much enthusiasm, in particular with regards to the new possibilities they offer for biological imaging. The recent years have seen a great advancement both in terms of new technological developments and exciting biological applications. Here, we review some of the important milestones in the field and highlight some recent biological applications. PMID:24616244

  4. Nucleic Acids as Information Molecules.

    ERIC Educational Resources Information Center

    McInerney, Joseph D.

    1996-01-01

    Presents an activity that aims at enabling students to recognize that DNA and RNA are information molecules whose function is to store, copy, and make available the information in biological systems, without feeling overwhelmed by the specialized vocabulary and the minutia of the central dogma. (JRH)

  5. How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

    PubMed Central

    Gierliński, Marek; Sherstnev, Alexander; Singh, Vijender; Wrobel, Nicola; Gharbi, Karim; Simpson, Gordon G.; Owen-Hughes, Tom; Blaxter, Mark

    2016-01-01

    RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%–40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools. PMID:27022035

  6. How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

    PubMed

    Schurch, Nicholas J; Schofield, Pietá; Gierliński, Marek; Cole, Christian; Sherstnev, Alexander; Singh, Vijender; Wrobel, Nicola; Gharbi, Karim; Simpson, Gordon G; Owen-Hughes, Tom; Blaxter, Mark; Barton, Geoffrey J

    2016-06-01

    RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%-40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools. PMID:27022035

  7. Specific ligation to double-stranded RNA for analysis of cellular RNA::RNA interactions.

    PubMed

    Faridani, Omid R; McInerney, Gerald M; Gradin, Katarina; Good, Liam

    2008-09-01

    Double-stranded RNA (dsRNA) is formed in cells as intra- and intermolecular RNA interactions and is involved in a range of biological processes including RNA metabolism, RNA interference and translation control mediated by natural antisense RNA and microRNA. Despite this breadth of activities, few molecular tools are available to analyse dsRNA as native hybrids. We describe a two-step ligation method for enzymatic joining of dsRNA adaptors to any dsRNA molecule in its duplex form without a need for prior sequence or termini information. The method is specific for dsRNA and can ligate various adaptors to label, map or amplify dsRNA sequences. When combined with reverse transcription-polymerase chain reaction, the method is sensitive and can detect low nanomolar concentrations of dsRNA in total RNA. As examples, we mapped dsRNA/single-stranded RNA junctions within Escherichia coli hok mRNA and the human immunodeficiency virus TAR element using RNA from bacteria and mammalian cells. PMID:18628292

  8. Si Shen Wan Inhibits mRNA Expression of Apoptosis-Related Molecules in p38 MAPK Signal Pathway in Mice with Colitis

    PubMed Central

    Zhao, Hai-Mei; Huang, Xiao-Ying; Zhou, Feng; Tong, Wen-Ting; Wan, Pan-Ting; Huang, Min-Fang; Ye, Qing; Liu, Duan-Yong

    2013-01-01

    Si Shen Wan (SSW) is used to effectively treat ulcerative colitis (UC) as a formula of traditional Chinese medicine. To explore the mechanism of SSW-inhibited apoptosis of colonic epithelial cell, the study observed mRNA expression of apoptosis-related molecules in p38 MAPK signal pathway in colonic mucosa in colitis mice treated with SSW. Experimental colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice; meanwhile, the mice were administrated daily either SSW (5 g/kg) or p38 MAPK inhibitor (2 mg/kg) or vehicle (physiological saline) for 10 days. While microscopical evaluation was observed, apoptosis rate of colonic epithelial cell and mRNA expression of apoptosis-related molecules were tested. Compared with colitis mice without treatment, SSW alleviated colonic mucosal injuries and decreased apoptosis rate of colonic epithelial cell, while the mRNA expressions of p38 MAPK, p53, caspase-3, c-jun, c-fos, Bax, and TNF-α were decreased in the colonic mucosa in colitis mice treated with SSW, and Bcl-2 mRNA and the ratio of Bcl-2/Bax were increased. The present study demonstrated that SSW inhibited mRNA expression of apoptosis-related molecules in p38 MAPK signal pathway to downregulate colonic epithelial cells apoptosis in colonic mucosa in mice with colitis. PMID:24223057

  9. In situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in calves with acute Pasteurella haemolytica pneumonia.

    PubMed

    Radi, Z A; Register, K B; Lee, E K; Kehrli, M E; Brogden, K A; Gallup, J M; Ackermann, M R

    1999-09-01

    The in situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in normal and pneumonic lung tissues of Holstein calves with bovine leukocyte adhesion deficiency (BLAD) was compared with that of age-matched non-BLAD Holstein calves by in situ hybridization. Twenty-four Holstein calves (both BLAD and non-BLAD) were randomly assigned to one of two experimental groups and inoculated intrabronchially with Pasteurella haemolytica or pyrogen-free saline. Lung tissues were collected and fixed in 10% neutral formalin at 2 or 4 hours postinoculation (PI). The expression and distribution of ICAM-1 mRNA in the different cell types of the lung tissue was detected by in situ hybridization with a 307-base-pair bovine ICAM-1 riboprobe. In lungs of both non-BLAD and BLAD saline-inoculated calves, ICAM-1 expression was present in epithelial cells but occurred in <30% of cells in bronchi, bronchioles, and alveoli. ICAM-1 expression in vascular endothelial cells was present in <30% of cells in pulmonary arteries and veins. The expression of ICAM-1 was significantly greater (>60% of cells) in bronchiolar and alveolar epithelial cells and pulmonary endothelial cells of arteries and veins in both BLAD and non-BLAD calves inoculated with P. haemolytica. Bronchiolar epithelium had the highest intensity of mRNA expression and highest percentage of cells that were stained, whereas bronchial epithelium had the lowest intensity and percentage of cells stained. Most alveolar macrophages and neutrophils in infected lungs also expressed ICAM-1. ICAM-1 expression was generally increased in infected BLAD calves at 2 hours PI as compared with non-BLAD calves but not at 4 hours PI. The increased expression of ICAM-1 during acute P. haemolytica pneumonia in calves suggests that ICAM-1 is upregulated and may play a role in leukocyte infiltration. The extent of ICAM-1 expression in P. haemolytica-inoculated calves with BLAD was initially enhanced but otherwise similar to that in non

  10. SASSIE: A program to study intrinsically disordered biological molecules and macromolecular ensembles using experimental scattering restraints

    NASA Astrophysics Data System (ADS)

    Curtis, Joseph E.; Raghunandan, Sindhu; Nanda, Hirsh; Krueger, Susan

    2012-02-01

    A program to construct ensembles of biomolecular structures that are consistent with experimental scattering data are described. Specifically, we generate an ensemble of biomolecular structures by varying sets of backbone dihedral angles that are then filtered using experimentally determined restraints to rapidly determine structures that have scattering profiles that are consistent with scattering data. We discuss an application of these tools to predict a set of structures for the HIV-1 Gag protein, an intrinsically disordered protein, that are consistent with small-angle neutron scattering experimental data. We have assembled these algorithms into a program called SASSIE for structure generation, visualization, and analysis of intrinsically disordered proteins and other macromolecular ensembles using neutron and X-ray scattering restraints. Program summaryProgram title: SASSIE Catalogue identifier: AEKL_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEKL_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU General Public License v3 No. of lines in distributed program, including test data, etc.: 3 991 624 No. of bytes in distributed program, including test data, etc.: 826 Distribution format: tar.gz Programming language: Python, C/C++, Fortran Computer: PC/Mac Operating system: 32- and 64-bit Linux (Ubuntu 10.04, Centos 5.6) and Mac OS X (10.6.6) RAM: 1 GB Classification: 3 External routines: Python 2.6.5, numpy 1.4.0, swig 1.3.40, scipy 0.8.0, Gnuplot-py-1.8, Tcl 8.5, Tk 8.5, Mac installation requires aquaterm 1.0 (or X window system) and Xcode 3 development tools. Nature of problem: Open source software to generate structures of disordered biological molecules that subsequently allow for the comparison of computational and experimental results is limiting the use of scattering resources. Solution method: Starting with an all atom model of a protein, for example, users can input

  11. Identification and biological activities of a new antiangiogenic small molecule that suppresses mitochondrial reactive oxygen species

    SciTech Connect

    Kim, Ki Hyun; Park, Ju Yeol; Jung, Hye Jin; Kwon, Ho Jeong

    2011-01-07

    Research highlights: {yields} YCG063 was screened as a new angiogenesis inhibitor which suppresses mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library. {yields} The compound inhibited in vitro and in vivo angiogenesis in a dose-dependent manner. {yields} This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions. -- Abstract: Mitochondrial reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. In particular, high levels of mitochondrial ROS in hypoxic cells regulate many angiogenesis-related diseases, including cancer and ischemic disorders. Here we report a new angiogenesis inhibitor, YCG063, which suppressed mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library with an ArrayScan HCS reader. YCG063 suppressed mitochondrial ROS generation under a hypoxic condition in a dose-dependent manner, leading to the inhibition of in vitro angiogenic tube formation and chemoinvasion as well as in vivo angiogenesis of the chorioallantoic membrane (CAM) at non-toxic doses. In addition, YCG063 decreased the expression levels of HIF-1{alpha} and its target gene, VEGF. Collectively, a new antiangiogenic small molecule that suppresses mitochondrial ROS was identified. This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions.

  12. Identification and validation of a novel microRNA-like molecule derived from a cytoplasmic RNA virus antigenome by bioinformatics and experimental approaches

    PubMed Central

    2014-01-01

    Background It is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs (miRNAs) due to nucleus inaccessibility. Recent studies have described cytoplasmic RNA virus genome-derived miRNAs in West Nile virus (WNV) and Dengue virus (DENV). However, naturally occurring miRNAs derived from the antigenome of a cytoplasmic RNA virus have not been described. Methods Hepatitis A virus (HAV) was served as a model virus to investigate whether the antigenome of a cytoplasmic RNA virus would be processed into miRNAs or miRNA-like small RNAs upon infection. HAV antigenome was queried for putative miRNA precursors (pre-miRNA) with the VMir analyzer program. Mature miRNA prediction was performed using MatureBayes and Bayes-SVM-MiRNA web server v1.0. Finally, multiple experimental approaches, including cloning and sequencing-, RNAi-, plasmid-based miRNA expression- and luciferase reporter assays, were performed to identify and validate naturally occurring viral antigenome-derived miRNAs. Results Using human HAV genotype IA (isolate H2) (HAVH2), a virally encoded miRNA-like small RNA was detected on the antigenome and named hav-miR-N1-3p. Transcription of viral pre-miRNA in KMB17 and HEK293T cells led to mature hav-miR-N1-3p production. In addition, silencing of the miRNA-processing enzyme Dicer or Drosha caused a dramatic reduction in miRNA levels. Furthermore, artificial target of hav-miR-N1-3p was silenced by synthesized viral miRNA mimics and the HAVH2 naturally-derived hav-miR-N1-3p. Conclusion These results suggested that the antigenome of a cytoplasmic RNA virus could be processed into functional miRNAs. Our findings provide new evidence supporting the hypothesis that cytoplasmic RNA viruses naturally encode miRNAs through cellular miRNA processing machinery. PMID:24981144

  13. microRNA-146a targets the L1 cell adhesion molecule and suppresses the metastatic potential of gastric cancer.

    PubMed

    Hou, Zhibo; Yin, Haitao; Chen, Cong; Dai, Xinzheng; Li, Xiaolin; Liu, Baorui; Fang, Xuefeng

    2012-09-01

    Recent studies have shown that microRNA-146a (miR-146a) is associated with cancer metastasis. However, the mechanisms underlying this process remain poorly understood. In this study, we aimed to investigate the potential role of miR-146a in gastric cancer metastasis. A wound-healing assay and a Transwell assay were used to investigate the impact of miR-146a on the migratory and invasive abilities of MKN-45 cells in vitro. MKN-45 cells stably expressing miR-146a or the negative control were transplanted into nude mice through the lateral tail vein to explore the effect of miR-146a on tumor metastasis in vivo. A luciferase reporter assay and western blot analysis were used to identify the potential target genes. Our results show that the overexpression of miR-146a inhibits the invasion and metastasis of MKN-45 cells in vitro and in vivo. Furthermore, the L1 cell adhesion molecule (L1CAM) was identified as a novel target of miR‑146a in gastric cancer. Taken together, our results provide evidence that miR-146a suppresses gastric cancer cell invasion and metastasis in vitro and in vivo, which may be in part due to the downregulation of L1CAM. miR-146a may have the therapeutic potential to suppress gastric cancer metastasis. PMID:22711166

  14. Novel epithelial cell adhesion molecule antibody conjugated polyethyleneimine-capped gold nanoparticles for enhanced and targeted small interfering RNA delivery to retinoblastoma cells

    PubMed Central

    Mitra, Moutushy; Kandalam, Mallikarjuna; Rangasamy, Judith; Shankar, Balaji; Maheswari, Uma K.; Swaminathan, Sethuraman

    2013-01-01

    Background Several nanoconjugates have been designed to deliver nucleic acids such as small interfering RNA (siRNA) and DNA to cells to study silencing and expression efficacies. In the present study, we prepared novel epithelial cell adhesion molecule (EpCAM) monoclonal antibody conjugated polyethyleneimine (PEI) capped gold nanoparticles (AuNPs) loaded with EpCAM-specific siRNA molecules to knock-down the EpCAM gene in retinoblastoma (RB) cells. We chose EpCAM as a target moiety to deliver siRNA because this molecule is highly expressed in various epithelial cancers and is an ideal target as it is highly expressed in the apical surface of tumor cells while showing basolateral expression in normal cells. Methods The EpCAM antibody was conjugated to AuNP-PEI loaded with siRNA molecules to specifically deliver siRNA to EpCAM-expressing RB cells. Conjugation efficiencies were confirmed with ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, and agarose and SDS–polyacrylamide gel electrophoresis. The size and zeta potential were measured using a Zeta sizer analyzer. Nanoparticle internalization and uptake were studied using fluorescent microscopy and flow cytometry. Gene silencing efficacy was monitored with western blot analysis and real-time quantitative PCR. Results Optimal size and neutral zeta potential properties of the AuNP-PEI- EpCAM antibody (EpAb) antibody were achieved for the transfection studies. The AuNP-PEI nanoparticles did not show any cytotoxicity to the cells, which means these nanomaterials are suitable for intracellular delivery of siRNA for therapeutic interventions. With EpCAM antibody conjugation, PEI-capped AuNPs loaded with EpCAM siRNA were significantly internalized in the Y79 cells as observed with fluorescence microscopy and flow cytometry and induced a highly significant reduction in the cell viability of the Y79 cells. Through increased binding of EpCAM antibody–conjugated AuNP-PEI nanoparticles

  15. PDMS-Glass bonding using grafted polymeric adhesive - Alternative process flow for compatibility with patterned biological molecules

    PubMed Central

    Beh, Cyrus Weijie; Zhou, Weizhuang

    2013-01-01

    We report a novel modification of silicone elastomer, polydimethylsiloxane (PDMS) with a polymer graft that allows interfacial bonding between elastomer and glass substrate to be performed without exposure of said substrate to harsh treatment conditions like oxygen plasma. Organic molecules can thus be patterned within microfluidic channels and still remain functional post-bonding. In addition, after polymer grafting the PDMS can be stored in a desiccator for at least 40 days, and activated upon exposure to acidic buffer for bonding. The bonded devices remain fully bonded in excess of 80 psi driving pressure, with no signs of compromise to the bond integrity. Finally, we demonstrate the compatibility of our method with biological molecules using a proof-of-concept DNA sensing device, in which fluorescently-labelled DNA targets are successfully captured by a patterned probe in a device sealed using our method, while the pattern on a plasma-treated device was completely destroyed. Therefore, this method provides a much-needed alternative bonding process for incorporation of biological molecules in microfluidic devices. PMID:22858861

  16. Nanometric Gap Structure with a Fluid Lipid Bilayer for the Selective Transport and Detection of Biological Molecules.

    PubMed

    Ando, Koji; Tanabe, Masashi; Morigaki, Kenichi

    2016-08-01

    The biological membrane is a natural biosensing platform that can detect specific molecules with extremely high sensitivity. We developed a biosensing methodology by combining a model biological membrane and a nanometer-sized gap structure on a glass substrate. The model membrane comprised lithographically patterned polymeric and fluid lipid bilayers. The polymeric bilayer was bonded to a poly(dimethylsiloxane) (PDMS) sheet by using an adhesion layer with a defined thickness (lipid vesicles). Extruded lipid vesicles having a biotin moiety on the surface were used as the adhesion layer in conjunction with the biotin-streptavidin linkage. A gap structure was formed between the fluid bilayer and PDMS (nanogap junction). The thickness of the gap structure was several tens of nanometers, as determined by the thickness of the adhesion layer. The nanogap junction acted as a sensitive biosensing platform. From a mixture of proteins (cholera toxin and albumin), the target protein (cholera toxin) was selectively transported into the gap by the specific binding to a glycolipid (GM1) in the fluid bilayer and lateral diffusion. The target protein molecules were then detected with an elevated signal-to-noise ratio due to the reduced background noise in the nanometric gap. The combination of selective transport and reduced background noise drastically enhanced the sensitivity toward the target protein. The nanogap junction should have broad biomedical applications by realizing highly selective and sensitive biosensing in samples having diverse coexisting molecules. PMID:27427950

  17. A transcribed ultraconserved noncoding RNA, Uc.173, is a key molecule for the inhibition of lead-induced neuronal apoptosis

    PubMed Central

    Chen, Lijian; Liu, Meiling; Zhang, Nan; Zhang, Li; Luo, Yuanwei; Liu, Zhenzhong; Dai, Lijun; Jiang, Yiguo

    2016-01-01

    As a common toxic metal, lead has significant neurotoxicity to brain development. Long non-coding RNAs (lncRNAs) function in multiple biological processes. However, whether lncRNAs are involved in lead-induced neurotoxicity remains unclear. Uc.173 is a lncRNA from a transcribed ultra-conservative region (T-UCR) of human, mouse and rat genomes. We established a lead-induced nerve injury mouse model. It showed the levels of Uc.173 decreased significantly in hippocampus tissue and serum of the model. We further tested the expression of Uc.173 in serum of lead-exposed children, which also showed a tendency to decrease. To explore the effects of Uc.173 on lead-induced nerve injury, we overexpressed Uc.173 in an N2a mouse nerve cell line and found Uc.173 had an inhibitory effect on lead-induced apoptosis of N2a. To investigate the molecular mechanisms of Uc.173 in apoptosis associated with lead-induced nerve injury, we predicted the target microRNAs of Uc.173 by using miRanda, TargetScan and RegRNA. After performing quantitative real-time PCR and bioinformatics analysis, we showed Uc.173 might inter-regulate with miR-291a-3p in lead-induced apoptosis and regulate apoptosis-associated genes. Our study suggests Uc.173 significantly inhibits the apoptosis of nerve cells, which may be mediated by inter-regulation with miRNAs in lead-induced nerve injury. PMID:26683706

  18. A transcribed ultraconserved noncoding RNA, Uc.173, is a key molecule for the inhibition of lead-induced neuronal apoptosis.

    PubMed

    Nan, Aruo; Zhou, Xinke; Chen, Lijian; Liu, Meiling; Zhang, Nan; Zhang, Li; Luo, Yuanwei; Liu, Zhenzhong; Dai, Lijun; Jiang, Yiguo

    2016-01-01

    As a common toxic metal, lead has significant neurotoxicity to brain development. Long non-coding RNAs (lncRNAs) function in multiple biological processes. However, whether lncRNAs are involved in lead-induced neurotoxicity remains unclear. Uc.173 is a lncRNA from a transcribed ultra-conservative region (T-UCR) of human, mouse and rat genomes. We established a lead-induced nerve injury mouse model. It showed the levels of Uc.173 decreased significantly in hippocampus tissue and serum of the model. We further tested the expression of Uc.173 in serum of lead-exposed children, which also showed a tendency to decrease. To explore the effects of Uc.173 on lead-induced nerve injury, we overexpressed Uc.173 in an N2a mouse nerve cell line and found Uc.173 had an inhibitory effect on lead-induced apoptosis of N2a. To investigate the molecular mechanisms of Uc.173 in apoptosis associated with lead-induced nerve injury, we predicted the target microRNAs of Uc.173 by using miRanda, TargetScan and RegRNA. After performing quantitative real-time PCR and bioinformatics analysis, we showed Uc.173 might inter-regulate with miR-291a-3p in lead-induced apoptosis and regulate apoptosis-associated genes. Our study suggests Uc.173 significantly inhibits the apoptosis of nerve cells, which may be mediated by inter-regulation with miRNAs in lead-induced nerve injury. PMID:26683706

  19. Monte Carlo simulation of several biologically relevant molecules and zwitterions in water

    NASA Astrophysics Data System (ADS)

    Patuwo, Michael Y.; Bettens, Ryan P. A.

    2012-02-01

    In this work, we study the hydration free energies of butane, zwitterionic alanine, valine, serine, threonine, and asparagine, and two neuraminidase inhibitors by means of Monte Carlo (MC) simulation. The solute molecule, represented in the form of distributed multipoles and modified 6-12 potential, was varied from a non-interacting 'ghost' molecule to its full potential functions in TIP4P water. Intermediate systems with soft-core solute-solvent interaction potentials are simulated separately and then subjected to Bennett's Acceptance ratio (BAR) for the free energy calculation. Hydration shells surrounding the solute particles were used to assess the quality of potential functions.

  20. Mapping of the active site of Escherichia coli methionyl-tRNA synthetase: Identification of amino acid residues labeled by periodate-oxidized tRNA sup fMet molecules having modified lengths at the 3 prime -acceptor end

    SciTech Connect

    Hountondji, C.; Schmitter, J.M.; Beauvallet, C.; Blanquet, S. )

    1990-09-04

    Initiator tRNA molecules modified at the 3{prime}-end and lacking either A{sub 76} (tRNA-C{sub 75}), the C{sub 75}-A{sub 76} (tRNA-C{sub 74}), the C{sub 74}-C{sub 75}-A{sub 76} (tRNA-A{sub 73}), or the A{sub 73}-C{sub 74}-C{sub 75}-A{sub 76} (tRNA-A{sub 72}) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments. When incubated with trypsin-modified methionyl-tRNA synthetase (MTS{sub T}), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C{sub 75}ox, tRNA-A{sub 73}ox, and tRNA-A{sub 72}ox) abolished both the isotopic ({sup 32}P)PP{sub i}ATP exchange and the tRNA aminoacylation activities of the enzyme. In the presence of limiting concentrations of the various tRNAox species, the relative extents of inactivation of the enzyme were consistent with the formation of 1:1 complexes of the reacting tRNAs with the monomeric modified synthetase. Specificity of the labeling was further established by demonstrating that tRNA-C{sub 75}ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C{sub 75}. The peptides of MTS{sub T} labeled with either tRNA-C{sub 75}ox or tRNA-C{sub 74}ox were identified. In a previous work all these peptides but one (peptide D) had been already found labeled upon MTS{sub T} incubation with ({sup 14}C)tRNA-A{sub 76}ox. According to the crystallographic structure of MTS{sub T}, the labeled residues K335, K61, K142, K147, and K149 are within a sphere of about 5.5-{angstrom} radius. The present results therefore argue for a marked flexibility of the 3{prime}-end of the enzyme-bound tRNA, enabling it to contact any of the identified reacting residues. Such a cluster of basic amino acids may reflect ionic requirements in the guiding of the negatively charged CCA arm of tRNA toward enzyme-bound methionyl-adenylate.

  1. 1,N6-etheno deoxy and ribo adenosine and 3,N4-etheno deoxy and ribo cytidine phosphoramidites. Strongly fluorescent structures for selective introduction in defined sequence DNA and RNA molecules.

    PubMed Central

    Srivastava, S C; Raza, S K; Misra, R

    1994-01-01

    Synthesis of 1,N6-etheno-2'-deoxyadenosine, 3,N4-etheno-2'-deoxycytidine, and further chemistry on both deoxy and ribo series etheno nucleosides produces the corresponding phosphoramidites. These novel phosphoramidites are introduced selectively, quantitatively, and at specific positions at single or multiple sites into DNA or RNA sequences. The purification and chemistry involved in the synthesis of these products has been optimized to achieve the purity in excess of 99%. The resulting phosphoramidites were tested for their ability to couple and produce poly deoxy and ribonucleotides by solid phase chemistry. The coupling efficiency achieved was greater than 99% per step. Due to the instability of these etheno compounds in acidic and basic medium, various criteria to obtain pure oligomers have been established. The selective introduction of these fluorescent nucleosides into defined sequence DNA and RNA molecule will greatly facilitate the structure-function studies of various RNAs, protein-RNA structures, and DNA-RNA based diagnostics applications. The characteristic and high fluorescent intensity (detection below 1 x 10(-9) M for adenosine sites and below 1 x 10(-7) M for cytidine sites) is particularly suited for the biochemical and biological research and product development applications. The usefulness of these etheno containing modified sequences as sequencing and amplification primers is demonstrated by their full participation in polymerase chain reaction experiments. Images PMID:7513082

  2. Unequal Activities of Enantiomers via Biological Receptors: Examples of Chiral Drug, Pesticide, and Fragrance Molecules

    ERIC Educational Resources Information Center

    Mannschreck, Albrecht; Kiesswetter, Roland; von Angerer, Erwin

    2007-01-01

    A molecule coming from outside an organism can form a ligand-receptor complex. Upon its formation, a message is transmitted, for example, to certain cells. In this way, two enantiomers can emit messages that differ, either quantitatively or qualitatively. In the present article, these facts are taken as a common basis for the actions of chiral…

  3. Integration of biological ion channels onto optically addressable micro-fluidic electrode arrays for single molecule characterization.

    SciTech Connect

    Brozik, Susan Marie; Frink, Laura J. Douglas; Bachand, George David; Keller, David J.; Patrick, Elizabeth L.; Marshall, Jason A.; Ortiz, Theodore P.; Meyer, Lauren A.; Davis, Ryan W.; Brozik, James A.; Flemming, Jeb Hunter

    2004-12-01

    The challenge of modeling the organization and function of biological membranes on a solid support has received considerable attention in recent years, primarily driven by potential applications in biosensor design. Affinity-based biosensors show great promise for extremely sensitive detection of BW agents and toxins. Receptor molecules have been successfully incorporated into phospholipid bilayers supported on sensing platforms. However, a collective body of data detailing a mechanistic understanding of membrane processes involved in receptor-substrate interactions and the competition between localized perturbations and delocalized responses resulting in reorganization of transmembrane protein structure, has yet to be produced. This report describes a systematic procedure to develop detailed correlation between (recognition-induced) protein restructuring and function of a ligand gated ion channel by combining single molecule fluorescence spectroscopy and single channel current recordings. This document is divided into three sections: (1) reported are the thermodynamics and diffusion properties of gramicidin using single molecule fluorescence imaging and (2) preliminary work on the 5HT{sub 3} serotonin receptor. Thirdly, we describe the design and fabrication of a miniaturized platform using the concepts of these two technologies (spectroscopic and single channel electrochemical techniques) for single molecule analysis, with a longer term goal of using the physical and electronic changes caused by a specific molecular recognition event as a transduction pathway in affinity based biosensors for biotoxin detection.

  4. RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation

    NASA Astrophysics Data System (ADS)

    Dawid, Alexandre; Cayrol, Bastien; Isambert, Hervé

    2009-06-01

    Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA-RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners.

  5. Coulomb explosion and binary encounter processes in collisions between slow ions and small molecules of biological interest

    SciTech Connect

    Juhasz, Z.; Sulik, B.

    2008-12-08

    In this work we study the ion impact induced fragmentation of small molecules, which are relevant for radiation damage studies in biological tissues. We present double differential ion emission yields for collisions of N{sup 6+} ions with water and methane molecules at 15 and 30 keV impact energies. The angular distribution of the fragment ions shows post-collision and nucleus-nucleus binary collision effects. In the multiple capture energy range, a strong interplay is indicated between the Coulomb explosion and the binary collision mechanisms. In the energy region, where triple capture is dominant, an unexpected angular distribution was found for water fragments, which may be attributed to orientation sensitivity of some of the capture channels. Such processes are relevant for astrophysics and radiation therapy.

  6. Effect of Co-Existing Biologically Relevant Molecules and Ions on DNA Photocleavage Caused by Pyrene and its Derivatives

    PubMed Central

    Wang, Shuguang; Yu, Hongtao

    2005-01-01

    Inorganic ions, coenzymes, amino acids, and saccharides could co-exist with toxic environmental chemicals, such as polycyclic aromatic hydrocarbons (PAHs), in the cell. The presence of these co-existing chemicals can modulate the toxicity of the PAHs. One of the genotoxic effects by PAHs is light-induced cleavage, or photocleavage, of DNA. The effect of inorganic ions I−, Na+, Ca2+, Mg2+, Fe3+, Mn2+, Cu2+, and Zn2+ and biological molecules riboflavin, histidine, mannitol, nicotinamide adenine dinucleotide (NAD), glutathione, and glutamic acid on the DNA photocleavage by pyrene, 1-hydroxypyrene (1-HP), and 1-aminopyrene (1-AP), is studied. The non-transition metal ions Na+, Ca2+, and Mg2+, usually have very little inhibitory effects, while the transition metal ions Fe3+, Cu2+, and Zn2+ enhance, Mn2+ inhibits the DNA photocleavage. The effect by biological molecules is complex, depending on the photochemical reaction mechanisms of the compounds tested (1-AP, 1-HP and pyrene) and on the chemical nature of the added biological molecules. Riboflavin, histidine, and mannitol enhance DNA photocleavage by all three compounds, except that mannitol has no effect on the photocleavage of DNA by pyrene. Glutathione inhibits the DNA photocleavage by 1-AP and 1-HP, but has no effect on that by pyrene. NAD enhances the DNA photocleavage by 1-AP, but has no effect on that by 1-HP and pyrene. Glutamic acid enhances the DNA photocleavage by 1-AP and pyrene, but inhibits that by 1-HP. These results show that the co-existing chemicals may have a profound effect on the toxicity of PAHs, or possibly on the toxicity of many other chemicals. Therefore, if one studies the toxic effects of PAHs or other toxic chemicals, the effect of the co-existing chemicals or ions needs to be considered. PMID:16705811

  7. High-throughput, dual probe biological assays based on single molecule detection

    DOEpatents

    Hollars, Christopher W.; Huser, Thomas R.; Lane, Stephen M.; Balhorn, Rodney L.; Bakajin, Olgica; Darrow, Christopher; 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.

  8. AT7519, A novel small molecule multi-cyclin-dependent kinase inhibitor, induces apoptosis in multiple myeloma via GSK-3beta activation and RNA polymerase II inhibition.

    PubMed

    Santo, L; Vallet, S; Hideshima, T; Cirstea, D; Ikeda, H; Pozzi, S; Patel, K; Okawa, Y; Gorgun, G; Perrone, G; Calabrese, E; Yule, M; Squires, M; Ladetto, M; Boccadoro, M; Richardson, P G; Munshi, N C; Anderson, K C; Raje, N

    2010-04-22

    Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM. PMID:20101221

  9. ToppMiR: ranking microRNAs and their mRNA targets based on biological functions and context

    PubMed Central

    Wu, Chao; Bardes, Eric E.; Jegga, Anil G.; Aronow, Bruce J.

    2014-01-01

    Identifying functionally significant microRNAs (miRs) and their correspondingly most important messenger RNA targets (mRNAs) in specific biological contexts is a critical task to improve our understanding of molecular mechanisms underlying organismal development, physiology and disease. However, current miR–mRNA target prediction platforms rank miR targets based on estimated strength of physical interactions and lack the ability to rank interactants as a function of their potential to impact a given biological system. To address this, we have developed ToppMiR (http://toppmir.cchmc.org), a web-based analytical workbench that allows miRs and mRNAs to be co-analyzed via biologically centered approaches in which gene function associated annotations are used to train a machine learning-based analysis engine. ToppMiR learns about biological contexts based on gene associated information from expression data or from a user-specified set of genes that relate to context-relevant knowledge or hypotheses. Within the biological framework established by the genes in the training set, its associated information content is then used to calculate a features association matrix composed of biological functions, protein interactions and other features. This scoring matrix is then used to jointly rank both the test/candidate miRs and mRNAs. Results of these analyses are provided as downloadable tables or network file formats usable in Cytoscape. PMID:24829448

  10. A recombinant, soluble, single-chain class I major histocompatibility complex molecule with biological activity.

    PubMed Central

    Mage, M G; Lee, L; Ribaudo, R K; Corr, M; Kozlowski, S; McHugh, L; Margulies, D H

    1992-01-01

    Heterodimeric class I major histocompatibility complex molecules, which consist of a 45-kDa heavy-chain and a 12-kDa beta 2-microglobulin (beta 2m) light chain, bind endogenously synthesized peptides for presentation to antigen-specific T cells. We have synthesized a gene encoding a single-chain, soluble class I molecule derived from mouse H-2Dd, in which the carboxyl terminus of beta 2m is linked via a peptide spacer to the amino terminus of the heavy chain. The chimeric protein is secreted efficiently from transfected L cells, is thermostable, and when loaded with an appropriate antigenic peptide, stimulates an H-2Dd-restricted antigen-specific T-cell hybridoma. Thus, functional binding of peptide does not require the complete dissociation of beta 2m, implying that a heavy chain/peptide complex is not an obligate intermediate in the assembly of the heavy-chain/beta 2m/peptide heterotrimer. Single-chain major histocompatibility complex molecules uniformly loaded with peptide have potential uses for structural studies, toxin or fluor conjugates, and vaccines. Images PMID:1438262

  11. The Formation, Destruction, and Spectra of Extraterrestrial Biological and Prebiological Molecules

    NASA Astrophysics Data System (ADS)

    Hudson, R.; Moore, M.

    Radio and IR observations have revealed that a rich organic chemistry exists in a variety of interstellar regions. Among the organic molecules detected are acids, alcohols, aldehydes, ketones, and nitriles. The simplest sugar, glycolaldehyde, has also been reported (Hollis et al., ApJ, 2000, 540, L107), as has an amino acid, glycine (Kuan et al., ApJ, 2003, 593, 848; but see Hollis et al., ApJ, 2003, 588, 353). Gas-phase reactions to produce many of these molecules are not well understood, and solid-phase chemistry is thought to make an important contribution. To better understand organic chemistry in cold cosmic environments, we have performed photo- and radiation chemical experiments on icy materials at 10 - 100 K. Gas-phase molecules, or molecular mixtures, are frozen in a vacuum chamber and then exposed to either a beam of MeV protons or vacuum-UV photons to mimic cosmic-ray bombardment or cosmic-UV exposure, respectively. Changes in ice composition are followed in situ with IR spectroscopy. In this COSPAR presentation we will describe new laboratory results for glycolaldehyde and glycine, as well as a few prebiological organics. Solid-state IR spectra and synthetic reaction pathways will be presented, and predictions will be made for not only interstellar chemistry, but also the chemistry of Solar System objects such as comets, icy moons (Europa), and Titan. -- This research is funded through NASA's Planetary Atmospheres and SARA programs.

  12. A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB

    PubMed Central

    Lu, Yanyan; Liang, Feng-Xia; Wang, Xiaozhong

    2014-01-01

    SUMMARY Signaling in the ancestral branch of the unfolded protein response (UPR) is initiated by unconventional splicing of HAC1/XBP1 mRNA during endoplasmic reticulum (ER) stress. In mammals, IRE1α has been known to cleave the XBP1 intron. However, the enzyme responsible for ligation of two XBP1 exons remains unknown. Using an XBP1 splicing-based synthetic circuit, we identify RtcB as the primary UPR RNA ligase. In RtcB knockout cells, XBP1 mRNA splicing is defective during ER stress. Genetic rescue and in vitro splicing show that the RNA ligase activity of RtcB is directly required for the splicing of XBP1 mRNA. Taken together, these data demonstrate that RtcB is the long sought RNA ligase that catalyzes unconventional RNA splicing during the mammalian UPR. PMID:25087875

  13. The RCSB PDB “Molecule of the Month”: Inspiring a Molecular View of Biology

    PubMed Central

    Goodsell, David S.; Dutta, Shuchismita; Zardecki, Christine; Voigt, Maria; Berman, Helen M.; Burley, Stephen K.

    2015-01-01

    The Research Collaboratory for Structural Bioinformatics (RCSB) Molecule of the Month series provides a curated introduction to the 3-D biomolecular structures available in the Protein Data Bank archive and the tools that are available at the RCSB website for accessing and exploring them. A variety of educational materials, such as articles, videos, posters, hands-on activities, lesson plans, and curricula, build on this series for use in a variety of educational settings as a general introduction to key topics, such as enzyme action, protein synthesis, and viruses. The series and associated educational materials are freely available at www.rcsb.org. PMID:25942442

  14. Regulation of platelet biology by platelet endothelial cell adhesion molecule-1.

    PubMed

    Jones, Chris I; Moraes, Leonardo A; Gibbins, Jonathan M

    2012-01-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoreceptor tyrosine-based inhibitory motif containing receptor, plays diverse and apparently contradictory roles in regulating the response of platelets to stimuli; inhibiting platelet response to immunoreceptor tyrosine-based activation motif and G protein-coupled receptor signalling following stimulation with collagen, adenosine diphosphate, and thrombin, as well as enhancing integrin outside-in signalling. These dual, and opposing, roles suggest an important and complex role for PECAM-1 in orchestrating platelet response to vascular damage. Indeed, during thrombus formation, the influence of PECAM-1 on the multiple signalling pathways combines leading to a relatively large inhibitory effect on thrombus formation. PMID:22035359

  15. The RCSB PDB "Molecule of the Month": Inspiring a Molecular View of Biology.

    PubMed

    Goodsell, David S; Dutta, Shuchismita; Zardecki, Christine; Voigt, Maria; Berman, Helen M; Burley, Stephen K

    2015-05-01

    The Research Collaboratory for Structural Bioinformatics (RCSB) Molecule of the Month series provides a curated introduction to the 3-D biomolecular structures available in the Protein Data Bank archive and the tools that are available at the RCSB website for accessing and exploring them. A variety of educational materials, such as articles, videos, posters, hands-on activities, lesson plans, and curricula, build on this series for use in a variety of educational settings as a general introduction to key topics, such as enzyme action, protein synthesis, and viruses. The series and associated educational materials are freely available at www.rcsb.org. PMID:25942442

  16. Patterning and characterization of surfaces with organic and biological molecules by the scanning electrochemical microscope.

    PubMed

    Turyan, I; Matsue, T; Mandler, D

    2000-08-01

    A novel approach for micropatterning of surfaces with organic and biological microstructures using the scanning electrochemical microscope (SECM) is described. The approach is based on the introduction of the spatial resolution by local deposition of gold particles followed by monolayer formation and functionalization. Specifically, gold patterns were deposited locally on silicon wafers with the SECM as a result of the controlled anodic dissolution of a gold microelectrode. The gold patterns were further used as microsubstrates for assembling cystamine monolayers to which either fluoresceine isothiocyanate (FIT) or glucose oxidase (GOD) were covalently attached. Characterization of the organic monolayers, as well as the biological activity of the enzyme patterns, was carried out by fluorescence microscopy and the SECM, respectively. PMID:10952523

  17. Detection of biological molecules using boronate-based chemical amplification and optical sensors

    SciTech Connect

    Antwerp, W.P. van; Mastrototaro, J.J.; Lane, S.M.; Satcher, J.H. Jr.; Darrow, C.B.; Peyser, T.A.; Harder, J.

    1999-12-14

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  18. Detection of biological molecules using boronate-based chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph; Lane, Stephen M.; Satcher, Jr., Joe H.; Darrow, Christopher B.; Peyser, Thomas A.; Harder, Jennifer

    1999-01-01

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  19. Detection of biological molecules using boronate-based chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph; Lane, Stephen M.; Satcher, Jr., Joe H.; Darrow, Christopher B.; Peyser, Thomas A.; Harder, Jennifer

    2004-06-15

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  20. Of Molecules and Models.

    ERIC Educational Resources Information Center

    Brinner, Bonnie

    1992-01-01

    Presents an activity in which models help students visualize both the DNA process and transcription. After constructing DNA, RNA messenger, and RNA transfer molecules; students model cells, protein synthesis, codons, and RNA movement. (MDH)

  1. A new strategy for introducing photoactivatable 4-thiouridine ((4S)U) into specific positions in a long RNA molecule.

    PubMed

    Yu, Y T; Steitz, J A

    1997-07-01

    We describe a new protocol, which does not require (4S)UpG, for introducing (4S)U into specific sites in a pre-mRNA substrate. A 5'-half and a full-length RNA are first synthesized by phage RNA polymerase. p(4S)Up, which is derived from (4S)UpU and can therefore be 32P-labeled, is then ligated to the 3' end of the 5'-half RNA with T4 RNA ligase. The 3' phosphate of the ligated product is removed subsequently by CIP (calf intestinal alkaline phosphatase) to produce a 3'-OH group. The 3'-half RNA with a 5' phosphate is produced by site-specific RNase H cleavage of the full-length pre-mRNA directed by a 2'-O-methyl RNA-DNA chimera. The two half RNAs are then aligned with a bridging oligonucleotide and ligated with T4 DNA ligase. Our results show that 32P-p(4S)Up ligation to the 3' end of the 5'-half RNA is comparable to 32P-pCp ligation. Also, the efficiency of the bridging oligonucleotide-mediated two-piece ligation is quite high, approximately 30-50%. This strategy has been applied to the P120 pre-mRNA containing an AT-AC intron, but should be applicable to many other RNAs. PMID:9214662

  2. Max Delbruck Prize in Biological Physics Lecture: Single-molecule protein folding and transition paths

    NASA Astrophysics Data System (ADS)

    Eaton, William

    2012-02-01

    The transition path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs by crossing the free energy barrier between two states. It is a uniquely single-molecule property, and has not yet been observed experimentally for any system in the condensed phase. The importance of the transition path in protein folding is that it contains all of the mechanistic information on how a protein folds. As a major step toward observing transition paths, we have determined the average transition-path time for a fast and a slow-folding protein from a photon-by-photon analysis of fluorescence trajectories in single-molecule FRET experiments. While the folding rate coefficients differ by 10,000-fold, surprisingly, the transition-path times differ by less than 5-fold, showing that a successful barrier crossing event takes almost the same time for a fast- and a slow-folding protein, i.e. almost the same time to fold when it actually happens.

  3. Biological activity of mRNA immobilized on nitrocellulose in NaI.

    PubMed Central

    Bresser, J; Hubbell, H R; Gillespie, D

    1983-01-01

    In 12.2 molal NaI and at 25 degrees C or below, mRNA bound to nitrocellulose while DNA and rRNA did not. Neither the poly(A) tract nor the cap were required for binding. The immobilized RNA could be translated, reverse transcribed, hybridized with radioactive probes, or released for further manipulation. mRNA was efficiently transferred from polyacrylamide to nitrocellulose in NaI. Baking was not required to fix NaI-immobilized mRNA to nitrocellulose. When cells dissolved in 12.2 molal NaI were filtered through nitrocellulose, mRNA became selectively bound (quickblot). The quick-blot system utilizing protease and detergents to prepare cells for NaI solubilization was especially suitable in quantitative, rapid screening of cells for expression of specific genes. Expression of highly repeated DNA sequences was detected in human leukemia cells. Images PMID:6579539

  4. Single-molecule fluorescence measurements reveal the reaction mechanisms of the core RISC, composed of human Argonaute 2 and a guide RNA.

    PubMed

    Jo, Myung Hyun; Song, Ji-Joon; Hohng, Sungchul

    2015-12-01

    In eukaryotes, small RNAs play important roles in both gene regulation and resistance to viral infection. Argonaute proteins have been identified as a key component of the effector complexes of various RNA-silencing pathways, but the mechanistic roles of Argonaute proteins in these pathways are not clearly understood. To address this question, we performed single-molecule fluorescence experiments using an RNA-induced silencing complex (core-RISC) composed of a small RNA and human Argonaute 2. We found that target binding of core-RISC starts at the seed region of the guide RNA. After target binding, four distinct reactions followed: target cleavage, transient binding, stable binding, and Argonaute unloading. Target cleavage required extensive sequence complementarity and accelerated core-RISC dissociation for recycling. In contrast, the stable binding of core-RISC to target RNAs required seed-match only, suggesting a potential explanation for the seed-match rule of microRNA (miRNA) target selection. PMID:26592935

  5. Crystallography Without Crystals: Determining the Structure of Individual Biological Molecules and Nanoparticles

    ScienceCinema

    Ourmazd, Abbas [University of Wisconsin, Milwaukee, Wisconsin, USA

    2010-01-08

    Ever shattered a valuable vase into 10 to the 6th power pieces and tried to reassemble it under a light providing a mean photon count of 10 minus 2 per detector pixel with shot noise? If you can do that, you can do single-molecule crystallography. This talk will outline how this can be done in principle. In more technical terms, the talk will describe how the combination of scattering physics and Bayesian algorithms can be used to reconstruct the 3-D diffracted intensity distribution from a collection of individual 2-D diffiraction patterns down to a mean photon count of 10 minus 2 per pixel, the signal level anticipated from the Linac Coherent Light Source, and hence determine the structure of individual macromolecules and nanoparticles.

  6. Identification of Small-Molecule Inhibitors of the HuR/RNA Interaction Using a Fluorescence Polarization Screening Assay Followed by NMR Validation

    PubMed Central

    Wang, Zhonghua; Bhattacharya, Akash; Ivanov, Dmitri N.

    2015-01-01

    The human antigen R (HuR) stabilizes many mRNAs of proto-oncogene, transcription factors, cytokines and growth factors by recognizing AU-rich elements (AREs) presented in their 3’ or 5’ untranslated region (UTR). Multiple lines of experimental evidence suggest that this process plays a key role in cancer development. Thus, destabilizing HuR/RNA interaction by small molecules presents an opportunity for cancer treatment/prevention. Here we present an integrated approach to identify inhibitors of HuR/RNA interaction using a combination of fluorescence-based and NMR-based high throughput screening (HTS). The HTS assay with fluorescence polarization readout and Z’-score of 0.8 was used to perform a screen of the NCI diversity set V library in a 384 well plate format. An NMR-based assay with saturation transfer difference (STD) detection was used for hits validation. Protein NMR spectroscopy was used to demonstrate that some hit compounds disrupt formation of HuR oligomer, whereas others block RNA binding. Thus, our integrated high throughput approach provides a new avenue for identification of small molecules targeting HuR/RNA interaction. PMID:26390015

  7. Impact of exogenous lipase supplementation on growth, intestinal function, mucosal immune and physical barrier, and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella).

    PubMed

    Liu, Sen; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Wu, Pei; Zeng, Yun-Yun; Xu, Shu-De; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

    2016-08-01

    This study investigated the effects of exogenous lipase supplementation on the growth performance, intestinal growth and function, immune response and physical barrier function, and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella). A total of 450 grass carp (255.02 ± 0.34 g) were fed five diets for 60 days. There were 5 dietary treatments that included a normal protein and lipid diet containing 30% crude protein (CP) with 5% ether extract (EE), and the low-protein and high-lipid diets (28% CP, 6% EE) supplemented with graded levels of exogenous lipase supplementation activity at 0, 1193, 2560 and 3730 U/kg diet. The results indicated that compared with a normal protein and lipid diet (30% CP, 5% EE), a low-protein and high-lipid diet (28% CP, 6% EE) (un-supplemented lipase) improved lysozyme activities and complement component 3 contents in the distal intestine (DI), interleukin 10 mRNA expression in the proximal intestine (PI), and glutathione S-transferases activity and glutathione content in the intestine of young grass carp. In addition, in low-protein and high-lipid diets, optimal exogenous lipase supplementation significantly increased acid phosphatase (ACP) activities and complement component 3 (C3) contents (P < 0.05), up-regulated the relative mRNA levels of antimicrobial peptides (liver expressed antimicrobial peptide 2 and hepcidin) and anti-inflammatory cytokines (interleukin 10 and transforming growth factor β1) and signaling molecules inhibitor protein-κBα (IκBα) and target of rapamycin (TOR) (P < 0.05), down-regulated the mRNA levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 8, interferon γ2, and interleukin 1β), and signaling molecules (nuclear factor kappa B p65, IκB kinase β, IκB kinase γ) (P < 0.05) in the intestine of young grass carp. Moreover, optimal exogenous lipase supplementation significantly decreased reactive oxygen species (ROS), malondialdehyde

  8. “Turn-on” fluorescence probe integrated polymer nanoparticles for sensing biological thiol molecules

    PubMed Central

    Ang, Chung Yen; Tan, Si Yu; Lu, Yunpeng; Bai, Linyi; Li, Menghuan; Li, Peizhou; Zhang, Quan; Selvan, Subramanian Tamil; Zhao, Yanli

    2014-01-01

    A “turn-on” thiol-responsive fluorescence probe was synthesized and integrated into polymeric nanoparticles for sensing intracellular thiols. There is a photo-induced electron transfer process in the off state of the probe, and this process is terminated upon the reaction with thiol compounds. Configuration interaction singles (CIS) calculation was performed to confirm the mechanism of this process. A series of sensing studies were carried out, showing that the probe-integrated nanoparticles were highly selective towards biological thiol compounds over non-thiolated amino acids. Kinetic studies were also performed to investigate the relative reaction rate between the probe and the thiolated amino acids. Subsequently, the Gibbs free energy of the reactions was explored by means of the electrochemical method. Finally, the detection system was employed for sensing intracellular thiols in cancer cells, and the sensing selectivity could be further enhanced with the use of a cancer cell-targeting ligand in the nanoparticles. This development paves a path for the sensing and detection of biological thiols, serving as a potential diagnostic tool in the future. PMID:25394758

  9. Galectin-3 is a marker of favorable prognosis and a biologically relevant molecule in neuroblastic tumors

    PubMed Central

    Veschi, V; Petroni, M; Bartolazzi, A; Altavista, P; Dominici, C; Capalbo, C; Boldrini, R; Castellano, A; McDowell, H P; Pizer, B; Frati, L; Screpanti, I; Gulino, A; Giannini, G

    2014-01-01

    Childhood neuroblastic tumors are characterized by heterogeneous clinical courses, ranging from benign ganglioneuroma (GN) to highly lethal neuroblastoma (NB). Although a refined prognostic evaluation and risk stratification of each tumor patient is becoming increasingly essential to personalize treatment options, currently only few biomolecular markers (essentially MYCN amplification, chromosome 11q status and DNA ploidy) are validated for this purpose in neuroblastic tumors. Here we report that Galectin-3 (Gal-3), a β-galactoside-binding lectin involved in multiple biological functions that has already acquired diagnostic relevance in specific clinical settings, is variably expressed in most differentiated and less aggressive neuroblastic tumors, such as GN and ganglioneuroblastoma, as well as in a subset of NB cases. Gal-3 expression is associated with the INPC histopathological categorization (P<0.001) and Shimada favorable phenotype (P=0.001), but not with other prognostically relevant features. Importantly, Gal-3 expression was associated with a better 5-year overall survival (P=0.003), and with improved cumulative survival in patient subsets at worse prognosis, such as older age at diagnosis, advanced stages or NB histopathological classification. In vitro, Gal-3 expression and nuclear accumulation accompanied retinoic acid-induced cell differentiation in NB cell lines. Forced Gal-3 overexpression increased phenotypic differentiation and substrate adherence, while inhibiting proliferation. Altogether, these findings suggest that Gal-3 is a biologically relevant player for neuroblastic tumors, whose determination by conventional immunohistochemistry might be used for outcome assessment and patient's risk stratification in the clinical setting. PMID:24603328

  10. Small Molecule Inhibitors of Staphylococcus aureus RnpA Alter Cellular mRNA Turnover, Exhibit Antimicrobial Activity, and Attenuate Pathogenesis

    PubMed Central

    Olson, Patrick D.; Kuechenmeister, Lisa J.; Anderson, Kelsi L.; Daily, Sonja; Beenken, Karen E.; Roux, Christelle M.; Reniere, Michelle L.; Lewis, Tami L.; Weiss, William J.; Pulse, Mark; Nguyen, Phung; Simecka, Jerry W.; Morrison, John M.; Sayood, Khalid; Asojo, Oluwatoyin A.; Smeltzer, Mark S.; Skaar, Eric P.; Dunman, Paul M.

    2011-01-01

    Methicillin-resistant Staphylococcus aureus is estimated to cause more U.S. deaths annually than HIV/AIDS. The emergence of hypervirulent and multidrug-resistant strains has further amplified public health concern and accentuated the need for new classes of antibiotics. RNA degradation is a required cellular process that could be exploited for novel antimicrobial drug development. However, such discovery efforts have been hindered because components of the Gram-positive RNA turnover machinery are incompletely defined. In the current study we found that the essential S. aureus protein, RnpA, catalyzes rRNA and mRNA digestion in vitro. Exploiting this activity, high through-put and secondary screening assays identified a small molecule inhibitor of RnpA-mediated in vitro RNA degradation. This agent was shown to limit cellular mRNA degradation and exhibited antimicrobial activity against predominant methicillin-resistant S. aureus (MRSA) lineages circulating throughout the U.S., vancomycin intermediate susceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) and other Gram-positive bacterial pathogens with high RnpA amino acid conservation. We also found that this RnpA-inhibitor ameliorates disease in a systemic mouse infection model and has antimicrobial activity against biofilm-associated S. aureus. Taken together, these findings indicate that RnpA, either alone, as a component of the RNase P holoenzyme, and/or as a member of a more elaborate complex, may play a role in S. aureus RNA degradation and provide proof of principle for RNA catabolism-based antimicrobial therapy. PMID:21347352

  11. Double-stranded RNA in the biological control of grain aphid (Sitobion avenae F.).

    PubMed

    Wang, Dahai; Liu, Qi; Li, Xia; Sun, Yongwei; Wang, Hui; Xia, Lanqin

    2015-03-01

    Grain aphid (Sitobion avenae F.) is the most dominant and destructive pest of wheat, which causes significant yield loss of cereal plants each year by inflicting damage both through the direct effects of feeding and by vectoring debilitating plant viruses. In this study, we performed de novo transcriptome sequencing of grain aphid via Roche 454 GS-FLX pyrosequencing. A total of 1,106,696 reads were obtained and assembled into 32,277 unigenes, of which 25,389, 21,635, and 16,211 unigenes matched the Nt, Nr, and Swiss-Prot databases, respectively. Functional annotation of these unigenes revealed not only the presence of genes that encode the key components of RNAi machinery such as Dicer and Argonaute but also the genes encoding the TAR RNA binding protein (TRBP) and the SID-1 protein, which function in assisting the RNA-induced silencing complex (RISC) formation in microRNA (miRNA) pathway and mediating a systemic RNA interference (RNAi) effect though a cellular uptake mechanism. Furthermore, among a set of 66 unigenes selected for a double-stranded RNA (dsRNA) artificial diet assay, four novel effective RNAi targets, which led to high mortality of aphids due to the down-regulation of the expression of the respective target gene, were identified. Moreover, the expansion of systemic RNAi effect in grain aphid was observed by adding the fluorescently labeled dsRNA in an artificial diet assay. PMID:25467938

  12. Single-Molecule Localization Microscopy allows for the analysis of cancer metastasis-specific miRNA distribution on the nanoscale.

    PubMed

    Oleksiuk, Olga; Abba, Mohammed; Tezcan, Kerem Can; Schaufler, Wladimir; Bestvater, Felix; Patil, Nitin; Birk, Udo; Hafner, Mathias; Altevogt, Peter; Cremer, Christoph; Allgayer, Heike

    2015-12-29

    We describe a novel approach for the detection of small non-coding RNAs in single cells by Single-Molecule Localization Microscopy (SMLM). We used a modified SMLM-setup and applied this instrument in a first proof-of-principle concept to human cancer cell lines. Our method is able to visualize single microRNA (miR)-molecules in fixed cells with a localization accuracy of 10-15 nm, and is able to quantify and analyse clustering and localization in particular subcellular sites, including exosomes. We compared the metastasis-site derived (SW620) and primary site derived (SW480) human colorectal cancer (CRC) cell lines, and (as a proof of principle) evaluated the metastasis relevant miR-31 as a first example. We observed that the subcellular distribution of miR-31 molecules in both cell lines was very heterogeneous with the largest subpopulation of optically acquired weakly metastatic cells characterized by a low number of miR-31 molecules, as opposed to a significantly higher number in the majority of the highly metastatic cells. Furthermore, the highly metastatic cells had significantly more miR-31-molecules in the extracellular space, which were visualized to co-localize with exosomes in significantly higher numbers. From this study, we conclude that miRs are not only aberrantly expressed and regulated, but also differentially compartmentalized in cells with different metastatic potential. Taken together, this novel approach, by providing single molecule images of miRNAs in cellulo can be used as a powerful supplementary tool in the analysis of miRNA function and behaviour and has far reaching potential in defining metastasis-critical subpopulations within a given heterogeneous cancer cell population. PMID:26561203

  13. Single-Molecule Localization Microscopy allows for the analysis of cancer metastasis-specific miRNA distribution on the nanoscale

    PubMed Central

    Tezcan, Kerem Can; Schaufler, Wladimir; Bestvater, Felix; Patil, Nitin; Birk, Udo; Hafner, Mathias; Altevogt, Peter; Cremer, Christoph; Allgayer, Heike

    2015-01-01

    We describe a novel approach for the detection of small non-coding RNAs in single cells by Single-Molecule Localization Microscopy (SMLM). We used a modified SMLM–setup and applied this instrument in a first proof-of-principle concept to human cancer cell lines. Our method is able to visualize single microRNA (miR)-molecules in fixed cells with a localization accuracy of 10–15 nm, and is able to quantify and analyse clustering and localization in particular subcellular sites, including exosomes. We compared the metastasis-site derived (SW620) and primary site derived (SW480) human colorectal cancer (CRC) cell lines, and (as a proof of principle) evaluated the metastasis relevant miR-31 as a first example. We observed that the subcellular distribution of miR-31 molecules in both cell lines was very heterogeneous with the largest subpopulation of optically acquired weakly metastatic cells characterized by a low number of miR-31 molecules, as opposed to a significantly higher number in the majority of the highly metastatic cells. Furthermore, the highly metastatic cells had significantly more miR-31-molecules in the extracellular space, which were visualized to co-localize with exosomes in significantly higher numbers. From this study, we conclude that miRs are not only aberrantly expressed and regulated, but also differentially compartmentalized in cells with different metastatic potential. Taken together, this novel approach, by providing single molecule images of miRNAs in cellulo can be used as a powerful supplementary tool in the analysis of miRNA function and behaviour and has far reaching potential in defining metastasis-critical subpopulations within a given heterogeneous cancer cell population. PMID:26561203

  14. Fluid Flows and Their Role in the Regulation of Biological Molecules in the Bloodstream

    NASA Astrophysics Data System (ADS)

    Sing, Charles; Alexander-Katz, Alfredo

    2010-03-01

    We use computer simulations to elucidate the physics underlying blood clotting mechanisms. A straightforward and general model for the behavior of proteins such as von Willebrand Factor (vWF) under various flow conditions has been developed. The particular case of vWF is considered in depth, since it demonstrates the counter-intuitive behavior of adsorbing to a surface at higher flow rates. We use the globule-stretch transition of a collapsed polymer to explain this phenomenon, and have identified the conditions necessary to induce this transition. We have also developed a theory to explain the mechanism of this transition, which is based on the nucleation and growth of large thermal protrusions. Upon the consideration of the specific length and time scales present under biological conditions, it is apparent that vWF is strongly regulated by elongational flows. We can show how phenomena from the molecular to physiological levels are supplemented by this understanding of vWF function.

  15. Small cantilevers for atomic force microscopy and force spectroscopy of biological molecules

    NASA Astrophysics Data System (ADS)

    Viani, M. B.; Schaffer, T. E.; Chand, A.; Smith, B. L.; Hansma, P. K.; Wendman, M.

    1998-03-01

    Small cantilevers offer new possibilities for high speed/low noise atomic force microscopy of soft, biological samples. We have used a novel process to fabricate metallic cantilevers that should maximize reflectivity and minimize thermal bending. We have fabricated and measured the properties of aluminum, nickel, silver, and 14-karat gold cantilevers that are 3-12 um long, 1-4 um wide, and 60-300 nm thick and have resonant frequencies of 0.5-2 MHz and spring constants of 0.1-3 N/m. We also have fabricated small cantilevers with ultra-low spring constants (1-10 mN/m) out of silicon nitride and used them for force spectroscopy of DNA. This work was supported by grant numbers NSF-DMR9622169 and NSF-DMR9632716 from the Materials Research Division of the National Science Foundation and by grant number DAAH04-96-1-004 from the Army Research Office.

  16. Diverse Biological Activities of the Vascular Non-Inflammatory Molecules - The Vanin Pantetheinases.

    PubMed Central

    Kaskow, Belinda J; Proffit, J Michael; Blangero, John; Moses, Eric K; Abraham, Lawrence J

    2011-01-01

    The Vanin genes are a family that encode pantetheinases involved in recycling Coenzyme A, caytalysing the breakdown of intermediate panetheine to vitamin B5 for reuse in CoA biosynthesis. The role of pantetheinase in this most fundamental of cellular processes, was substantially characterised by the 1970s. The next 20 years saw little further interest in pantetheinase until various genetic studies implicated the vanin locus in a range of normal and disease phenotypes, and a consequent interest in the other product of pantetheinase activity, cysteamine. This report seeks to bring together the early biochemical studies with recent biological data implicating cysteamine as a regulator of the oxidative state of a cell. Numerous studies now report a role for Vanin in inflammation, oxidative stress, cell migration and numerous diseases including cardiovascular disease. PMID:22155241

  17. The Origin of Universal Scaling Laws in Biology from Molecules and Cells to Whales

    NASA Astrophysics Data System (ADS)

    West, Geoffrey B.

    1999-10-01

    Even though biological systems are the most complex physical systems known, they satisfy remarkably simple scaling laws. For example, metabolic rate (the power needed to sustain life) scales like the -power of mass over 26 orders of magnitude ranging from the molecular respiratory complex within mitochondria up through the smallest unicellular organism (mycoplasma) to the largest animals (whales) and plants (giant sequoia). Other scaling laws relate how organismal features change with size over many orders of magnitude; these include time-scales (such as lifespan and heart-rate) and sizes (such as the radius of a tree trunk or the aorta). All of these can be expressed as power laws with exponents which are typically simple multiples of . Their phenomenology will be discussed and a quantitative, unified model presented that can explain their origin, including that of the universal -power.

  18. Rotational dynamics of water molecules near biological surfaces with implications for nuclear quadrupole relaxation.

    PubMed

    Braun, Daniel; Schmollngruber, Michael; Steinhauser, Othmar

    2016-09-21

    Based on Molecular Dynamics simulations of two different systems, the protein ubiquitin dissolved in water and an AOT reverse micelle, we present a broad analysis of the single particle rotational dynamics of water. A comprehensive connection to NQR, which is a prominent experimental method in this field, is developed, based on a reformulation of its theoretical framework. Interpretation of experimental NQR results requires a model which usually assumes that the NQR experiences retardation only in the first hydration shell. Indeed, the present study shows that this first-shell model is correct. Moreover, previous experimental retardation factors are quantitatively reproduced. All of this is seemingly contradicted by results of other methods, e.g., dielectric spectroscopy, responsible for a long-standing debate in this field. Our detailed analysis shows that NQR omits important information contained in overall water dynamics, most notably, the retardation of the water dipole axis in the electric field exerted by a biological surface. PMID:27546227

  19. Directed Evolution of RebH for Site Selective Halogenation of Large, Biologically Active Molecules**

    PubMed Central

    Payne, James T.; Poor, Catherine B.

    2015-01-01

    We recently characterized the substrate scope of wild-type RebH and evolved variants of this enzyme with improved stability for biocatalysis. The substrate scopes of both RebH and the stabilized variants, however, are limited primarily to compounds similar in size to tryptophan. We have now used a substrate walking approach to further evolve RebH variants with expanded substrate scope. Two particularly notable variants were identified: 3-SS, which provides high conversion of tricyclic tryptoline derivatives; and 4-V, which accepts a broad range of large indoles and carbazoles. This constitutes the first reported use of directed evolution to enable functionalization of substrates not accepted by wild-type RebH and demonstrates the utility of RebH variants for site-selective halogenation of biologically active compounds. PMID:25678465

  20. Electron flow through biological molecules: Does hole hopping protect proteins from oxidative damage?

    PubMed Central

    Winkler, Jay R.; Gray, Harry B.

    2016-01-01

    Biological electron transfers often occur between metal-containing cofactors that are separated by very large molecular distances. Employing photosensitizer-modified iron and copper proteins, we have shown that single-step electron tunneling can occur on nanosecond to microsecond timescales at distances between 15 and 20 angstroms. We also have shown that charge transport can occur over even longer distances by hole hopping (multistep tunneling) through intervening tyrosines and tryptophans. In this Perspective, we advance the hypothesis that such hole hopping through Tyr/Trp chains could protect oxygenase, dioxygenase, and peroxidase enzymes from oxidative damage. In support of this view, by examining the structures of P450 (CYP102A) and 2OG-Fe (TauD) enzymes, we have identified candidate Tyr/Trp chains that could transfer holes from uncoupled high-potential intermediates to reductants in contact with protein surface sites. PMID:26537399

  1. Detection of nerve agents and biological molecules using embedded piezoresistive microcantilever sensors.

    NASA Astrophysics Data System (ADS)

    Porter, Timothy; Vail, Tim; Wooley, Amanda

    2008-03-01

    Embedded piezoresistive microcantilever (EPM) sensors have been used in the detection of a variety of analyte species. EPM sensors utilize a tiny piezoresistive microcantilever partially embedded into a sensing material to produce a sensing element that is compact, simple, resistant to movement and shock, and suitable for remote sensing applications. In the current project, we have used sensing materials comprised of an immobilizing polymer functionalized with either target enzymes or antibodies to detect two biological agents, bacillus globigi (BG) and Diisopropyl fluorophosphate (DFP). DFP is an organophosphate used as a simulant for organophosphate nerve agents, while BG is a large bacterial spore used as a simulant for other bacterial spores such as bacillus anthracis. Sensing results are presented for both types of EPM sensors.

  2. Screening and characterization of molecules that modulate the biological activity of IFNs-I.

    PubMed

    Bürgi, Milagros; Zapol'skii, Viktor A; Hinkelmann, Bettina; Köster, Mario; Kaufmann, Dieter E; Sasse, Florenz; Hauser, Hansjörg; Etcheverrigaray, Marina; Kratje, Ricardo; Bollati-Fogolín, Mariela; Oggero, Marcos

    2016-09-10

    Type I Interferons (IFNs-I) are species-specific glycoproteins which play an important role as primary defence against viral infections and that can also modulate the adaptive immune system. In some autoimmune diseases, interferons (IFNs) are over-produced. IFNs are widely used as biopharmaceuticals for a variety of cancer indications, chronic viral diseases, and for their immuno-modulatory action in patients with multiple sclerosis; therefore, increasing their therapeutic efficiency and decreasing their side effects is of high clinical value. In this sense, it is interesting to find molecules that can modulate the activity of IFNs. In order to achieve that, it was necessary to establish a simple, fast and robust assay to analyze numerous compounds simultaneously. We developed four reporter gene assays (RGAs) to identify IFN activity modulator compounds by using WISH-Mx2/EGFP, HeLa-Mx2/EGFP, A549-Mx2/EGFP, and HEp2-Mx2/EGFP reporter cell lines (RCLs). All of them present a Z' factor higher than 0.7. By using these RGAs, natural and synthetic compounds were analyzed simultaneously. A total of 442 compounds were studied by the Low Throughput Screening (LTS) assay using the four RCLs to discriminate between their inhibitory or enhancing effects on IFN activity. Some of them were characterized and 15 leads were identified. Finally, one promising candidate with enhancing effect on IFN-α/-β activity and five compounds with inhibitory effect were described. PMID:27346232

  3. Design, Synthesis, and Biological Evaluation of Novel Selenium (Se-NSAID) Molecules as Anticancer Agents.

    PubMed

    Plano, Daniel; Karelia, Deepkamal N; Pandey, Manoj K; Spallholz, Julian E; Amin, Shantu; Sharma, Arun K

    2016-03-10

    The synthesis and anticancer evaluation of novel selenium-nonsteroidal anti-inflammatory drug (Se-NSAID) hybrid molecules are reported. The Se-aspirin analogue 8 was identified as the most effective agent in reducing the viability of different cancer cell lines, particularly colorectal cancer (CRC) cells, was more selective toward cancer cells than normal cells, and was >10 times more potent than 5-FU, the current therapy for CRC. Compound 8 inhibits CRC growth via the inhibition of the cell cycle in G1 and G2/M phases and reduces the cell cycle markers like cyclin E1 and B1 in a dose dependent manner; the inhibition of the cell cycle may be dependent on the ability of 8 to induce p21 expression. Furthermore, 8 induces apoptosis by activating caspase 3/7 and PARP cleavage, and its longer exposure causes increase in intracellular ROS levels in CRC cells. Taken together, 8 has the potential to be developed further as a chemotherapeutic agent for CRC. PMID:26750401

  4. Detecting and Identifying Organic Molecules in Space - The AstroBiology Explorer (ABE) MIDEX Mission Concept

    NASA Technical Reports Server (NTRS)

    Sandford, Scott A.

    2001-01-01

    Infrared spectroscopy in the 2.5-16 micron (4000-625/cm) range is a principle means by which organic compounds are detected and identified in space. Ground-based, airborne, and spaceborne IR spectral studies have already demonstrated that a significant fraction of the carbon in the interstellar medium (ISM) resides in the form of complex organic molecular species. Unfortunately, neither the distribution of these materials nor their genetic and evolutionary relationships with each other or their environments are well understood. The Astrobiology Explorer (ABE) is a MIDEX (Medium-class Explorer) mission concept currently under study at NASA's Ames Research Center in collaboration with Ball Aerospace and Technologies Corporation. ABE will conduct IR spectroscopic observations to address outstanding important problems in astrobiology, astrochemistry, and astrophysics. The core observational program would make fundamental scientific progress in understanding (1) the evolution of ices and organic matter in dense molecular clouds and young forming stellar systems, (2) the chemical evolution of organic molecules in the ISM as they transition from AGB outflows to planetary nebulae to the general diffuse ISM to H II regions and dense clouds, (3) the distribution of organics in the diffuse ISM, (4) the nature of organics in the Solar System (in comets, asteroids, satellites), and (5) the nature and distribution of organics in local galaxies. Both the scientific goals of the mission and how they would be achieved will be discussed.

  5. Identifying Organic Molecules in Space: The AstroBiology Explorer (ABE) MIDEX Mission Concept

    NASA Technical Reports Server (NTRS)

    Sandford, Scott A.; Allamandola, Louis; Bregman, Jesse; Ennico, Kimberly; Greene, Thomas; Hudgins, Douglas; Strecker, Donald; DeVincenzi, Donald (Technical Monitor)

    2001-01-01

    Infrared spectroscopy in the 2.5-16 micron range is a principle means by which organic compounds are detected and identified in space. Ground-based, airborne, and spaceborne IR spectral studies have already demonstrated that a significant fraction of the carbon in the interstellar medium (ISM) resides in the form of complex organic molecular species. Unfortunately, neither the distribution of these materials nor their genetic and evolutionary relationships with each other or their environments are well understood. The Astrobiology Explorer (ABE) is a MIDEX mission concept currently under study at NASA's Ames Research Center in collaboration with Ball Aerospace and Technologies Corporation. ABE will conduct IR spectroscopic observations to address outstanding important problems in astrobiology, astrochemistry, and astrophysics. The core observational program would make fundamental scientific progress in understanding (1) the evolution of ices and organic matter in dense molecular clouds and young forming stellar systems, (2) the chemical evolution of organic molecules in the ISM as they transition from AGB outflows to planetary nebulae to the general diffuse ISM to H II regions and dense clouds, (3) the distribution of organics in the diffuse ISM, (4) the nature of organics in the Solar System (in comets, asteroids, satellites), and (5) the nature and distribution of organics in local galaxies. The technical considerations of achieving these science objectives in a MIDEX-sized mission will be described.

  6. Detecting and Identifying Organic Molecules in Space: The AstroBiology Explorer (ABE) MIDEX Mission Concept

    NASA Technical Reports Server (NTRS)

    Sandford, Scott A.; DeVincenzi, Donald (Technical Monitor)

    2001-01-01

    Infrared spectroscopy in the 2.5-16 microns (4000-625/cm) range is a principle means by which organic compounds are detected and identified in space. Ground-based, airborne, and spaceborne IR spectral studies have already demonstrated that a significant fraction of the carbon in the interstellar medium (ISM) resides in the form of complex organic molecular species. Unfortunately, neither the distribution of these materials nor their genetic and evolutionary relationships with each other or their environments are well understood. The Astrobiology Explorer (ABE) is a MIDEX (Medium-class Explorer) mission concept currently under study at NASA's Ames Research Center in collaboration with Ball Aerospace and Technologies Corporation. ABE will conduct IR spectroscopic observations to address outstanding important problems in astrobiology, astrochemistry, and astrophysics. The core observational program would make fundamental scientific progress in understanding (1) the evolution of ices and organic matter in dense molecular clouds and young forming stellar systems, (2) the chemical evolution of organic molecules in the ISM as they transition from AGB outflows to planetary nebulae to the general diffuse ISM to H II regions and dense clouds, (3) the distribution of organics in the diffuse ISM, (4) the nature of organics in the Solar System (in comets, asteroids, satellites), and (5) the nature and distribution of organics in local galaxies. Both the scientific goals of the mission and how they would be achieved will be discussed.

  7. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  8. Evolution of conformational changes in the dynamics of small biological molecules: a hybrid MD/RRK approach.

    PubMed

    Segev, Elad; Grumbach, Mikael; Gerber, Robert Benny

    2006-11-14

    The dynamics of long timescale evolution of conformational changes in small biological molecules is described by a hybrid molecular dynamics/RRK algorithm. The approach employs classical trajectories for transitions between adjacent structures separated by a low barrier, and the classical statistical RRK approximation when the barrier involved is high. In determining the long-time dynamics from an initial structure to a final structure of interest, an algorithm is introduced for determining the most efficient pathways (sequence of the intermediate conformers). This method uses the Dijkstra algorithm for finding optimal paths on networks. Three applications of the method using an AMBER force field are presented: a detailed study of conformational transitions in a blocked valine dipeptide; a multiple reaction path study of the blocked valine tripeptide; and the evolution in time from the beta hairpin to alpha helix structure of a blocked alanine hexapeptide. Advantages and limitations of the method are discussed in light of the results. PMID:17066182

  9. Biological functions of hCG and hCG-related molecules

    PubMed Central

    2010-01-01

    Background hCG is a term referring to 4 independent molecules, each produced by separate cells and each having completely separate functions. These are hCG produced by villous syncytiotrophoblast cells, hyperglycosylated hCG produced by cytotrophoblast cells, free beta-subunit made by multiple primary non-trophoblastic malignancies, and pituitary hCG made by the gonadotrope cells of the anterior pituitary. Results and discussion hCG has numerous functions. hCG promotes progesterone production by corpus luteal cells; promotes angiogenesis in uterine vasculature; promoted the fusion of cytotrophoblast cell and differentiation to make syncytiotrophoblast cells; causes the blockage of any immune or macrophage action by mother on foreign invading placental cells; causes uterine growth parallel to fetal growth; suppresses any myometrial contractions during the course of pregnancy; causes growth and differentiation of the umbilical cord; signals the endometrium about forthcoming implantation; acts on receptor in mother's brain causing hyperemesis gravidarum, and seemingly promotes growth of fetal organs during pregnancy. Hyperglycosylated hCG functions to promote growth of cytotrophoblast cells and invasion by these cells, as occurs in implantation of pregnancy, and growth and invasion by choriocarcinoma cells. hCG free beta-subunit is produced by numerous non-trophoblastic malignancies of different primaries. The detection of free beta-subunit in these malignancies is generally considered a sign of poor prognosis. The free beta-subunit blocks apoptosis in cancer cells and promotes the growth and malignancy of the cancer. Pituitary hCG is a sulfated variant of hCG produced at low levels during the menstrual cycle. Pituitary hCG seems to mimic luteinizing hormone actions during the menstrual cycle. PMID:20735820

  10. In vivo biochemistry: Applications for small molecule biosensors in plant biology

    PubMed Central

    Jones, Alexander; Grossmann, Guido; Danielson, Jonas Å.H.; Sosso, Davide; Chen, Li-Qing; Ho, Cheng Hsun; Frommer, Wolf B.

    2013-01-01

    Summary Revolutionary new technologies, namely in the areas of DNA sequencing and molecular imaging, continue to impact new discoveries in plant science and beyond. For decades we have been able to determine properties of enzymes, receptors and transporters in vitro or in heterologous systems, and more recently been able to analyze their regulation at the transcriptional level, use GFP reporters to obtain insights into cellular and subcellular localization, and measure ion and metabolite levels with unprecedented precision using mass spectrometry. However, we lack key information on location and dynamics of the substrates of enzymes, receptors and transporters, and on the regulation of these proteins in their cellular environment. Such information can now be obtained by transitioning from in vitro to in vivo biochemistry using biosensors. Genetically encoded fluorescent protein-based sensors for ion and metabolite dynamics provide highly resolved spatial and temporal information, and are complemented by sensors for pH, redox, voltage, and tension. They serve as powerful tools for identifying missing processes (e.g. glucose transport across ER membranes), components (e.g. SWEET sugar transporters for cellular sugar efflux), and signaling networks (e.g. from systematic screening of mutants that affect sugar transport or cytosolic and vacuolar pH). Combined with the knowledge of properties of enzymes and transporters and their interactions with the regulatory machinery, biosensors promise to be key diagnostic tools for systems and synthetic biology. PMID:23587939

  11. Identifying Organic Molecules in Space: The AstroBiology Explorer (ABE) Mission Concept

    NASA Technical Reports Server (NTRS)

    Ennico, K. A.; Sandford, S. A.; Allamandola, L.; Bregman, J.; Cohen, M.; Cruikshank, D.; Dumas, C.; Greene, T.; Hudgins, D.; Kwok, S.

    2004-01-01

    The AstroBiology Explorer (ABE) mission concept consists of a dedicated space observatory having a 60 cm class primary mirror cooled to T < 50 K equipped with medium resolution cross-dispersed spectrometers having cooled large format near- and mid-infrared detector arrays. Such a system would be capable of addressing outstanding problems in Astrochemistry and Astrophysics that are particularly relevant to Astrobiology and addressable via astronomical observation. The mission s observational program would make fundamental scientific progress in establishing the nature, distribution, formation and evolution of organic and other molecular materials in the following extra-terrestrial environments: 1) The Outflow of Dying Stars, 2) The Diffuse Interstellar Medium, 3) Dense Molecular Clouds, Star Formation Regions, and Young StellarPlanetary Systems, 4) Planets, Satellites, and Small Bodies within the Solar System, and 5 ) The Interstellar Media of Other Galaxies. ABE could make fundamental progress in all of these areas by conducting a 1 to 2 year mission to obtain a coordinated set of infrared spectroscopic observations over the 2.5-20 micron spectral range at a spectral resolution of R > 2000 of about 1500 objects including galaxies, stars, planetary nebulae, young stellar objects, and solar system objects. Keywords: Astrobiology, infrared, Explorers, interstellar organics, telescope, spectrometer, space, infrared detectors

  12. From molecule to man: integrating molecular biology with whole organ physiology in studying respiratory disease.

    PubMed

    Königshoff, Melanie; Uhl, Franziska; Gosens, Reinoud

    2011-10-01

    Chronic lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) are all characterized by structural changes of the airways and/or lungs that limit airflow and/or gas exchange. Currently, there is no therapy available that adequately targets the structural remodeling of the airways and lungs in these diseases. This underscores the great need for insight into the mechanisms that underpin the development of airway remodeling, fibrosis and emphysema in these diseases, in order to identify suitable drug targets. It is increasingly evident that structural cell-cell communication within the lung is central to the development of remodeling, indicating that a more integrative approach should be considered when studying molecular and cellular mechanisms of remodeling. Therefore, there is a great need to study molecular and cellular physiological and pathophysiological mechanisms in as much detail as possible, but with as little as possible loss of the physiological context. Here, we will review the use of models such as cellular co-culture, tissue culture, and lung slice culture, in which cell-cell communication and tissue architecture are better preserved or mimicked than in cell culture, and zoom in on the usefulness of molecular and cellular biological tools in these complex model systems to read out or control signaling and gene/protein regulation. PMID:21356323

  13. Identifying Organic Molecules in Space: The AstroBiology Explorer (ABE) Mission Concept

    NASA Technical Reports Server (NTRS)

    Ennico, Kimberly; Sandford, S.; Allamandola, L.; Bregman, J.; Cohen, M.; Cruikshank, D.; Dumas, C.; Greene, T.; Hudgins, D.; Kwok, S.

    2004-01-01

    The AstroBiology Explorer (ABE) mission concept consists of a modest dedicated space observatory having a 60 cm class primary mirror cooled to T less than 50 K equipped with medium resolution cross-dispersed spectrometers having cooled large format near- and mid-infrared detector arrays. Such a system would be capable of addressing outstanding problems in Astrochemistry and Astrophysics that are particularly relevant to Astrobiology and addressable via astronomical observation. The mission's observaticxiai program woiild make fundamental scieztific: prngress in establishing the nature, distribution, formation and evolution of organic and other molecular materials in the following extra-terrestrial environments: 1) The Outflow of Dying Stars; 2) The Diffuse Interstellar Medium (DISM); 3) Dense Molecular Clouds, Star Formation Regions, and Young Stellar/Planetary Systems; 4) Planets, Satellites, and Small Bodies within the Solar System; and 5) The Interstellar Media of Other Galaxies ABE could make fundamental progress in all of these area by conducting a 1 to 2 year mission to obtain a coordinated set of infrared spectroscopic observations over the 2.5 - 20 micron spectral range at a spectral resolution of R greater than 2500 of about 1500 galaxies, stars, planetary nebulae, young stellar objects, and solar system objects.

  14. Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

    SciTech Connect

    Taylor, R.C.

    1991-11-01

    This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese's group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a group of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.

  15. Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

    SciTech Connect

    Taylor, R.C.

    1991-11-01

    This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese`s group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a group of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.

  16. Short RNA Molecules with High Binding Affinity to the KH Motif of A-Kinase Anchoring Protein 1 (AKAP1): Implications for the Regulation of Steroidogenesis

    PubMed Central

    Grozdanov, Petar N.

    2012-01-01

    One of the key regulators of acute steroid hormone biosynthesis in steroidogenic tissues is the steroidogenic acute regulatory (STAR) protein. Acute regulation of STAR production on the transcriptional level is mainly achieved through a cAMP-dependent mechanism, which is well understood. However, less is known about the posttranscriptional regulation of STAR synthesis, specifically the factors influencing the destiny of the Star mRNA after it leaves the nucleus. Here, we show that the 3′-untranslated region of Star mRNA interacts with the heterogeneous nuclear ribonucleoprotein K-homology (KH) motif of the mitochondrial scaffold A-kinase anchoring protein 1 (AKAP1) in vitro with a moderate affinity as measured by EMSAs. A mutation that mimics the phosphorylation state of the KH motif at a specific serine either did not alter, or had a negative impact on, protein-RNA binding under these conditions. The KH motif of AKAP1 binds short pyrimidine-rich RNA molecules with a stable hairpin structure as demonstrated by in vitro selection. AKAP1 also interacts with STAR mRNA in a dibutyryl-cAMP-stimulated human steroidogenic adrenocortical carcinoma cell line in vivo. Therefore, we propose a model in which AKAP1 anchors Star mRNA at the mitochondria, thus stabilizing the translational complex at this organelle, a situation that might affect STAR production and steroidogenesis. In addition, we suggest that the last 216 amino acid residues of AKAP1 might participate in the degradation of STAR and other nuclear-encoded mitochondrial mRNAs through interaction with a RNA-induced silencing complex, specifically with the argonaute 2 protein. PMID:23077346

  17. The Diversity of Ribonuclease P: Protein and RNA Catalysts with Analogous Biological Functions.

    PubMed

    Klemm, Bradley P; Wu, Nancy; Chen, Yu; Liu, Xin; Kaitany, Kipchumba J; Howard, Michael J; Fierke, Carol A

    2016-01-01

    Ribonuclease P (RNase P) is an essential endonuclease responsible for catalyzing 5' end maturation in precursor transfer RNAs. Since its discovery in the 1970s, RNase P enzymes have been identified and studied throughout the three domains of life. Interestingly, RNase P is either RNA-based, with a catalytic RNA subunit, or a protein-only (PRORP) enzyme with differential evolutionary distribution. The available structural data, including the active site data, provides insight into catalysis and substrate recognition. The hydrolytic and kinetic mechanisms of the two forms of RNase P enzymes are similar, yet features unique to the RNA-based and PRORP enzymes are consistent with different evolutionary origins. The various RNase P enzymes, in addition to their primary role in tRNA 5' maturation, catalyze cleavage of a variety of alternative substrates, indicating a diversification of RNase P function in vivo. The review concludes with a discussion of recent advances and interesting research directions in the field. PMID:27187488

  18. Co-expression of RNA–protein complexes in Escherichia coli and applications to RNA biology

    PubMed Central

    Ponchon, Luc; Catala, Marjorie; Seijo, Bili; El Khouri, Marguerite; Dardel, Frédéric; Nonin-Lecomte, Sylvie; Tisné, Carine

    2013-01-01

    RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA–protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA–His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins. PMID:23804766

  19. Pegylation of biological molecules and potential benefits: pharmacological properties of certolizumab pegol.

    PubMed

    Pasut, Gianfranco

    2014-04-01

    PEGylation of biological proteins, defined as the covalent conjugation of proteins with polyethylene glycol (PEG), leads to a number of biopharmaceutical improvements, including increased half-life, increased solubility and reduced aggregation, and reduced immunogenicity. Since their introduction in 1990, PEGylated proteins have significantly improved the management of various chronic diseases, including rheumatoid arthritis (RA) and Crohn's disease. Certolizumab pegol is the only PEGylated anti-tumour necrosis factor (TNF)-α agent. It is a PEGylated, humanised, antigen-binding fragment of an anti-TNF monoclonal antibody. Unlike other anti-TNF agents, it has no crystallisable fragment (Fc) domain. Because of its novel structure, certolizumab pegol may have a different mechanism of action to the other anti-TNF agents, and also has different pharmacodynamic properties, which could possibly translate to a different safety profile. Pharmacodynamic studies have shown that certolizumab pegol binds to TNF with a higher affinity than adalimumab and infliximab. Certolizumab pegol is also more potent at neutralising soluble TNF-mediated signalling than adalimumab and infliximab, and has similar or lesser potency to etanercept. Certolizumab pegol does not cause detrimental in vitro effects such as degranulation, loss of cell integrity, apoptosis, complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. Certolizumab pegol may also penetrate more effectively into inflamed arthritic tissue than other anti-TNF agents, and is not actively transported across the placenta during pregnancy. Pharmacokinetic studies in healthy volunteers demonstrated that single intravenous and subcutaneous doses of certolizumab pegol had predictable pharmacokinetics. The pharmacokinetics of certolizumab pegol in patients with RA and Crohn's disease were consistent with pharmacokinetics in healthy volunteers. PMID:24687235

  20. Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA

    PubMed Central

    Dueck, Kevin J.; Hu, YuanShen (Sandy); Chen, Peter; Deschambault, Yvon; Lee, Jocelyn; Varga, Jessie

    2015-01-01

    ABSTRACT Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity. IMPORTANCE dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they

  1. Effects of siRNA-Mediated Knockdown of HDAC1 on the Biological Behavior of Esophageal Carcinoma Cell Lines

    PubMed Central

    Wang, Xing; Guo, Haisheng; Liu, Weixin; Yang, Chunmei; Yang, Lei; Wang, Dongguan; Wang, Xunguo

    2016-01-01

    Background HDAC1 has been shown to be closely associated with the occurrence of tumors. We aimed to investigate the effects of siRNA-mediated HDAC1 knockdown on the biological behavior of esophageal carcinoma cell lines. Material/Methods HDAC1 expression in esophageal cancer cell lines TE-1, Eca109, and EC9706 was compared by Western blot analysis. These cells were transfected with siRNA-HDAC1 and cell proliferation was evaluated by MTT assay to select the optimum cell line for subsequent experiments. The effects of siRNA-HDAC1 on the migration and invasion of the selected cell line were assessed by transwell assay. The expression of cell cycle-related proteins cyclinD1, p21 and p27, and epithelial-mesenchymal transition (EMT)-related protein zonula occludens-1 (ZO-1), E-cadherin and vimentin was determined by Western blot analysis. Results HDAC1 expression in TE-1, Eca109 and EC9706 cells was significantly higher compared with normal esophageal cell line HEEC (P<0.01). MTT assay, Western blot and RT-PCR analyses demonstrated that the inhibitory effects of siRNA on HDAC1 expression and cell viability in TE-1 cells were the highest among all cell lines, which was therefore used in subsequent experiments. After TE-1 cells were transfected with siRNA-HDAC1, their migration and invasion were significantly lower compared with the controls (P<0.01). CyclinD1 and vimentin expression was significantly lower compared with the controls (P<0.01), whereas the expression of p21, p27, ZO-1 and E-cadherin was significantly higher (P<0.01). Conclusions The siRNA-mediated HDAC1 knockdown significantly inhibited the proliferation, migration and invasion of TE-1 cells probably by regulating the expression of cell cycle- and EMT-related proteins. PMID:27086779

  2. Novel hypovirulence-associated RNA mycovirus in the plant-pathogenic fungus Botrytis cinerea: molecular and biological characterization.

    PubMed

    Yu, Lin; Sang, Wen; Wu, Ming-De; Zhang, Jing; Yang, Long; Zhou, Ying-Jun; Chen, Wei-Dong; Li, Guo-Qing

    2015-04-01

    Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A -1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The -1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1. PMID:25595766

  3. Novel Hypovirulence-Associated RNA Mycovirus in the Plant-Pathogenic Fungus Botrytis cinerea: Molecular and Biological Characterization

    PubMed Central

    Yu, Lin; Sang, Wen; Wu, Ming-De; Zhang, Jing; Yang, Long; Zhou, Ying-Jun; Chen, Wei-Dong

    2015-01-01

    Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A −1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The −1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1. PMID:25595766

  4. Design and Fabrication of Nano Biologically Sensitive Field-Effect Transistor (Nano Bio-FET) for Bio-Molecule Detection

    NASA Astrophysics Data System (ADS)

    Foo, K. L.; Kashif, M.; Hashim, U.

    2011-05-01

    Biologically Sensitive Field-Effect Transistor (Bio-FET) based on silicon nanowires (SiNWs), so called Nano Bio-FET, promise ultrasensitive dynamic label-free electrical detection of bio-molecule. Therefore, Nano Bio-FET which is one type of biosensor is mainly used for biological element detection such as nucleic acid, antibodies and enzyme. SiNWs which is act as the transducer is one type of the Nanowire where it can be produced using a top-down or bottom-up approach. As to minimize the cost of fabrication process, conventional CMOS process was chosen. For this reason, SiNWs, as small as 40 nm in width, is fabricated using spacer patterning lithography (SPL) technique. SPL is one type of top-down technique used to fabricate nanostructure especially nanowire. It is a low-cost and compatible to standard CMOS fabrication process. SPL, in general is a combination of conventional photolithography, anisotropic etchings and the excellent homogeneity and reproducibility of conformal chemical vapor deposition processes.

  5. RNA catalysis and the origins of life

    NASA Technical Reports Server (NTRS)

    Orgel, Leslie E.

    1986-01-01

    The role of RNA catalysis in the origins of life is considered in connection with the discovery of riboszymes, which are RNA molecules that catalyze sequence-specific hydrolysis and transesterification reactions of RNA substrates. Due to this discovery, theories positing protein-free replication as preceding the appearance of the genetic code are more plausible. The scope of RNA catalysis in biology and chemistry is discussed, and it is noted that the development of methods to select (or predict) RNA sequences with preassigned catalytic functions would be a major contribution to the study of life's origins.

  6. Peptide-coated semiconductor quantum dots and their applications in biological imaging of single molecules in live cells and organisms

    NASA Astrophysics Data System (ADS)

    Pinaud, Fabien Florent

    2007-12-01

    A new surface chemistry has been developed for the solubilization and biofunctionalization of inorganic semiconductor nanocrystals fluorescent probes, also known as quantum dots. This chemistry is based on the surface coating of quantum dots with custom-designed polycysteine peptides and yields water-soluble, small, monodispersed and colloidally stable probes that remain bright and photostable in complex biological milieus. This peptide coating strategy was successfully tested on several types of core and core-shell quantum dots emitting from the visible (e.g. CdSe/ZnS) to the NIR spectrum range (e.g. CdTe/CdSe/ZnS). By taking advantage of the versatile physico-chemical properties of peptides, a peptide "toolkit" was designed and employed to impart several biological functions to individual quantum dots and control their biochemical activity at the nanometer scale. These biofunctionalized peptide-coated quantum dots were exploited in very diverse biological applications. Near-infrared emitting quantum dot probes were engineered with optimized blood circulation and biodistribution properties for in vivo animal imaging. Visible emitting quantum dots were used for single molecule tracking of raft-associated GPI-anchored proteins in live cells. This last application revealed the presence of discrete and non-caveolar lipid microdomains capable of impeding free lateral diffusions in the plasma membrane of Hela cells. Imaging and tracking of peptide-coated quantum dots provided the first direct evidence that microdomains having the composition and behavior expected for lipid rafts can induce molecular compartmentalization in the membrane of living cells.

  7. Conceptual Model-Based Systems Biology: Mapping Knowledge and Discovering Gaps in the mRNA Transcription Cycle

    PubMed Central

    Somekh, Judith; Choder, Mordechai; Dori, Dov

    2012-01-01

    We propose a Conceptual Model-based Systems Biology framework for qualitative modeling, executing, and eliciting knowledge gaps in molecular biology systems. The framework is an adaptation of Object-Process Methodology (OPM), a graphical and textual executable modeling language. OPM enables concurrent representation of the system's structure—the objects that comprise the system, and behavior—how processes transform objects over time. Applying a top-down approach of recursively zooming into processes, we model a case in point—the mRNA transcription cycle. Starting with this high level cell function, we model increasingly detailed processes along with participating objects. Our modeling approach is capable of modeling molecular processes such as complex formation, localization and trafficking, molecular binding, enzymatic stimulation, and environmental intervention. At the lowest level, similar to the Gene Ontology, all biological processes boil down to three basic molecular functions: catalysis, binding/dissociation, and transporting. During modeling and execution of the mRNA transcription model, we discovered knowledge gaps, which we present and classify into various types. We also show how model execution enhances a coherent model construction. Identification and pinpointing knowledge gaps is an important feature of the framework, as it suggests where research should focus and whether conjectures about uncertain mechanisms fit into the already verified model. PMID:23308089

  8. A microRNA-1280/JAG2 network comprises a novel biological target in high-risk medulloblastoma.

    PubMed

    Wang, Fengfei; Remke, Marc; Bhat, Kruttika; Wong, Eric T; Zhou, Shuang; Ramaswamy, Vijay; Dubuc, Adrian; Fonkem, Ekokobe; Salem, Saeed; Zhang, Hongbing; Hsieh, Tze-Chen; O'Rourke, Stephen T; Wu, Lizi; Li, David W; Hawkins, Cynthia; Kohane, Isaac S; Wu, Joseph M; Wu, Min; Taylor, Michael D; Wu, Erxi

    2015-02-20

    Over-expression of PDGF receptors (PDGFRs) has been previously implicated in high-risk medulloblastoma (MB) pathogenesis. However, the exact biological functions of PDGFRα and PDGFRβ signaling in MB biology remain poorly understood. Here, we report the subgroup specific expression of PDGFRα and PDGFRβ and their associated biological pathways in MB tumors. c-MYC, a downstream target of PDGFRβ but not PDGFRα, is involved in PDGFRβ signaling associated with cell proliferation, cell death, and invasion. Concurrent inhibition of PDGFRβ and c-MYC blocks MB cell proliferation and migration synergistically. Integrated analysis of miRNA and miRNA targets regulated by both PDGFRβ and c-MYC reveals that increased expression of JAG2, a target of miR-1280, is associated with high metastatic dissemination at diagnosis and a poor outcome in MB patients. Our study may resolve the controversy on the role of PDGFRs in MB and unveils JAG2 as a key downstream effector of a PDGFRβ-driven signaling cascade and a potential therapeutic target. PMID:25576913

  9. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions

    PubMed Central

    Long, Yicheng; Jackman, Jane E.

    2015-01-01

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from A. castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing. PMID:26143376

  10. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions.

    PubMed

    Long, Yicheng; Jackman, Jane E

    2015-07-22

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from Acanthamoeba castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing. PMID:26143376

  11. Structure and Biological Function of the RNA Pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus

    SciTech Connect

    Messing, S.; Gabelli, S; Liu, Q; Celesnik, H; Belasco, J; Pineiro, S; Amzel, L

    2009-01-01

    Until recently, the mechanism of mRNA decay in bacteria was thought to be different from that of eukaryotes. This paradigm changed with the discovery that RppH (ORF176/NudH/YgdP), an Escherichia coli enzyme that belongs to the Nudix superfamily, is an RNA pyrophosphohydrolase that initiates mRNA decay by cleaving pyrophosphate from the 5?-triphosphate. Here we report the 1.9 A resolution structure of the Nudix hydrolase BdRppH from Bdellovibrio bacteriovorus, a bacterium that feeds on other Gram-negative bacteria. Based on the structure of the enzyme alone and in complex with GTP-Mg2+, we propose a mode of RNA binding similar to that of the nuclear decapping enzyme from Xenopus laevis, X29. In additional experiments, we show that BdRppH can indeed function in vitro and in vivo as an RNA pyrophosphohydrolase. These findings set the basis for the identification of possible decapping enzymes in other bacteria.

  12. tRNA Core Hypothesis for the Transition from the RNA World to the Ribonucleoprotein World

    PubMed Central

    de Farias, Savio T.; Rêgo, Thais G.; José, Marco V.

    2016-01-01

    Herein we present the tRNA core hypothesis, which emphasizes the central role of tRNAs molecules in the origin and evolution of fundamental biological processes. tRNAs gave origin to the first genes (mRNA) and the peptidyl transferase center (rRNA), proto-tRNAs were at the core of a proto-translation system, and the anticodon and operational codes then arose in tRNAs molecules. Metabolic pathways emerged from evolutionary pressures of the decoding systems. The transitions from the RNA world to the ribonucleoprotein world to modern biological systems were driven by three kinds of tRNAs transitions, to wit, tRNAs leading to both mRNA and rRNA. PMID:27023615

  13. tRNA Core Hypothesis for the Transition from the RNA World to the Ribonucleoprotein World.

    PubMed

    de Farias, Savio T; Rêgo, Thais G; José, Marco V

    2016-01-01

    Herein we present the tRNA core hypothesis, which emphasizes the central role of tRNAs molecules in the origin and evolution of fundamental biological processes. tRNAs gave origin to the first genes (mRNA) and the peptidyl transferase center (rRNA), proto-tRNAs were at the core of a proto-translation system, and the anticodon and operational codes then arose in tRNAs molecules. Metabolic pathways emerged from evolutionary pressures of the decoding systems. The transitions from the RNA world to the ribonucleoprotein world to modern biological systems were driven by three kinds of tRNAs transitions, to wit, tRNAs leading to both mRNA and rRNA. PMID:27023615

  14. Omics of Brucella: Species-Specific sRNA-Mediated Gene Ontology Regulatory Networks Identified by Computational Biology.

    PubMed

    Vishnu, Udayakumar S; Sankarasubramanian, Jagadesan; Gunasekaran, Paramasamy; Sridhar, Jayavel; Rajendhran, Jeyaprakash

    2016-06-01

    Brucella is an intracellular bacterium that causes the zoonotic infectious disease, brucellosis. Brucella species are currently intensively studied with a view to developing novel global health diagnostics and therapeutics. In this context, small RNAs (sRNAs) are one of the emerging topical areas; they play significant roles in regulating gene expression and cellular processes in bacteria. In the present study, we forecast sRNAs in three Brucella species that infect humans, namely Brucella melitensis, Brucella abortus, and Brucella suis, using a computational biology analysis. We combined two bioinformatic algorithms, SIPHT and sRNAscanner. In B. melitensis 16M, 21 sRNA candidates were identified, of which 14 were novel. Similarly, 14 sRNAs were identified in B. abortus, of which four were novel. In B. suis, 16 sRNAs were identified, and five of them were novel. TargetRNA2 software predicted the putative target genes that could be regulated by the identified sRNAs. The identified mRNA targets are involved in carbohydrate, amino acid, lipid, nucleotide, and coenzyme metabolism and transport, energy production and conversion, replication, recombination, repair, and transcription. Additionally, the Gene Ontology (GO) network analysis revealed the species-specific, sRNA-based regulatory networks in B. melitensis, B. abortus, and B. suis. Taken together, although sRNAs are veritable modulators of gene expression in prokaryotes, there are few reports on the significance of sRNAs in Brucella. This report begins to address this literature gap by offering a series of initial observations based on computational biology to pave the way for future experimental analysis of sRNAs and their targets to explain the complex pathogenesis of Brucella. PMID:27223678

  15. Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

    PubMed Central

    Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

    2013-01-01

    Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

  16. RNA-Seq: Improving Our Understanding of Retinal Biology and Disease.

    PubMed

    Farkas, Michael H; Au, Elizabeth D; Sousa, Maria E; Pierce, Eric A

    2015-01-01

    Over the past several years, rapid technological advances have allowed for a dramatic increase in our knowledge and understanding of the transcriptional landscape, because of the ability to study gene expression in greater depth and with more detail than previously possible. To this end, RNA-Seq has quickly become one of the most widely used methods for studying transcriptomes of tissues and individual cells. Unlike previously favored analysis methods, RNA-Seq is extremely high-throughput, and is not dependent on an annotated transcriptome, laying the foundation for novel genetic discovery. Additionally, RNA-Seq derived transcriptomes provide a basis for widening the scope of research to identify potential targets in the treatment of retinal disease. PMID:25722474

  17. Dual roles of DNA repair enzymes in RNA biology/post-transcriptional control.

    PubMed

    Vohhodina, Jekaterina; Harkin, D Paul; Savage, Kienan I

    2016-09-01

    Despite consistent research into the molecular principles of the DNA damage repair pathway for almost two decades, it has only recently been found that RNA metabolism is very tightly related to this pathway, and the two ancient biochemical mechanisms act in alliance to maintain cellular genomic integrity. The close links between these pathways are well exemplified by examining the base excision repair pathway, which is now well known for dual roles of many of its members in DNA repair and RNA surveillance, including APE1, SMUG1, and PARP1. With additional links between these pathways steadily emerging, this review aims to provide a summary of the emerging roles for DNA repair proteins in the post-transcriptional regulation of RNAs. WIREs RNA 2016, 7:604-619. doi: 10.1002/wrna.1353 For further resources related to this article, please visit the WIREs website. PMID:27126972

  18. Small molecule-mediated up-regulation of microRNA targeting a key cell death modulator BNIP3 improves cardiac function following ischemic injury

    PubMed Central

    Lee, Se-Yeon; Lee, Seahyoung; Choi, Eunhyun; Ham, Onju; Lee, Chang Youn; Lee, Jiyun; Seo, Hyang-Hee; Cha, Min-Ji; Mun, Bohyun; Lee, Yunmi; Yoon, Cheesoon; Hwang, Ki-Chul

    2016-01-01

    Genetic ablation of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), an essential regulator of cardiac cell death, is an effective way to prevent cardiac cell death triggered by pathologic conditions. However, currently there exists no known means, such as inhibitors, to down-regulate BNIP3 in mature heart. Here, we report that a small molecule inducer of microRNA-182 (miR-182) suppressed ischemia/reperfusion (I/R)-induced cardiac cell death by down-regulating BNIP3. We first selected miR-182 as a potent BNIP3-targeting miRNA based on miRNA-target prediction databases and empirical data. The subsequent screening of small molecules for inducing miR-182 expression identified Kenpaullone as a hit compound. Both exogenous miR-182 and Kenpaullone significantly suppressed hypoxia-induced cardiomyocyte death in vitro. To investigate the effect of changing substituents of Kenpaullone on miR-182 expression, we synthesized 9 derivatives of Kenpaullone. Among these derivatives, compound 5 showed significantly improved ability to induce miR-182 expression. The results of the in vivo study showed that compound 5 significantly improved heart function following I/R-injury in rats. Our study provides strong evidence that the small molecule-mediated up-regulation of miRNAs is a viable strategy to down-regulate target proteins with no known chemical inhibitor and that compound 5 may have potential to prevent I/R-inflicted cardiac cell death. PMID:27008992

  19. Small molecule-mediated up-regulation of microRNA targeting a key cell death modulator BNIP3 improves cardiac function following ischemic injury.

    PubMed

    Lee, Se-Yeon; Lee, Seahyoung; Choi, Eunhyun; Ham, Onju; Lee, Chang Youn; Lee, Jiyun; Seo, Hyang-Hee; Cha, Min-Ji; Mun, Bohyun; Lee, Yunmi; Yoon, Cheesoon; Hwang, Ki-Chul

    2016-01-01

    Genetic ablation of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), an essential regulator of cardiac cell death, is an effective way to prevent cardiac cell death triggered by pathologic conditions. However, currently there exists no known means, such as inhibitors, to down-regulate BNIP3 in mature heart. Here, we report that a small molecule inducer of microRNA-182 (miR-182) suppressed ischemia/reperfusion (I/R)-induced cardiac cell death by down-regulating BNIP3. We first selected miR-182 as a potent BNIP3-targeting miRNA based on miRNA-target prediction databases and empirical data. The subsequent screening of small molecules for inducing miR-182 expression identified Kenpaullone as a hit compound. Both exogenous miR-182 and Kenpaullone significantly suppressed hypoxia-induced cardiomyocyte death in vitro. To investigate the effect of changing substituents of Kenpaullone on miR-182 expression, we synthesized 9 derivatives of Kenpaullone. Among these derivatives, compound 5 showed significantly improved ability to induce miR-182 expression. The results of the in vivo study showed that compound 5 significantly improved heart function following I/R-injury in rats. Our study provides strong evidence that the small molecule-mediated up-regulation of miRNAs is a viable strategy to down-regulate target proteins with no known chemical inhibitor and that compound 5 may have potential to prevent I/R-inflicted cardiac cell death. PMID:27008992

  20. The Diversity of Ribonuclease P: Protein and RNA Catalysts with Analogous Biological Functions

    PubMed Central

    Klemm, Bradley P.; Wu, Nancy; Chen, Yu; Liu, Xin; Kaitany, Kipchumba J.; Howard, Michael J.; Fierke, Carol A.

    2016-01-01

    Ribonuclease P (RNase P) is an essential endonuclease responsible for catalyzing 5’ end maturation in precursor transfer RNAs. Since its discovery in the 1970s, RNase P enzymes have been identified and studied throughout the three domains of life. Interestingly, RNase P is either RNA-based, with a catalytic RNA subunit, or a protein-only (PRORP) enzyme with differential evolutionary distribution. The available structural data, including the active site data, provides insight into catalysis and substrate recognition. The hydrolytic and kinetic mechanisms of the two forms of RNase P enzymes are similar, yet features unique to the RNA-based and PRORP enzymes are consistent with different evolutionary origins. The various RNase P enzymes, in addition to their primary role in tRNA 5’ maturation, catalyze cleavage of a variety of alternative substrates, indicating a diversification of RNase P function in vivo. The review concludes with a discussion of recent advances and interesting research directions in the field. PMID:27187488

  1. Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes

    PubMed Central

    Katz, Zachary B; English, Brian P; Lionnet, Timothée; Yoon, Young J; Monnier, Nilah; Ovryn, Ben; Bathe, Mark; Singer, Robert H

    2016-01-01

    Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality. DOI: http://dx.doi.org/10.7554/eLife.10415.001 PMID:26760529

  2. The microRNA-200 family: small molecules with novel roles in cancer development, progression and therapy

    PubMed Central

    Humphries, Brock; Yang, Chengfeng

    2015-01-01

    MicroRNAs (miRNAs) are a large family of small non-coding RNAs that negatively regulate protein-coding gene expression post-transcriptionally via base pairing between the 5′ seed region of a miRNA and the 3′ untranslated region (3′UTR) of a messenger RNA (mRNA). Recent evidence has supported the critical role that miRNAs play in many diseases including cancer. The miR-200 family consisting of 5 members (miR-200a, -200b, -200c, -141, -429) is an emerging miRNA family that has been shown to play crucial roles in cancer initiation and metastasis, and potentially be important for the diagnosis and treatment of cancer. While miR-200s were found to be critically involved in the metastatic colonization to the lungs in mouse mammary xenograft tumor models, a large number of studies demonstrated their strong suppressive effects on cell transformation, cancer cell proliferation, migration, invasion, tumor growth and metastasis. This review aims to discuss research findings about the role of the miR-200 family in cancer initiation, each step of cancer metastatic cascade, cancer diagnosis and treatment. A comprehensive summary of currently validated miR-200 targets is also presented. It is concluded that miR-200 family may serve as novel targets for the therapy of multiple types of cancer. PMID:25762624

  3. lncRNA-RNA Interactions across the Human Transcriptome

    PubMed Central

    Szcześniak, Michał Wojciech; Makałowska, Izabela

    2016-01-01

    Long non-coding RNAs (lncRNAs) represent a numerous class of non-protein coding transcripts longer than 200 nucleotides. There is possibility that a fraction of lncRNAs are not functional and represent mere transcriptional noise but a growing body of evidence shows they are engaged in a plethora of molecular functions and contribute considerably to the observed diversification of eukaryotic transcriptomes and proteomes. Still, however, only ca. 1% of lncRNAs have well established functions and much remains to be done towards decipherment of their biological roles. One of the least studied aspects of lncRNAs biology is their engagement in gene expression regulation through RNA-RNA interactions. By hybridizing with mate RNA molecules, lncRNAs could potentially participate in modulation of pre-mRNA splicing, RNA editing, mRNA stability control, translation activation, or abrogation of miRNA-induced repression. Here, we implemented a similarity-search based method for transcriptome-wide identification of RNA-RNA interactions, which enabled us to find 18,871,097 lncRNA-RNA base-pairings in human. Further analyses showed that the interactions could be involved in processing, stability control and functions of 57,303 transcripts. An extensive use of RNA-Seq data provided support for approximately one third of the interactions, at least in terms of the two RNA components being co-expressed. The results suggest that lncRNA-RNA interactions are broadly used to regulate and diversify the human transcriptome. PMID:26930590

  4. Cleavage of highly structured viral RNA molecules by combinatorial libraries of hairpin ribozymes. The most effective ribozymes are not predicted by substrate selection rules.

    PubMed

    Yu, Q; Pecchia, D B; Kingsley, S L; Heckman, J E; Burke, J M

    1998-09-01

    Combinatorial libraries of hairpin ribozymes representing all possible cleavage specificities (>10(5)) were used to evaluate all ribozyme cleavage sites within a large (4.2-kilobase) and highly structured viral mRNA, the 26 S subgenomic RNA of Sindbis virus. The combinatorial approach simultaneously accounts for target site structure and dynamics, together with ribozyme folding, and the sequences that result in a ribozyme-substrate complex with maximal activity. Primer extension was used to map and rank the relative activities of the ribozyme pool against individual sites and revealed two striking findings. First, only a small fraction of potential recognition sites are effectively cleaved (activity-selected sites). Second, nearly all of the most effectively cleaved sites deviated substantially from the established consensus selection rules for the hairpin ribozyme and were not predicted by examining the sequence, or through the use of computer-assisted predictions of RNA secondary structure. In vitro selection methods were used to isolate ribozymes with increased activity against substrates that deviate from the GUC consensus sequence. trans-Acting ribozymes targeting nine of the activity-selected sites were synthesized, together with ribozymes targeting four sites with a perfect match to the cleavage site consensus (sequence-selected sites). Activity-selected ribozymes have much higher cleavage activity against the long, structured RNA molecules than do sequence-selected ribozymes, although the latter are effective in cleaving oligoribonucleotides, as predicted. These results imply that, for Sindbis virus 26 S RNA, designing ribozymes based on matches to the consensus sequence may be an ineffective strategy. PMID:9722591

  5. Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells

    SciTech Connect

    Komm, B.S.; Terpening, C.M.; Benz, D.J.; Graeme, K.A.; Gallegos, A.; Korc, M.; Greene, G.L.; O'Malley, B.W.; Haussler, M.R.

    1988-07-01

    High specific activity estradiol labeled with iodine-125 was used to detect approximately 200 saturable, high-affinity (dissociation constant approximately equal to 1.0 nM) nuclear binding sites in rat (ROS 17/2.8) and human (HOS TE85) clonal osteoblast-like osteosarcoma cells. Of the steroids tested, only testosterone exhibited significant cross-reactivity with estrogen binding. RNA blot analysis with a complementary DNA probe to the human estrogen receptor revealed putative receptor transcripts of 6 to 6.2 kilobases in both rat and human osteosarcoma cells. Type I procollagen and transforming growth factor-beta messenger RNA levels were enhanced in cultured human osteoblast-like cells treated with 1 nM estradiol. Thus, estrogen can act directly on osteoblasts by a receptor-mediated mechanism and thereby modulate the extracellular matrix and other proteins involved in the maintenance of skeletal mineralization and remodeling.

  6. Supramolecular assembly of biological molecules purified from bovine nerve cells: from microtubule bundles and necklaces to neurofilament networks

    NASA Astrophysics Data System (ADS)

    Needleman, Daniel J.; Jones, Jayna B.; Raviv, Uri; Ojeda-Lopez, Miguel A.; Miller, H. P.; Li, Y.; Wilson, L.; Safinya, C. R.

    2005-11-01

    With the completion of the human genome project, the biosciences community is beginning the daunting task of understanding the structures and functions of a large number of interacting biological macromolecules. Examples include the interacting molecules involved in the process of DNA condensation during the cell cycle, and in the formation of bundles and networks of filamentous actin proteins in cell attachment, motility and cytokinesis. In this proceedings paper we present examples of supramolecular assembly based on proteins derived from the vertebrate nerve cell cytoskeleton. The axonal cytoskeleton in vertebrate neurons provides a rich example of bundles and networks of neurofilaments, microtubules (MTs) and filamentous actin, where the nature of the interactions, structures, and structure-function correlations remains poorly understood. We describe synchrotron x-ray diffraction, electron microscopy, and optical imaging data, in reconstituted protein systems purified from bovine central nervous system, which reveal unexpected structures not predicted by current electrostatic theories of polyelectrolyte bundling, including three-dimensional MT bundles and two-dimensional MT necklaces.

  7. Bioorganic chemistry à la baguette: studies on molecular recognition in biological systems using rigid-rod molecules.

    PubMed

    Matile, S

    2001-01-01

    Initial studies using rigid-rod molecules or "baguettes" to address bioorganic topics of current scientific concern are reported. It is illustrated how transmembrane oligo(p-phenylene)s as representative model rods can be tuned to recognize lipid bilayer membranes either by their thickness or polarization. The construction of otherwise problematic hydrogen-bonded chains along transmembrane rods yields "proton wires," which act by a mechanism that is central in bioenergetics but poorly explored by means of synthetic models. Another example focuses on multivalent ligands assembling rigid-rod cell-surface receptors into transmembrane dynamic arene arrays. The potassium transport mediated by these ligand-receptor complexes provides experimental support for the potential biological importances of the controversial cation-pi mechanism. More complex supramolecular architecture is portrayed in the first artificial beta-barrels. It is shown how programmed assembly of toroidal rigid-rod supramolecules in detergent-free water permits control of diameter of the chemical nature of their interior. Reversed rigid-rod beta-barrels are assembled to function as self-assembled ionophores, ion channel models, and transmembrane nanopores. The potential of future intratoroidal chemistry is exemplified by encapsulation and planarization of beta-carotene in water and the construction of transmembrane B-DNA at the center of a second-sphere host-guest complex à al baguette. PMID:11893065

  8. Biological and Molecular Effects of Small Molecule Kinase Inhibitors on Low-Passage Human Colorectal Cancer Cell Lines

    PubMed Central

    Lange, Falko; Franz, Benjamin; Maletzki, Claudia; Linnebacher, Michael; Hühns, Maja; Jaster, Robert

    2014-01-01

    Low-passage cancer cell lines are versatile tools to study tumor cell biology. Here, we have employed four such cell lines, established from primary tumors of colorectal cancer (CRC) patients, to evaluate effects of the small molecule kinase inhibitors (SMI) vemurafenib, trametinib, perifosine, and regorafenib in an in vitro setting. The mutant BRAF (V600E/V600K) inhibitor vemurafenib, but also the MEK1/2 inhibitor trametinib efficiently inhibited DNA synthesis, signaling through ERK1/2 and expression of genes downstream of ERK1/2 in BRAF mutant cells only. In case of the AKT inhibitor perifosine, three cell lines showed a high or intermediate responsiveness to the drug while one cell line was resistant. The multikinase inhibitor regorafenib inhibited proliferation of all CRC lines with similar efficiency and independent of the presence or absence of KRAS, BRAF, PIK3CA, and TP53 mutations. Regorafenib action was associated with broad-range inhibitory effects at the level of gene expression but not with a general inhibition of AKT or MEK/ERK signaling. In vemurafenib-sensitive cells, the antiproliferative effect of vemurafenib was enhanced by the other SMI. Together, our results provide insights into the determinants of SMI efficiencies in CRC cells and encourage the further use of low-passage CRC cell lines as preclinical models. PMID:25309914

  9. Prospect of detection and recognition of single biological molecules using ultrafast coherent dynamics in quantum dot-metallic nanoparticle systems

    NASA Astrophysics Data System (ADS)

    Sadeghi, S. M.

    2015-08-01

    Conventional plasmonic sensors are based on the intrinsic resonances of metallic nanoparticles. In such sensors wavelength shift of such resonances are used to detect biological molecules. Recently we introduced ultra-sensitive timedomain nanosensors based on the way variations in the environmental conditions influence coherent dynamics of hybrid systems consisting of metallic nanoparticles and quantum dots. Such dynamics are generated via interaction of these systems with a laser field, generating quantum coherence and coherent exciton-plasmon coupling. These sensors are based on impact of variations of the refractive index of the environment on such dynamics, generating time-dependent changes in the emission of the QDs. In this paper we study the impact of material properties of the metallic nanoparticles on this process and demonstrate the key role played by the design of the quantum dots. We show that Ag nanoparticles, even in a simple spherical shape, may allow these sensors to operate at room temperature, owing to the special properties of quantum dot-metallic nanoparticle systems that may allow coherent effects utilized in such sensors happen in the presence of the ultrafast polarization dephasing of quantum dots.

  10. Small molecules that interact with RNA: riboswitch-based gene control and its involvement in metabolic regulation in plants and algae.

    PubMed

    Bocobza, Samuel E; Aharoni, Asaph

    2014-08-01

    Riboswitches are RNA elements that bind small molecules and in turn regulate gene expression. This mechanism allows the cell to sense the intracellular concentration of these small molecules. A particular riboswitch typically regulates its adjacent gene by altering the transcription, the translation or the splicing of this gene. Recently, a riboswitch that binds thiamin pyrophosphate (TPP) was characterized and found to regulate thiamin biosynthesis in plants and algae. Furthermore, it appears that this element is an essential regulator of primary metabolism in plants. Manipulation of endogenous riboswitch activity resulted in metabolic phenotypes that underlined the role of these elements and their ligands in preserving metabolic homeostasis. This situation supports the hypothesis that riboswitches could be remnants of the most ancient metabolic regulators. Here, we review the mode of action of the plant and algal TPP riboswitch and its relevance to the metabolic network. We also discuss the potential engineering of riboswitches as metabolite sensors in plants and platforms for gene control. Whether additional such RNA-based mechanisms exist in plants and in algae is still an open question, yet, the importance of these elements to metabolic regulation is beyond doubt. PMID:24773387

  11. Volatile evolution of long noncoding RNA repertoires: mechanisms and biological implications

    PubMed Central

    Kapusta, Aurélie; Feschotte, Cédric

    2014-01-01

    Thousands of genes encoding long noncoding RNAs (lncRNAs) have been identified in all vertebrate genomes thus far examined. The list of lncRNAs partaking in arguably important biochemical, cellular, and developmental activities is steadily growing. However, it is increasingly clear that lncRNA repertoires are subject to weak functional constraint and rapid turnover during vertebrate evolution. Here we discuss some of the factors that may explain this apparent paradox, including relaxed constraint on sequence to maintain lncRNA structure/function, extensive redundancy in the regulatory circuits in which lncRNAs act, as well as adaptive and non-adaptive forces such as genetic drift. We explore the molecular mechanisms promoting the birth and rapid evolution of lncRNA genes with an emphasis on the influence of bidirectional transcription and transposable elements, two pervasive features of vertebrate genomes. Together these properties reveal a remarkably dynamic and malleable noncoding transcriptome, which may represent an important source of robustness and evolvability. PMID:25218058

  12. Cofactors in the RNA World

    NASA Technical Reports Server (NTRS)

    Ditzler, Mark A.

    2014-01-01

    RNA world theories figure prominently in many scenarios for the origin and early evolution of life. These theories posit that RNA molecules played a much larger role in ancient biology than they do now, acting both as the dominant biocatalysts and as the repository of genetic information. Many features of modern RNA biology are potential examples of molecular fossils from an RNA world, such as the pervasive involvement of nucleotides in coenzymes, the existence of natural aptamers that bind these coenzymes, the existence of natural ribozymes, a biosynthetic pathway in which deoxynucleotides are produced from ribonucleotides, and the central role of ribosomal RNA in protein synthesis in the peptidyl transferase center of the ribosome. Here, we uses both a top-down approach that evaluates RNA function in modern biology and a bottom-up approach that examines the capacities of RNA independent of modern biology. These complementary approaches exploit multiple in vitro evolution techniques coupled with high-throughput sequencing and bioinformatics analysis. Together these complementary approaches advance our understanding of the most primitive organisms, their early evolution, and their eventual transition to modern biochemistry.

  13. Phage-mediated counting by the naked eye of miRNA molecules at attomolar concentrations in a Petri dish

    NASA Astrophysics Data System (ADS)

    Zhou, Xin; Cao, Peng; Zhu, Ye; Lu, Wuguang; Gu, Ning; Mao, Chuanbin

    2015-10-01

    The ability to count biomolecules such as cancer-biomarker miRNAs with the naked eye is seemingly impossible in molecular diagnostics. Here, we show an ultrasensitive naked-eye-counting strategy for quantifying miRNAs by employing T7 phage--a bacteria-specific virus nanoparticle--as a surrogate. The phage is genetically engineered to become fluorescent and capable of binding a miRNA-capturing gold nanoparticle (GNP) in a one-to-one manner. Target miRNAs crosslink the resultant phage-GNP couple and miRNA-capturing magnetic microparticles, forming a sandwich complex containing equimolar phage and miRNA. The phage is then released from the complex and developed into one macroscopic fluorescent plaque in a Petri dish by plating it in a host bacterial medium. Counting the plaques by the naked eye enables the quantification of miRNAs with detection limits of ~3 and ~5 aM for single-target and two-target miRNAs, respectively. This approach offers ultrasensitive and convenient quantification of disease biomarkers by the naked eye.

  14. Ontogenic expression of microRNA in bovine mammary gland

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MicroRNAs (miR) are small RNA molecules (~22 nucleotides) that are important regulators of numerous biological processes, including organ and tissue morphogenesis and function. In this capacity, most miR inhibit protein synthesis by binding to the 3’-untranslated region of targeted mRNA species. H...

  15. LigandRNA: computational predictor of RNA–ligand interactions

    PubMed Central

    Philips, Anna; Milanowska, Kaja; Łach, Grzegorz; Bujnicki, Janusz M.

    2013-01-01

    RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA–small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA–ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a “meta-predictor” leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl. PMID:24145824

  16. RNA based evolutionary optimization

    NASA Astrophysics Data System (ADS)

    Schuster, Peter

    1993-12-01

    The notion of an RNA world has been introduced for a prebiotic scenario that is dominated by RNA molecules and their properties, in particular their capabilities to act as templates for reproduction and as catalysts for several cleavage and ligation reactions of polynucleotides and polypeptides. This notion is used here also for simple experimental assays which are well suited to study evolution in the test tube. In molecular evolution experiments fitness is determined in essence by the molecular structures of RNA molecules. Evidence is presented for adaptation to environment in cell-free media. RNA based molecular evolution experiments have led to interesting spin-offs in biotechnology, commonly called ‘applied molecular evolution’, which make use of Darwinian trial-and-error strategies in order to synthesize new pharmacological compounds and other advanced materials on a biological basis. Error-propagation in RNA replication leads to formation of mutant spectra called ‘quasispecies’. An increase in the error rate broadens the mutant spectrum. There exists a sharply defined threshold beyond which heredity breaks down and evolutionary adaptation becomes impossible. Almost all RNA viruses studied so far operate at conditions close to this error threshold. Quasispecies and error thresholds are important for an understanding of RNA virus evolution, and they may help to develop novel antiviral strategies. Evolution of RNA molecules can be studied and interpreted by considering secondary structures. The notion of sequence space introduces a distance between pairs of RNA sequences which is tantamount to counting the minimal number of point mutations required to convert the sequences into each other. The mean sensitivity of RNA secondary structures to mutation depends strongly on the base pairing alphabet: structures from sequences which contain only one base pair (GC or AU are much less stable against mutation than those derived from the natural (AUGC) sequences

  17. Design, synthesis and biological evaluation of dinucleotide mRNA cap analog containing propargyl moiety.

    PubMed

    Shanmugasundaram, Muthian; Charles, Irudaya; Kore, Anilkumar R

    2016-03-15

    The first example of the synthesis of new dinucleotide cap analog containing propargyl group such as m(7,3'-)(O)(-propargyl)G[5']ppp[5']G is reported. The effect of propargyl cap analog with standard cap was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured HeLa cells. It is noteworthy that propargyl cap analog outperforms standard cap by 3.1 fold in terms of translational properties. The propargyl cap analog forms a more stable complex with translation initiation factor eIF4E based on the molecular modeling studies. PMID:26899596

  18. antaRNA: ant colony-based RNA sequence design

    PubMed Central

    Kleinkauf, Robert; Mann, Martin; Backofen, Rolf

    2015-01-01

    Motivation: RNA sequence design is studied at least as long as the classical folding problem. Although for the latter the functional fold of an RNA molecule is to be found, inverse folding tries to identify RNA sequences that fold into a function-specific target structure. In combination with RNA-based biotechnology and synthetic biology, reliable RNA sequence design becomes a crucial step to generate novel biochemical components. Results: In this article, the computational tool antaRNA is presented. It is capable of compiling RNA sequences for a given structure that comply in addition with an adjustable full range objective GC-content distribution, specific sequence constraints and additional fuzzy structure constraints. antaRNA applies ant colony optimization meta-heuristics and its superior performance is shown on a biological datasets. Availability and implementation: http://www.bioinf.uni-freiburg.de/Software/antaRNA Contact: backofen@informatik.uni-freiburg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26023105

  19. microRNA and Wound Healing.

    PubMed

    Banerjee, Jaideep; Sen, Chandan K

    2015-01-01

    microRNAs (miRNAs) are small noncoding RNA molecules which play pivotal roles in wound healing. The increased expression of certain genes and expression of some others represent a key component of the wound biology and are largely under the regulation of naturally occurring miRNAs. Understanding the dysregulated miRNAs in chronic wound biology will therefore enable the development of newer therapies. This chapter focuses on the miRNAs that can be potentially targeted for improving skin wound healing and the challenges in miRNA therapy, including considerations in miRNA target identification and delivery. PMID:26663189

  20. Evolution in an RNA World

    PubMed Central

    Joyce, Gerald F.

    2009-01-01

    A longstanding research goal has been to develop a self-sustained chemical system that is capable of undergoing Darwinian evolution. The notion of primitive RNA-based life suggests this goal might be achieved by constructing an RNA enzyme that catalyzes the replication of RNA molecules, including the RNA enzyme itself. This reaction recently was demonstrated in a cross-catalytic system involving two RNA enzymes that catalyze each other’s synthesis from a total of four component substrates. The cross-replicating RNA enzymes undergo self-sustained exponential amplification at a constant temperature in the absence of proteins or other biological materials. Amplification occurs with a doubling time of 30–60 min, and can be continued indefinitely. Small populations of cross-replicating RNA enzymes can be made to compete for limited resources within a common environment. The molecules reproduce with high fidelity, but occasionally give rise to recombinants that also can replicate. Over the course of many “generations” of selective amplification, novel variants arise and grow to dominate the population based on their relative fitness under the chosen reaction conditions. This is the first example, outside of biology, of evolutionary adaptation in a molecular genetic system. PMID:19667013

  1. Local Kinetic Measures of Macromolecular Structure Reveal Partitioning Among Multiple Parallel Pathways from the Earliest Steps in the Folding of a Large RNA Molecule

    SciTech Connect

    Laederach,A.; Shcherbakova, I.; Liang, M.; Brenowitz, M.; Altman, R.

    2006-01-01

    At the heart of the RNA folding problem is the number, structures, and relationships among the intermediates that populate the folding pathways of most large RNA molecules. Unique insight into the structural dynamics of these intermediates can be gleaned from the time-dependent changes in local probes of macromolecular conformation (e.g. reports on individual nucleotide solvent accessibility offered by hydroxyl radical ({center_dot}OH) footprinting). Local measures distributed around a macromolecule individually illuminate the ensemble of separate changes that constitute a folding reaction. Folding pathway reconstruction from a multitude of these individual measures is daunting due to the combinatorial explosion of possible kinetic models as the number of independent local measures increases. Fortunately, clustering of time progress curves sufficiently reduces the dimensionality of the data so as to make reconstruction computationally tractable. The most likely folding topology and intermediates can then be identified by exhaustively enumerating all possible kinetic models on a super-computer grid. The folding pathways and measures of the relative flux through them were determined for Mg{sup 2+} and Na{sup +}-mediated folding of the Tetrahymena thermophila group I intron using this combined experimental and computational approach. The flux during Mg{sup 2+}-mediated folding is divided among numerous parallel pathways. In contrast, the flux during the Na{sup +}-mediated reaction is predominantly restricted through three pathways, one of which is without detectable passage through intermediates. Under both conditions, the folding reaction is highly parallel with no single pathway accounting for more than 50% of the molecular flux. This suggests that RNA folding is non-sequential under a variety of different experimental conditions even at the earliest stages of folding. This study provides a template for the systematic analysis of the time-evolution of RNA structure

  2. RNA interference in Entamoeba histolytica: implications for parasite biology and gene silencing

    PubMed Central

    Zhang, Hanbang; Pompey, Justine M; Singh, Upinder

    2011-01-01

    Entamoeba histolytica is a major health threat to people in developing countries, where it causes invasive diarrhea and liver abscesses. The study of this important human pathogen has been hindered by a lack of tools for genetic manipulation. Recently, a number of genetic approaches based on variations of the RNAi method have been successfully developed and cloning of endogenous small-interfering RNAs from E. histolytica revealed an abundant population of small RNAs with an unusual 5′-polyphosphate structure. However, little is known about the implications of these findings to amebic biology or the mechanisms of gene silencing in this organism. In this article we review the literature relevant to RNAi in E. histolytica, discuss its implications for advances in gene silencing in this organism and outline potential future directions towards understanding the repertoire of RNAi and its impact on the biology of this deep-branching eukaryotic parasite. PMID:21162639

  3. RNA as an Enzyme.

    ERIC Educational Resources Information Center

    Cech, Thomas R.

    1986-01-01

    Reviews current findings that explain RNA's function as an enzyme in addition to being an informational molecule. Highlights recent research efforts and notes changes in the information base on RNA activity. Includes models and diagrams of RNA activity. (ML)

  4. RNA-Mediated Epigenetic Programming of Genome Rearrangements

    PubMed Central

    Nowacki, Mariusz; Shetty, Keerthi; Landweber, Laura F.

    2012-01-01

    RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa. Maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transport somatic mutations to the next generation. This opportunity for RNA-mediated genome rearrangement and DNA repair is profound in the ciliate Oxytricha, which deletes 95% of its germline genome during development in a process that severely fragments its chromosomes and then sorts and reorders the hundreds of thousands of pieces remaining. Oxytricha’s somatic nuclear genome is therefore an epigenome formed through RNA templates and signals arising from the previous generation. Furthermore, this mechanism of RNA-mediated epigenetic inheritance can function across multiple generations, and the discovery of maternal template RNA molecules has revealed new biological roles for RNA and has hinted at the power of RNA molecules to sculpt genomic information in cells. PMID:21801022

  5. Understanding gene regulatory mechanisms by integrating ChIP-seq and RNA-seq data: statistical solutions to biological problems

    PubMed Central

    Angelini, Claudia; Costa, Valerio

    2014-01-01

    The availability of omic data produced from international consortia, as well as from worldwide laboratories, is offering the possibility both to answer long-standing questions in biomedicine/molecular biology and to formulate novel hypotheses to test. However, the impact of such data is not fully exploited due to a limited availability of multi-omic data integration tools and methods. In this paper, we discuss the interplay between gene expression and epigenetic markers/transcription factors. We show how integrating ChIP-seq and RNA-seq data can help to elucidate gene regulatory mechanisms. In particular, we discuss the two following questions: (i) Can transcription factor occupancies or histone modification data predict gene expression? (ii) Can ChIP-seq and RNA-seq data be used to infer gene regulatory networks? We propose potential directions for statistical data integration. We discuss the importance of incorporating underestimated aspects (such as alternative splicing and long-range chromatin interactions). We also highlight the lack of data benchmarks and the need to develop tools for data integration from a statistical viewpoint, designed in the spirit of reproducible research. PMID:25364758

  6. An RNA motif that binds ATP

    NASA Technical Reports Server (NTRS)

    Sassanfar, M.; Szostak, J. W.

    1993-01-01

    RNAs that contain specific high-affinity binding sites for small molecule ligands immobilized on a solid support are present at a frequency of roughly one in 10(10)-10(11) in pools of random sequence RNA molecules. Here we describe a new in vitro selection procedure designed to ensure the isolation of RNAs that bind the ligand of interest in solution as well as on a solid support. We have used this method to isolate a remarkably small RNA motif that binds ATP, a substrate in numerous biological reactions and the universal biological high-energy intermediate. The selected ATP-binding RNAs contain a consensus sequence, embedded in a common secondary structure. The binding properties of ATP analogues and modified RNAs show that the binding interaction is characterized by a large number of close contacts between the ATP and RNA, and by a change in the conformation of the RNA.

  7. Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies

    PubMed Central

    Sharma, Amit; Leach, Robert N.; Gell, Christopher; Zhang, Nan; Burrows, Patricia C.; Shepherd, Dale A.; Wigneshweraraj, Sivaramesh; Smith, David Alastair; Zhang, Xiaodong; Buck, Martin; Stockley, Peter G.; Tuma, Roman

    2014-01-01

    Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ70 or σ54, that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ54 version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ70 and σ54, the domain movements of the latter have evolved to require an activator ATPase. PMID:24553251

  8. Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies.

    PubMed

    Sharma, Amit; Leach, Robert N; Gell, Christopher; Zhang, Nan; Burrows, Patricia C; Shepherd, Dale A; Wigneshweraraj, Sivaramesh; Smith, David Alastair; Zhang, Xiaodong; Buck, Martin; Stockley, Peter G; Tuma, Roman

    2014-04-01

    Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ(70) or σ(54), that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ(54) version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ(70) and σ(54), the domain movements of the latter have evolved to require an activator ATPase. PMID:24553251

  9. Laser-Assisted Single Molecule Refolding

    NASA Astrophysics Data System (ADS)

    Zhao, Rui; Marshall, Myles; Aleman, Elvin; Lamichhane, Rajan; Rueda, David

    2010-03-01

    In vivo, many RNA molecules can adopt multiple conformations depending on their biological context such as the HIV Dimerization Initiation Sequence (DIS) or the DsrA RNA in bacteria. It is quite common that the initial interaction between the two RNAs takes place via complementary unpaired regions, thus forming a so-called kissing complex. However, the exact kinetic mechanism by which the two RNA molecules reach the dimerized state is still not well understood. To investigate the refolding energy surface of RNA molecules, we have developed new technology based on the combination of single molecule spectroscopy with laser induced temperature jump kinetics, called Laser Assisted Single-molecule Refolding (LASR). LASR enables us to induce folding reactions of otherwise kinetically trapped RNAs at the single molecule level, and to characterize their folding landscape. LASR provides an exciting new approach to study molecular memory effects and kinetically trapped RNAs in general. LASR should be readily applicable to study DNA and protein folding as well.

  10. RNA helicases

    PubMed Central

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes. PMID:23093803

  11. Phenotypic MicroRNA Microarrays

    PubMed Central

    Kwon, Yong-Jun; Heo, Jin Yeong; Kim, Hi Chul; Kim, Jin Yeop; Liuzzi, Michel; Soloveva, Veronica

    2013-01-01

    Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models.

  12. Chemical evolution of life-like system under hydrothermal environments: prebiotic formation, degradation, and functions regarding protein-like molecules and RNA

    NASA Astrophysics Data System (ADS)

    Kawamura, Kunio

    The accumulation of biopolymers without enzymes is an essential step for the chemical evolu-tion towards a primitive life-like system. Previously, we discussed the relationship between the RNA world hypothesis and the hydrothermal origin of life hypothesis on the basis of the em-pirical data of RNA behaviors under the hydrothermal environments examined using real-time monitoring technique for hydrothermal reactions within the millisecond to second time scale. On the other hand, we have also examined the stabilities and behaviors of amino acids, pep-tides, and proteins under the hydrothermal environments. These observations have shown the possibility that oligopeptides could have been accumulated under near submarine hydrother-mal vent environments on primitive Earth within the relatively short time scale. However, the formation of oligopeptides under the simulated hydrothermal conditions is not so effective in the absence of catalysts and condensation agents. Thus, the investigation of the roles of min-eral catalysis and condensation reagents are very important since these materials could have enhanced efficiently the formation of peptides and stabilize primitive protein-like molecules. Recently, we investigated the roles of condensation reagents for the elongation of oligopeptides in the presence of minerals. In addition, we have designed a mineral-mediated hydrothermal flow reactor system (MHFR), which enables monitoring hydrothermal reactions in the presence of solid particles. By using MHFR, we attempted to examine naturally occurring minerals, such as apatite and quartz, for the elongation of oligopeptides at temperatures over 200 o C within 10 -30 sec. According to these data, the chemical evolution of protein-like molecules on primitive Earth will be discussed.

  13. The centrality of RNA for engineering gene expression

    PubMed Central

    Chappell, James; Takahashi, Melissa K; Meyer, Sarai; Loughrey, David; Watters, Kyle E; Lucks, Julius

    2013-01-01

    Synthetic biology holds promise as both a framework for rationally engineering biological systems and a way to revolutionize how we fundamentally understand them. Essential to realizing this promise is the development of strategies and tools to reliably and predictably control and characterize sophisticated patterns of gene expression. Here we review the role that RNA can play towards this goal and make a case for why this versatile, designable, and increasingly characterizable molecule is one of the most powerful substrates for engineering gene expression at our disposal. We discuss current natural and synthetic RNA regulators of gene expression acting at key points of control – transcription, mRNA degradation, and translation. We also consider RNA structural probing and computational RNA structure predication tools as a way to study RNA structure and ultimately function. Finally, we discuss how next-generation sequencing methods are being applied to the study of RNA and to the characterization of RNA's many properties throughout the cell. PMID:24124015

  14. Structural Alignment of RNA with Complex Pseudoknot Structure

    NASA Astrophysics Data System (ADS)

    Wong, Thomas K. F.; Lam, T. W.; Sung, Wing-Kin; Yiu, S. M.

    The secondary structure of an ncRNA molecule is known to play an important role in its biological functions. Aligning a known ncRNA to a target candidate to determine the sequence and structural similarity helps in identifying de novo ncRNA molecules that are in the same family of the known ncRNA. However, existing algorithms cannot handle complex pseudoknot structures which are found in nature. In this paper, we propose algorithms to handle two types of complex pseudoknots: simple non-standard pseudoknots and recursive pseudoknots. Although our methods are not designed for general pseudoknots, it already cover all known ncRNAs in both Rfam and PseudoBase databases. A preliminary evaluation on our algorithms show that it is useful to identify ncRNA molecules in other species which are in the same family of a known ncRNA.

  15. Characterization of RNA-Like Oligomers from Lipid-Assisted Nonenzymatic Synthesis: Implications for Origin of Informational Molecules on Early Earth.

    PubMed

    Mungi, Chaitanya V; Rajamani, Sudha

    2015-01-01

    Prebiotic polymerization had to be a nonenzymatic, chemically driven process. These processes would have been particularly favored in scenarios which push reaction regimes far from equilibrium. Dehydration-rehydration (DH-RH) cycles are one such regime thought to have been prevalent on prebiotic Earth in niches like volcanic geothermal pools. The present study defines the optimum DH-RH reaction conditions for lipid-assisted polymerization of nucleotides. The resultant products were characterized to understand their chemical makeup. Primarily, our study demonstrates that the resultant RNA-like oligomers have abasic sites, which means these oligomers lack information-carrying capability because of losing most of their bases during the reaction process. This results from low pH and high temperature conditions, which, importantly, also allows the formation of sugar-phosphate oligomers when ribose 5'-monophosphates are used as the starting monomers instead. Formation of such oligomers would have permitted sampling of a large variety of bases on a preformed polymer backbone, resulting in "prebiotic phosphodiester polymers" prior to the emergence of modern RNA-like molecules. This suggests that primitive genetic polymers could have utilized bases that conferred greater N-glycosyl bond stability, a feature crucial for information propagation in low pH and high temperature regimes of early Earth. PMID:25569237

  16. Targeting L1 cell adhesion molecule expression using liposome-encapsulated siRNA suppresses prostate cancer bone metastasis and growth

    PubMed Central

    Sung, Shian-Ying; Petros, John A.; Wu, Hsi-Chin; Zeng, Hong-Jie; Huang, Wei-Chien; Chung, Leland W. K.; Hsieh, Chia-Ling

    2014-01-01

    The L1 cell adhesion molecule (L1CAM) has been implicated in tumor progression of many types of cancers, but its role in prostate cancer and its application in targeted gene therapy have not been investigated. Herein, we demonstrated that the L1CAM was expressed in androgen-insensitive and highly metastatic human prostate cancer cell lines. The correlation between L1CAM expression and prostate cancer metastasis was also validated in serum samples of prostate cancer patients. Knockdown of L1CAM expression in prostate cancer cells by RNA interference significantly decreased their aggressive behaviors, including colony formation, migration and invasion in vitro, and tumor formation in a metastatic murine model. These anti-malignant phenotypes of L1CAM-knockdown cancer cells were accompanied by G0/G1 cell cycle arrest and suppression of matrix metalloproteinase (MMP)-2 and MMP-9 expression and nuclear factor NF-κB activation. In vivo targeting of L1CAM expression using liposome-encapsulated L1CAM siRNAs effectively inhibited prostate cancer growth in mouse bone, which was associated with decreased L1CAM expression and cell proliferation by tumor cells. These results provide the first evidence for L1CAM being a major contributor to prostate cancer metastasis and translational application of siRNA-based L1CAM-targeted therapy. PMID:25294816

  17. Toll-Like Receptor and Accessory Molecule mRNA Expression in Humans and Mice as Well as in Murine Autoimmunity, Transient Inflammation, and Progressive Fibrosis

    PubMed Central

    Ramaiah, Santhosh Kumar Vankayala; Günthner, Roman; Lech, Maciej; Anders, Hans-Joachim

    2013-01-01

    The cell type-, organ-, and species-specific expression of the Toll-like receptors (TLRs) are well described, but little is known about the respective expression profiles of their accessory molecules. We therefore determined the mRNA expression levels of LBP, MD2, CD36, CD14, granulin, HMGB1, LL37, GRP94, UNC93b1, TRIL, PRAT4A, AP3B1, AEP and the respective TLRs in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. In addition, the expression profiles in transient tissue inflammation upon renal ischemia-reperfusion injury, in spleens and kidneys from mice with lupus-like systemic autoimmunity, and in progressive tissue fibrosis upon unilateral ureteral obstruction were studied. Several TLR co-factors were specifically regulated during the different phases of these disease entities, suggesting a functional involvement in the disease process. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to TLR-mediated innate immunity, which seems to be involved in the tissue injury phase, in the phase of tissue regeneration, and in progressive tissue remodelling. PMID:23803655

  18. Characterization of RNA-Like Oligomers from Lipid-Assisted Nonenzymatic Synthesis: Implications for Origin of Informational Molecules on Early Earth

    PubMed Central

    Mungi, Chaitanya V.; Rajamani, Sudha

    2015-01-01

    Prebiotic polymerization had to be a nonenzymatic, chemically driven process. These processes would have been particularly favored in scenarios which push reaction regimes far from equilibrium. Dehydration-rehydration (DH-RH) cycles are one such regime thought to have been prevalent on prebiotic Earth in niches like volcanic geothermal pools. The present study defines the optimum DH-RH reaction conditions for lipid-assisted polymerization of nucleotides. The resultant products were characterized to understand their chemical makeup. Primarily, our study demonstrates that the resultant RNA-like oligomers have abasic sites, which means these oligomers lack information-carrying capability because of losing most of their bases during the reaction process. This results from low pH and high temperature conditions, which, importantly, also allows the formation of sugar-phosphate oligomers when ribose 5'-monophosphates are used as the starting monomers instead. Formation of such oligomers would have permitted sampling of a large variety of bases on a preformed polymer backbone, resulting in “prebiotic phosphodiester polymers” prior to the emergence of modern RNA-like molecules. This suggests that primitive genetic polymers could have utilized bases that conferred greater N-glycosyl bond stability, a feature crucial for information propagation in low pH and high temperature regimes of early Earth. PMID:25569237

  19. Systems biology approach to identify transcriptome reprogramming and candidate microRNA targets during the progression of polycystic kidney disease

    PubMed Central

    2011-01-01

    Background Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation throughout the kidney parenchyma. It is caused by mutations in either of two genes, PKD1 and PKD2. Mice that lack functional Pkd1 (Pkd1-/-), develop rapidly progressive cystic disease during embryogenesis, and serve as a model to study human ADPKD. Genome wide transcriptome reprogramming and the possible roles of micro-RNAs (miRNAs) that affect the initiation and progression of cyst formation in the Pkd1-/- have yet to be studied. miRNAs are small, regulatory non-coding RNAs, implicated in a wide spectrum of biological processes. Their expression levels are altered in several diseases including kidney cancer, diabetic nephropathy and PKD. Results We examined the molecular pathways that modulate renal cyst formation and growth in the Pkd1-/- model by performing global gene-expression profiling in embryonic kidneys at days 14.5 and 17.5. Gene Ontology and gene set enrichment analysis were used to identify overrepresented signaling pathways in Pkd1-/- kidneys. We found dysregulation of developmental, metabolic, and signaling pathways (e.g. Wnt, calcium, TGF-β and MAPK) in Pkd1-/- kidneys. Using a comparative transcriptomics approach, we determined similarities and differences with human ADPKD: ~50% overlap at the pathway level among the mis-regulated pathways was observed. By using computational approaches (TargetScan, miRanda, microT and miRDB), we then predicted miRNAs that were suggested to target the differentially expressed mRNAs. Differential expressions of 9 candidate miRNAs, miRs-10a, -30a-5p, -96, -126-5p, -182, -200a, -204, -429 and -488, and 16 genes were confirmed by qPCR. In addition, 14 candidate miRNA:mRNA reciprocal interactions were predicted. Several of the highly regulated genes and pathways were predicted as targets of miRNAs. Conclusions We have described global transcriptional reprogramming during the progression of PKD in the Pkd1-/- model. We

  20. Distinctive expression of extracellular matrix molecules at mRNA and protein levels during formation of cellular and acellular cementum in the rat.

    PubMed

    Sasano, Y; Maruya, Y; Sato, H; Zhu, J X; Takahashi, I; Mizoguchi, I; Kagayama, M

    2001-02-01

    Little is known about differential expression of extracellular matrices secreted by cementoblasts between cellular and acellular cementum. We hypothesize that cementoblasts lining acellular cementum express extracellular matrix genes differently from those lining cellular cementum, thereby forming two distinct types of extracellular matrices. To test this hypothesis, we investigated spatial and temporal gene expression of selected extracellular matrix molecules, that is type I collagen, bone sialoprotein, osteocalcin and osteopontin, during formation of both cellular and acellular cementum using in situ hybridization. In addition, their extracellularly deposited and accumulated proteins were examined immunohistochemically. The mRNA transcripts of pro-alpha1 (I) collagen were primarily localized in cementoblasts of cellular cementum and cementocytes, while those of bone sialoprotein were predominantly seen in cementoblasts lining acellular cementum. In contrast, osteocalcin was expressed by both types of cementoblasts and cementocytes and so was osteopontin but only transiently. Our immunohistochemical examination revealed that translated proteins were localized extracellularly where the genes had been expressed intracellularly. The present study demonstrated the distinctive expression of genes and proteins of the extracellular matrix molecules between cellular and acellular cementum. PMID:11432645

  1. A novel method developed for estimating mineralization efficiencies and its application in PC and PEC degradations of large molecule biological compounds with unknown chemical formula.

    PubMed

    Li, Guiying; Liu, Xiaolu; An, Taicheng; Wong, Po Keung; Zhao, Huijun

    2016-05-15

    A new method to estimate the photocatalytic (PC) and photoelectrocatalytic (PEC) mineralization efficiencies of large molecule biological compounds with unknown chemical formula in water was firstly developed and experimentally validated. The method employed chemical oxidation under the standard dichromate chemical oxygen demand (COD) conditions to obtain QCOD values of model compounds with unknown chemical formula. The measured QCOD values were used as the reference to replace QCOD values of model compounds for calculation of the mineralization efficiencies (in %) by assuming the obtained QCOD values are the measure of the theoretical charge required for the complete mineralization of organic pollutants. Total organic carbon (TOC) was also employed as a reference to confirm the mineralization capacity of dichromate chemical oxidation. The developed method was applied to determine the degradation extent of model compounds, such as bovine serum albumin (BSA), lecithin and bacterial DNA, by PC and PEC. Incomplete PC mineralization of all large molecule biological compounds was observed, especially for BSA. But the introduction of electrochemical technique into a PC oxidation process could profoundly improve the mineralization efficiencies of model compounds. PEC mineralization efficiencies of bacterial DNA was the highest, while that of lecithin was the lowest. Overall, PEC degradation method was found to be much effective than PC method for all large molecule biological compounds investigated, with PEC/PC mineralization ratios followed an order of BSA > lecithin > DNA. PMID:26994335

  2. Nanoscale imaging of RNA with expansion microscopy.

    PubMed

    Chen, Fei; Wassie, Asmamaw T; Cote, Allison J; Sinha, Anubhav; Alon, Shahar; Asano, Shoh; Daugharthy, Evan R; Chang, Jae-Byum; Marblestone, Adam; Church, George M; Raj, Arjun; Boyden, Edward S

    2016-08-01

    The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine. PMID:27376770

  3. Prognostic and Biological Significance of MicroRNA-127 Expression in Human Breast Cancer

    PubMed Central

    Wang, Shaohua; Li, Hanjun; Wang, Jingjie; Wang, Dan; Yao, Anlong; Li, Qiurong

    2014-01-01

    The purpose of this study was to determine the expression of miR-127 and analyze its prognostic and biological significance in breast cancer (BC). A quantitative reverse transcription PCR assay was performed to detect the expression of miR-127 in 15 pairs of BC and corresponding noncancerous tissues. The expression of miR-127 was detected in another 110 BC tissues and its correlations with clinicopathological factors of patients were examined. Univariate and multivariate analyses were performed to analyze the prognostic significance of miR-127 expression. The effects of miR-127 expression on malignant phenotypes of BC cells and its possible molecular mechanisms were further determined. miR-127 was significantly downregulated in BC tissues, and low miR-127 expression was significantly correlated with lymph node metastasis and advanced clinical stage. Patients with low miR-127 showed poorer overall survival than those with high miR-127. Multivariate analyses indicated that status of miR-127 was an independent prognostic factor for patients. Functional analyses showed that upregulation of miR-127 significantly inhibited growth, enhanced apoptosis, and reduced migration and invasion in BC cells by targeting the protooncogene BCL-6. Therefore, miR-127 may be a potential biomarker for predicting the survival of BC patients and might be a molecular target for treatment of human BCs. PMID:25477702

  4. Prebiologically Important Interstellar Molecules

    NASA Astrophysics Data System (ADS)

    Kuan, Y.-J.; Huang, H.-C.; Charnley, S. B.; Tseng, W.-L.; Snyder, L. E.; Ehrenfreund, P.; Kisiel, Z.; Thorwirth, S.; Bohn, R. K.; Wilson, T. L.

    2004-06-01

    Understanding the organic chemistry of molecular clouds, particularly the formation of biologically important molecules, is fundamental to the study of the processes which lead to the origin, evolution and distribution of life in the Galaxy. Determining the level of molecular complexity attainable in the clouds, and the nature of the complex organic material available to protostellar disks and the planetary systems that form from them, requires an understanding of the possible chemical pathways and is therefore a central question in astrochemistry. We have thus searched for prebiologically important molecules in the hot molecular cloud cores: Sgr B2(N-LMH), W51 e1/e2 and Orion-KL. Among the molecules searched: Pyrimidine is the unsubstituted ring analogue for three of the DNA and RNA bases. 2H-Azirine and Aziridine are azaheterocyclic compounds. And Glycine is the simplest amino acid. Detections of these interstellar organic molecular species will thus have important implications for Astrobiology. Our preliminary results indicate a tentative detection of interstellar glycine. If confirmed, this will be the first detection of an amino acid in interstellar space and will greatly strengthen the thesis that interstellar organic molecules could have played a pivotal role in the prebiotic chemistry of the early Earth.

  5. Hydration Structures and Thermodynamic Properties of Cationized Biologically Relevant Molecules, M+(Indole)(H2O)n (M = Na, K; n = 3-6)

    NASA Astrophysics Data System (ADS)

    Ke, Haochen; Lisy, James

    2015-03-01

    The balance between various noncovalent interactions plays a key role in determining the hydration structures and thermodynamic properties of biologically relevant molecules in biological mediums. Such properties of biologically relevant molecules are closely related to their often unique biological functionalities. The indole moiety is a basic functional group of many important neurotransmitters and hormones and has been used as tractable model for more complex biomolecules. The cationized indole water cluster is a perfect system for the quantitative and systematic study of the competition and cooperation of noncovalent interactions, as electrostatic interactions can be adjusted by introducing different monovalent cations and hydrogen bonding interactions can be adjusted by varying the level of hydration. IRPD spectra with isotopic (H/D) analysis helped unravel the overlapping N-H and O-H stretching modes, a major challenge of earlier studies. Thermodynamic analysis using relative Gibbs free energies, for energy ordering, together with spectral analysis provided unambiguous assignment of spectral features and structural configurations. A systematic hydration model with an in-depth account of noncovalent interactions is presented.

  6. Computational Design of a Circular RNA with Prionlike Behavior.

    PubMed

    Badelt, Stefan; Flamm, Christoph; Hofacker, Ivo L

    2016-01-01

    RNA molecules engineered to fold into predefined conformations have enabled the design of a multitude of functional RNA devices in the field of synthetic biology and nanotechnology. More complex designs require efficient computational methods, which need to consider not only equilibrium thermodynamics but also the kinetics of structure formation. Here we present a novel type of RNA design that mimics the behavior of prions, that is, sequences capable of interaction-triggered autocatalytic replication of conformations. Our design was computed with the ViennaRNA package and is based on circular RNA that embeds domains amenable to intermolecular kissing interactions. PMID:26934089

  7. [Relationships between microRNA expressions and prognosis in patients with tongue squamous cell carcinoma and the mechanisms microRNA regulating tongue squamous cell carcinoma biological behavior].

    PubMed

    Jia, Ling-fei; Gan, Ye-hua; Yu, Guang-yan

    2016-02-18

    Tongue squamous cell carcinoma (TSCC) is the most common type of oral cancer and is well known for its high rate of proliferation and lymph nodal metastasis. Exploring the underlying pathways regulating TSCC could provide novel ideas for diagnosis and prognosis of TSCC patients, as well as molecular targets for treatment of TSCC. MicroRNAs (miRNAs) are small noncoding RNAs that inhibit gene expression through the 3' untranslated regions (3'UTRs) of their target messenger RNAs. They play crucial roles in numerous biological processes, including cancer progression. Although great efforts have been made, what role miRNAs may play in the early detection and diagnosis of TSCC is not fully understood. Recently, our team has performed a series of basic and clinical researches in an attempt to investigate the relationships between miRNA expressions and prognosis of patients with TSCC and the mechanisms under regulation of TSCC. The results showed that miR-195, miR-34a, miR-29b, miR-375 and miR-26a could inhibit TSCC cells progression and development via a sophisticated network of genes. Specifically, the anti-tumor effects of miR-195 in TSCC may be partially mediated by its inhibition of CyclinD1 and Bcl-2 expression. The expression of miR-34a could inhibit migration and invasion of TSCC cell lines via targeting MMP9 and MMP14. The function of miR-29b may be through the miR-29b/Sp1/PTEN/AKT axis. Overexpression of miR-375 inhibited Sp1 expression by targeting the 3' untranslated region of the Sp1 transcript. MEG3 and miR-26a inhibited TSCC cell proliferation, cycle progression and promoted cell apoptosis and miR-26a could increase the MEG3 expression through reduction of the expression of DNMT3B in TSCC. In light of the role of those miRNAs in diagnosis and prognosis of TSCC, we reported that decreased miR-195 and miR-375 expression was associated with poor overall survival rate of the TSCC patients, while miR-34a expression was negatively correlated with cervical lymph

  8. Electron Collisions with Large Molecules

    NASA Astrophysics Data System (ADS)

    McKoy, Vincent

    2006-10-01

    In recent years, interest in electron-molecule collisions has increasingly shifted to large molecules. Applications within the semiconductor industry, for example, require electron collision data for molecules such as perfluorocyclobutane, while almost all biological applications involve macromolecules such as DNA. A significant development in recent years has been the realization that slow electrons can directly damage DNA. This discovery has spurred studies of low-energy collisions with the constituents of DNA, including the bases, deoxyribose, the phosphate, and larger moieties assembled from them. In semiconductor applications, a key goal is development of electron cross section sets for plasma chemistry modeling, while biological studies are largely focused on understanding the role of localized resonances in inducing DNA strand breaks. Accurate calculations of low-energy electron collisions with polyatomic molecules are computationally demanding because of the low symmetry and inherent many-electron nature of the problem; moreover, the computational requirements scale rapidly with the size of the molecule. To pursue such studies, we have adapted our computational procedure, known as the Schwinger multichannel method, to run efficiently on highly parallel computers. In this talk, we will present some of our recent results for fluorocarbon etchants used in the semiconductor industry and for constituents of DNA and RNA. In collaboration with Carl Winstead, California Institute of Technology.

  9. Dysregulated co-stimulatory molecule expression in a Sjögren's syndrome mouse model with potential implications by microRNA-146a.

    PubMed

    Gauna, Adrienne E; Park, Yun-Jong; Nayar, Gautam; Onate, Marelys; Jin, Jun-o; Stewart, Carol M; Yu, Qing; Cha, Seunghee

    2015-12-01

    Sjögren's syndrome (SjS) is an autoimmune condition that primarily affects salivary and lacrimal glands, causing loss of secretion. We have previously shown that microRNA-146a (miR-146a) is over-expressed in the salivary glands and peripheral blood mononuclear cells (PBMC) of SjS-prone mice (C57BL/6.NOD-Aec1Aec2, B6DC) and in PBMC of SjS patients. The purpose of this research was to identify a target molecule of miR-146a and identify subpopulations of cells affected by altered miR-146a in the salivary glands of SjS-prone mice. In silico analyses identified costimulatory molecule CD80 as a potential target of miR-146a. Luciferase assay of the human CD80 3'untranslated region demonstrated miR-146a directly inhibited CD80 protein expression as indicated by reduced luciferase reporter expression and an examination of B6DC salivary glands revealed a reduction in CD80 protein. More interestingly, the specific reduction in CD80 protein was detected from the salivary gland epithelial cell population and in interstitial dendritic cells in the glands as well. The reduction in CD80 protein levels in salivary gland epithelial cells were negatively associated with elevated miR-146a expression. Therefore, this study provides the first indication that salivary gland epithelial cells may be critically involved in SjS progression by altering CD86:CD80 protein ratio in response to miR-146a upregulation. PMID:26505653

  10. Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy

    PubMed Central

    Sun, Shiyu; Zhao, Guangxu; Huang, Yibing; Cai, Mingjun; Shan, Yuping; Wang, Hongda; Chen, Yuxin

    2016-01-01

    In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers. PMID:27363513

  11. Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy.

    PubMed

    Sun, Shiyu; Zhao, Guangxu; Huang, Yibing; Cai, Mingjun; Shan, Yuping; Wang, Hongda; Chen, Yuxin

    2016-01-01

    In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers. PMID:27363513

  12. RNA editing, epitranscriptomics, and processing in cancer progression

    PubMed Central

    Witkin, Keren L; Hanlon, Sean E; Strasburger, Jennifer A; Coffin, John M; Jaffrey, Samie R; Howcroft, T Kevin; Dedon, Peter C; Steitz, Joan A; Daschner, Phil J; Read-Connole, Elizabeth

    2015-01-01

    The transcriptome is extensively and dynamically regulated by a network of RNA modifying factors. RNA editing enzymes APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) and ADAR (adenosine deaminase, RNA-specific) irreversibly recode primary RNA sequences, whereas newly described methylases (writers) and de-methylases (erasers) dynamically alter RNA molecules in response to environmental conditions. RNA modifications can affect RNA splicing, nuclear-cytoplasmic transport, translation, and regulation of gene expression by RNA interference. In addition, tRNA base modifications, processing, and regulated cleavage have been shown to alter global patterns of mRNA translation in response to cellular stress pathways. Recent studies, some of which were discussed at this workshop, have rekindled interest in the emerging roles of RNA modifications in health and disease. On September 10th, 2014, the Division of Cancer Biology, NCI sponsored a workshop to explore the role of epitranscriptomic RNA modifications and tRNA processing in cancer progression. The workshop attendees spanned a scientific range including chemists, virologists, and RNA and cancer biologists. The goal of the workshop was to explore the interrelationships between RNA editing, epitranscriptomics, and RNA processing and the enzymatic pathways that regulate these activities in cancer initiation and progression. At the conclusion of the workshop, a general discussion focused on defining the major challenges and opportunities in this field, as well as identifying the tools, technologies, resources and community efforts required to accelerate research in this emerging area. PMID:25455629

  13. Human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation mediated by vascular cell adhesion molecule-1: evidence for involvement of cell adhesion molecules in HTLV-1 biology.

    PubMed Central

    Hildreth, J E; Subramanium, A; Hampton, R A

    1997-01-01

    While studying the potential role of vascular cell adhesion molecule-1 (VCAM-1) in infection of endothelial cells by human immunodeficiency virus (HIV), we found that VCAM-1 can mediate human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation. Both expression-vector-encoded and endogenously expressed VCAM-1 supported fusion of uninfected cells with HTLV-1-infected cells. Fusion was obtained with cell lines carrying the HTLV-1 genome and expressing viral proteins but not with an HTLV-1-transformed cell line that does not express viral proteins. In clones of VCAM-1-transfected cells, the degree of syncytium formation observed directly reflected the level of VCAM-1 expression. Syncytium formation between HTLV-1-expressing cells and VCAM-1+ cells could be blocked with antiserum against HTLV-1 gp46 and with a monoclonal antibody (MAb) against VCAM-1. Fusion was not blocked by antiserum against HIV or a MAb against VLA-4, the physiological counter-receptor for VCAM-1. The results indicate that VCAM-1 can serve as an accessory molecule or potential coreceptor for HTLV-1-induced cell fusion and provide direct evidence of a role for cell adhesion molecules in the biology of HTLV-1. PMID:8995639

  14. Isothermal titration calorimetry of RNA.

    PubMed

    Salim, Nilshad N; Feig, Andrew L

    2009-03-01

    Isothermal titration calorimetry (ITC) is a fast and robust method to study the physical basis of molecular interactions. A single well-designed experiment can provide complete thermodynamic characterization of a binding reaction, including K(a), DeltaG, DeltaH, DeltaS and reaction stoichiometry (n). Repeating the experiment at different temperatures allows determination of the heat capacity change (DeltaC(P)) of the interaction. Modern calorimeters are sensitive enough to probe even weak biological interactions making ITC a very popular method among biochemists. Although ITC has been applied to protein studies for many years, it is becoming widely applicable in RNA biochemistry as well, especially in studies which involve RNA folding and RNA interactions with small molecules, proteins and with other RNAs. This review focuses on best practices for planning, designing and executing effective ITC experiments when one or more of the reactants is an RNA. PMID:18835447

  15. Regulatory effects of cotranscriptional RNA structure formation and transitions.

    PubMed

    Liu, Sheng-Rui; Hu, Chun-Gen; Zhang, Jin-Zhi

    2016-09-01

    RNAs, which play significant roles in many fundamental biological processes of life, fold into sophisticated and precise structures. RNA folding is a dynamic and intricate process, which conformation transition of coding and noncoding RNAs form the primary elements of genetic regulation. The cellular environment contains various intrinsic and extrinsic factors that potentially affect RNA folding in vivo, and experimental and theoretical evidence increasingly indicates that the highly flexible features of the RNA structure are affected by these factors, which include the flanking sequence context, physiochemical conditions, cis RNA-RNA interactions, and RNA interactions with other molecules. Furthermore, distinct RNA structures have been identified that govern almost all steps of biological processes in cells, including transcriptional activation and termination, transcriptional mutagenesis, 5'-capping, splicing, 3'-polyadenylation, mRNA export and localization, and translation. Here, we briefly summarize the dynamic and complex features of RNA folding along with a wide variety of intrinsic and extrinsic factors that affect RNA folding. We then provide several examples to elaborate RNA structure-mediated regulation at the transcriptional and posttranscriptional levels. Finally, we illustrate the regulatory roles of RNA structure and discuss advances pertaining to RNA structure in plants. WIREs RNA 2016, 7:562-574. doi: 10.1002/wrna.1350 For further resources related to this article, please visit the WIREs website. PMID:27028291

  16. Signal transduction pathways mediated by PECAM-1: new roles for an old molecule in platelet and vascular cell biology.

    PubMed

    Newman, Peter J; Newman, Debra K

    2003-06-01

    Recent studies of platelet endothelial cell adhesion molecule-1 (PECAM-1 [CD31])-deficient mice have revealed that this molecule plays an important role in controlling the activation and survival of cells on which it is expressed. In this review, we focus on the complex cytoplasmic domain of PECAM-1 and describe what is presently known about its structure, posttranslational modifications, and binding partners. In addition, we summarize findings that implicate PECAM-1 as an inhibitor of cellular activation via protein tyrosine kinase-dependent signaling pathways, an activator of integrins, and a suppressor of cell death via pathways that depend on damage to the mitochondria. The challenge of future research will be to bridge our understanding of the functional and biochemical properties of PECAM-1 by establishing mechanistic links between signals transduced by the PECAM-1 cytoplasmic domain and discrete cellular responses. PMID:12689916

  17. 1H NMR analysis of complexation of hydrotropic agents nicotinamide and caffeine with aromatic biologically active molecules in aqueous solution

    NASA Astrophysics Data System (ADS)

    Lantushenko, Anastasia O.; Mukhina, Yulia V.; Veselkov, Kyrill A.; Davies, David B.; Veselkov, Alexei N.

    2004-07-01

    NMR spectroscopy has been used to elucidate the molecular mechanism of solubilization action of hydrotropic agents nicotinamide (NA) and caffeine (CAF). Hetero-association of NA with riboflavine-mononucleotide (FMN) and CAF with low soluble in aqueous solution synthetic analogue of antibiotic actinomycin D, actinocyl-bis-(3-dimethylaminopropyl) amine (Actill), has been investigated by 500 MHz 1H NMR spectroscopy. Concentration and temperature dependences of proton chemical shifts have been analysed in terms of a statistical-thermodynamic model of indefinite self- and heteroassociation of aromatic molecules. The obtained results enable to conclude that NA-FMN and CAF-Actill intermolecular complexes are mainly stabilized by the stacking interactions of the aromatic chromophores. Hetero-association of the investigated molecules plays an important role in solubilization of aromatic drugs by hydrotropic agents nicotinamide and caffeine.

  18. Interaction of biological molecules with clay minerals: a combined spectroscopic and sorption study of lysozyme on saponite.

    PubMed

    Johnston, Cliff T; Premachandra, Gnanasiri S; Szabo, Tamas; Lok, Joyce; Schoonheydt, Robert A

    2012-01-10

    The interaction of hen egg white lysozyme (HEWL) with Na- and Cs-exchanged saponite was investigated using sorption, structural, and spectroscopic methods as a model system to study clay-protein interactions. HEWL sorption to Na- and Cs-saponite was determined using the bicinchoninic acid (BCA) assay, thermogravimetric analysis, and C and N analysis. For Na-saponite, the TGA and elemental analysis-derived sorption maximum was 600 mg/g corresponding to a surface coverage of 0.85 ng/mm(2) with HEWL occupying 526 m(2)/g based on a cross-sectional area of 13.5 nm(2)/molecule. HEWL sorption on Na-saponite was accompanied by the release of 9.5 Na(+) ions for every molecule of HEWL sorbed consistent with an ion exchange mechanism between the positively charged HEWL (IEP 11) and the negatively charged saponite surface. The d-spacing of the HEWL-Na-saponite complex increased to a value of 4.4 nm consistent with the crystallographic dimensions of HEWL of 3 × 3 × 4.5 nm. In the case of Cs-saponite, there was no evidence of interlayer sorption; however, sorption of HEWL to the "external" surface of Cs-saponite showed a high affinity isotherm. FTIR and Raman analysis of the amide I region of the HEWL-saponite films prepared from water and D(2)O showed little perturbation to the secondary structure of the protein. The overall hydrophilic nature of the HEWL-Na-saponite complex was determined by water vapor sorption measurements. The clay retained its hydrophilic character with a water content of 18% at high humidity corresponding to 240 H(2)O molecules per molecule of HEWL. PMID:22047516

  19. Micromanipulation system for handling of biological molecule and screening of microbes in a microchannel by electric field and laser tweezers

    NASA Astrophysics Data System (ADS)

    Morishima, Keisuke; Arai, Fumihito; Fukuda, Toshio; Matsuura, Hideo; Yoshikawa, Kenichi

    1998-09-01

    In this paper, we propose a novel methodology on noncontact transportation of DNA molecules by dielectrophoretic force and high throughput screening of microbes. First, we utilize the conformational transition in the higher order structure of DNA for transportation. We designed a simple micro electrode-flow system. Experimental demonstration of DNA transportation in the globule state using dielectrophoretic force and direct observation of the DNA molecule in a non- uniform electric field were carried out with fluorescence microscopy. We discuss the experimental results on the motion of the DNA molecule. We show that transportation of DNA with the state of compacted globule is profitable in the future practical application for the separation of giant DNAs such as human gene. Next, we have developed a prototype of Microchannel system for high throughput screening of Escherichia coli. Experimental demonstration of noncontact transportation and manipulation of Escherichia coli by dielectrophoretic force and radiation pressure of laser tweezers were carried out with laser manipulator system. We discussed the basic strategies to improve the working efficiency and the operability of the micromanipulation and presented a new direction in this field. In experiments, we show that transportation and separation of E. coli cells by dielectrophoretic force and optical trapping is useful for future practical application to the high throughput screening of microbes. We showed the possibility of the Microchannel system as one of the biomanipulation and automation systems for DNA sequencing and pharmaceutical field.

  20. Analysis of the landscape of biologically-derived pharmaceuticals in Europe: dominant production systems, molecule types on the rise and approval trends.

    PubMed

    Kyriakopoulos, Sarantos; Kontoravdi, Cleo

    2013-02-14

    A thorough sort of the human drugs approved by the European Medicines Agency (EMA) between its establishment in 1995 until June 2012 is presented herein with a focus on biologically-derived pharmaceuticals. Over 200 (33%) of the 640 approved therapeutic drugs are derived from natural sources, produced via recombinant DNA technology, or generated through virus propagation. A breakdown based on production method, type of molecule and therapeutic category is presented. Current EMA approvals demonstrate that mammalian cells are the only choice for glycoprotein drugs, with Chinese hamster ovary cells being the dominant hosts for their production. On the other hand, bacterial cells and specifically Escherichia coli are the dominant hosts for protein-based drugs, followed by the yeast Saccharomyces cerevisiae. The latter is the dominant host for recombinant vaccine production, although egg-based production is still the main platform of vaccine provision. Our findings suggest that the majority of biologically-derived drugs are prescribed for cancer and related conditions, as well as the treatment of diabetes. The approval rate for biologically-derived drugs shows a strong upward trend for monoclonal antibodies and fusion proteins since 2009, while hormones, antibodies and growth factors remain the most populous categories. Despite a clear pathway for the approval of biosimilars set by the EMA and their potential to drive sales growth, we have only found approved biosimilars for three molecules. In 2012 there appears to be a slow-down in approvals, which coincides with a reported decline in the growth rate of biologics sales. PMID:23262060

  1. Arrest of human mitochondrial RNA polymerase transcription by the biological aldehyde adduct of DNA, M1dG

    PubMed Central

    Cline, Susan D.; Lodeiro, M. Fernanda; Marnett, Lawrence J.; Cameron, Craig E.; Arnold, Jamie J.

    2010-01-01

    The biological aldehydes, malondialdehyde and base propenal, react with DNA to form a prevalent guanine adduct, M1dG. The exocyclic ring of M1dG opens to the acyclic N2-OPdG structure when paired with C but remains closed in single-stranded DNA or when mispaired with T. M1dG is a target of nucleotide excision repair (NER); however, NER is absent in mitochondria. An in vitro transcription system with purified human mitochondrial RNA polymerase (POLRMT) and transcription factors, mtTFA and mtTFB2, was used to determine the effect of M1dG on POLRMT elongation. DNA templates contained a single adduct opposite either C or T downstream of either the light-strand (LSP) or heavy-strand (HSP1) promoter for POLRMT. M1dG in the transcribed strand arrested 60–90% POLRMT elongation complexes with greater arrest by the adduct when opposite T. POLRMT was more sensitive to N2-OPdG and M1dG after initiation at LSP, which suggests promoter-specific differences in the function of POLRMT complexes. A closed-ring analog of M1dG, PdG, blocked ≥95% of transcripts originating from either promoter regardless of base pairing, and the transcripts remained associated with POLRMT complexes after stalling at the adduct. This work suggests that persistent M1dG adducts in mitochondrial DNA hinder the transcription of mitochondrial genes. PMID:20671026

  2. DARWIN REVIEW: microRNA, seeds and Darwin? – Diverse Function of miRNA in Seed Biology and Plant Responses to Stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MicroRNAs (miRNAs) are small, single-stranded RNAs that downregulate target genes at the post-transcriptional level. miRNAs regulate target genes by guiding mRNA cleavage or by repressing translation. miRNAs play crucial roles in a broad range of developmental processes in plants. Multiple miRNAs ar...

  3. RSRE: RNA structural robustness evaluator.

    PubMed

    Shu, Wenjie; Bo, Xiaochen; Zheng, Zhiqiang; Wang, Shengqi

    2007-07-01

    Biological robustness, defined as the ability to maintain stable functioning in the face of various perturbations, is an important and fundamental topic in current biology, and has become a focus of numerous studies in recent years. Although structural robustness has been explored in several types of RNA molecules, the origins of robustness are still controversial. Computational analysis results are needed to make up for the lack of evidence of robustness in natural biological systems. The RNA structural robustness evaluator (RSRE) web server presented here provides a freely available online tool to quantitatively evaluate the structural robustness of RNA based on the widely accepted definition of neutrality. Several classical structure comparison methods are employed; five randomization methods are implemented to generate control sequences; sub-optimal predicted structures can be optionally utilized to mitigate the uncertainty of secondary structure prediction. With a user-friendly interface, the web application is easy to use. Intuitive illustrations are provided along with the original computational results to facilitate analysis. The RSRE will be helpful in the wide exploration of RNA structural robustness and will catalyze our understanding of RNA evolution. The RSRE web server is freely available at http://biosrv1.bmi.ac.cn/RSRE/ or http://biotech.bmi.ac.cn/RSRE/. PMID:17567615

  4. Serratia Secondary Metabolite Prodigiosin Inhibits Pseudomonas aeruginosa Biofilm Development by Producing Reactive Oxygen Species that Damage Biological Molecules

    PubMed Central

    Kimyon, Önder; Das, Theerthankar; Ibugo, Amaye I.; Kutty, Samuel K.; Ho, Kitty K.; Tebben, Jan; Kumar, Naresh; Manefield, Mike

    2016-01-01

    Prodigiosin is a heterocyclic bacterial secondary metabolite belonging to the class of tripyrrole compounds, synthesized by various types of bacteria including Serratia species. Prodigiosin has been the subject of intense research over the last decade for its ability to induce apoptosis in several cancer cell lines. Reports suggest that prodigiosin promotes oxidative damage to double-stranded DNA (dsDNA) in the presence of copper ions and consequently leads to inhibition of cell-cycle progression and cell death. However, prodigiosin has not been previously implicated in biofilm inhibition. In this study, the link between prodigiosin and biofilm inhibition through the production of redox active metabolites is presented. Our study showed that prodigiosin (500 μM) (extracted from Serratia marcescens culture) and a prodigiosin/copper(II) (100 μM each) complex have strong RNA and dsDNA cleaving properties while they have no pronounced effect on protein. Results support a role for oxidative damage to biomolecules by H2O2 and hydroxyl radical generation. Further, it was demonstrated that reactive oxygen species scavengers significantly reduced the DNA and RNA cleaving property of prodigiosin. P. aeruginosa cell surface hydrophobicity and biofilm integrity were significantly altered due to the cleavage of nucleic acids by prodigiosin or the prodigiosin/copper(II) complex. In addition, prodigiosin also facilitated the bactericidal activity. The ability of prodigiosinto cause nucleic acid degradation offers novel opportunities to interfere with extracellular DNA dependent bacterial biofilms. PMID:27446013

  5. Serratia Secondary Metabolite Prodigiosin Inhibits Pseudomonas aeruginosa Biofilm Development by Producing Reactive Oxygen Species that Damage Biological Molecules.

    PubMed

    Kimyon, Önder; Das, Theerthankar; Ibugo, Amaye I; Kutty, Samuel K; Ho, Kitty K; Tebben, Jan; Kumar, Naresh; Manefield, Mike

    2016-01-01

    Prodigiosin is a heterocyclic bacterial secondary metabolite belonging to the class of tripyrrole compounds, synthesized by various types of bacteria including Serratia species. Prodigiosin has been the subject of intense research over the last decade for its ability to induce apoptosis in several cancer cell lines. Reports suggest that prodigiosin promotes oxidative damage to double-stranded DNA (dsDNA) in the presence of copper ions and consequently leads to inhibition of cell-cycle progression and cell death. However, prodigiosin has not been previously implicated in biofilm inhibition. In this study, the link between prodigiosin and biofilm inhibition through the production of redox active metabolites is presented. Our study showed that prodigiosin (500 μM) (extracted from Serratia marcescens culture) and a prodigiosin/copper(II) (100 μM each) complex have strong RNA and dsDNA cleaving properties while they have no pronounced effect on protein. Results support a role for oxidative damage to biomolecules by H2O2 and hydroxyl radical generation. Further, it was demonstrated that reactive oxygen species scavengers significantly reduced the DNA and RNA cleaving property of prodigiosin. P. aeruginosa cell surface hydrophobicity and biofilm integrity were significantly altered due to the cleavage of nucleic acids by prodigiosin or the prodigiosin/copper(II) complex. In addition, prodigiosin also facilitated the bactericidal activity. The ability of prodigiosinto cause nucleic acid degradation offers novel opportunities to interfere with extracellular DNA dependent bacterial biofilms. PMID:27446013

  6. Molecular dynamics simulations of solvated yeast tRNA(Asp).

    PubMed Central

    Auffinger, P; Louise-May, S; Westhof, E

    1999-01-01

    Transfer RNA molecules are involved in a variety of biological processes, implying complex recognition events with proteins and other RNAs. From a structural point of view, tRNAs constitute a reference system for studying RNA folding and architecture. A deeper understanding of their structural and functional properties will derive from our ability to model accurately their dynamical behavior. We present the first dynamical model of a fully neutralized and solvated tRNA molecule over a 500-ps time scale. Starting from the crystallographic structure of yeast tRNA(Asp), the 75-nucleotide molecule was modeled with 8055 water molecules and 74 NH4+ counterions, using the AMBER4.1 program and the particle mesh Ewald (PME) method for the treatment of long-range electrostatic interactions. The calculations led to a dynamically stable model of the tRNA molecule. During the simulation, all secondary and tertiary base pairs are maintained while a certain lability of base triples in the tRNA core is observed. This lability was interpreted as resulting from intrinsic factors associated with the "weaker" hydrogen bonding patterns seen in these base triples and from an altered ionic environment of the tRNA molecule. Calculated thermal factors are used to compare the dynamics of the tRNA in solution and in the crystal. The present molecular dynamics simulation of a complex and highly charged nucleic acid molecule attests to the fact that simulation methods are now able to investigate not only the dynamics of proteins, but also that of large RNA molecules. Thus they also provide a basis for further investigations on the structural and functional effects of chemical and posttranscriptionally modified nucleotides as well as on ionic environmental effects. PMID:9876122

  7. Biologic activity of the novel small molecule STAT3 inhibitor LLL12 against canine osteosarcoma cell lines

    PubMed Central

    2012-01-01

    Background STAT3 [1] has been shown to be dysregulated in nearly every major cancer, including osteosarcoma (OS). Constitutive activation of STAT3, via aberrant phosphorylation, leads to proliferation, cell survival and resistance to apoptosis. The present study sought to characterize the biologic activity of a novel allosteric STAT3 inhibitor, LLL12, in canine OS cell lines. Results We evaluated the effects of LLL12 treatment on 4 canine OS cell lines and found that LLL12 inhibited proliferation, induced apoptosis, reduced STAT3 phosphorylation, and decreased the expression of several transcriptional targets of STAT3 in these cells. Lastly, LLL12 exhibited synergistic anti-proliferative activity with the chemotherapeutic doxorubicin in the OS lines. Conclusion LLL12 exhibits biologic activity against canine OS cell lines through inhibition of STAT3 related cellular functions supporting its potential use as a novel therapy for OS. PMID:23244668

  8. Charge density of the biologically active molecule (2-oxo-1,3-benzoxazol-3(2H)-yl)acetic acid.

    PubMed

    Wang, Ai; Ashurov, Jamshid; Ibragimov, Aziz; Wang, Ruimin; Mouhib, Halima; Mukhamedov, Nasir; Englert, Ulli

    2016-02-01

    (2-Oxo-1,3-benzoxazol-3(2H)-yl)acetic acid is a member of a biologically active class of compounds. Its molecular structure in the crystal has been determined by X-ray diffraction, and its gas phase structure was obtained by quantum chemical calculations at the B3LYP/6-311++G(d,p) level of theory. In order to understand the dynamics of the molecule, two presumably soft degrees of freedom associated with the relative orientation of the planar benzoxazolone system and its substituent at the N atom were varied systematically. Five conformers have been identified as local minima on the resulting two-dimensional potential energy surface within an energy window of 27 kJ mol(-1). The energetically most favourable minimum closely matches the conformation observed in the crystal. Based on high-resolution diffraction data collected at low temperature, the experimental electron density of the compound was determined. Comparison with the electron density established by theory for the isolated molecule allowed the effect of intermolecular interactions to be addressed, in particular a moderately strong O-H...O hydrogen bond with a donor...acceptor distance of 2.6177 (9) Å: the oxygen acceptor is clearly polarized in the extended solid. The hydrogen bond connects consecutive molecules to chains, and the pronounced charge separation leads to stacking between neighburs with antiparallel dipole moments perpendicular to the chain direction. PMID:26830806

  9. Revealing G-protein-coupled receptor oligomerization at the single-molecule level through a nanoscopic lens: methods, dynamics and biological function.

    PubMed

    Scarselli, Marco; Annibale, Paolo; McCormick, Peter J; Kolachalam, Shivakumar; Aringhieri, Stefano; Radenovic, Aleksandra; Corsini, Giovanni U; Maggio, Roberto

    2016-04-01

    The introduction of super-resolution fluorescence microscopy has allowed the visualization of single proteins in their biological environment. Recently, these techniques have been applied to determine the organization of class A G-protein-coupled receptors (GPCRs), and to determine whether they exist as monomers, dimers and/or higher-order oligomers. On this subject, this review highlights recent evidence from photoactivated localization microscopy (PALM), which allows the visualization of single molecules in dense samples, and single-molecule tracking (SMT), which determines how GPCRs move and interact in living cells in the presence of different ligands. PALM has demonstrated that GPCR oligomerization depends on the receptor subtype, the cell type, the actin cytoskeleton, and other proteins. Conversely, SMT has revealed the transient dynamics of dimer formation, whereby receptors show a monomer-dimer equilibrium characterized by rapid association and dissociation. At steady state, depending on the subtype, approximately 30-50% of receptors are part of dimeric complexes. Notably, the existence of many GPCR dimers/oligomers is also supported by well-known techniques, such as resonance energy transfer methodologies, and by approaches that exploit fluorescence fluctuations, such as fluorescence correlation spectroscopy (FCS). Future research using single-molecule methods will deepen our knowledge related to the function and druggability of homo-oligomers and hetero-oligomers. PMID:26509747

  10. Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines.

    PubMed

    Squires, Matthew S; Feltell, Ruth E; Wallis, Nicola G; Lewis, E Jonathan; Smith, Donna-Michelle; Cross, David M; Lyons, John F; Thompson, Neil T

    2009-02-01

    Cyclin-dependent kinases (CDK), and their regulatory cyclin partners, play a central role in eukaryotic cell growth, division, and death. This key role in cell cycle progression, as well as their deregulation in several human cancers, makes them attractive therapeutic targets in oncology. A series of CDK inhibitors was developed using Astex's fragment-based medicinal chemistry approach, linked to high-throughput X-ray crystallography. A compound from this series, designated AT7519, is currently in early-phase clinical development. We describe here the biological characterization of AT7519, a potent inhibitor of several CDK family members. AT7519 showed potent antiproliferative activity (40-940 nmol/L) in a panel of human tumor cell lines, and the mechanism of action was shown here to be consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines. AT7519 caused cell cycle arrest followed by apoptosis in human tumor cells and inhibited tumor growth in human tumor xenograft models. Tumor regression was observed following twice daily dosing of AT7519 in the HCT116 and HT29 colon cancer xenograft models. We show that these biological effects are linked to inhibition of CDKs in vivo and that AT7519 induces tumor cell apoptosis in these xenograft models. AT7519 has an attractive biological profile for development as a clinical candidate, and the tolerability and efficacy in animal models compare favorably with other CDK inhibitors in clinical development. Studies described here formed the biological rationale for investigating the potential therapeutic benefit of AT7519 in cancer patients. PMID:19174555

  11. The AstroBiology Explorer (ABE) MIDEX Mission: Using Infrared Spectroscopy to Identify Organic Molecules in Space

    NASA Technical Reports Server (NTRS)

    Sandford, S. A.

    2002-01-01

    The AstroBiology Explorer (ABE) mission is one of four selected for Phase A Concept Study in NASA's current call for MIDEX class missions. ABE is a cooled space telescope equipped with spectrographs covering the 2.5-20 micron spectral range. The ABE mission is devoted to the detection and identification of organic and related molecular species in space. ABE is currently under study at NASA's Ames Research Center in collaboration with Ball Aerospace.

  12. RNA Structures as Mediators of Neurological Diseases and as Drug Targets.

    PubMed

    Bernat, Viachaslau; Disney, Matthew D

    2015-07-01

    RNAs adopt diverse folded structures that are essential for function and thus play critical roles in cellular biology. A striking example of this is the ribosome, a complex, three-dimensionally folded macromolecular machine that orchestrates protein synthesis. Advances in RNA biochemistry, structural and molecular biology, and bioinformatics have revealed other non-coding RNAs whose functions are dictated by their structure. It is not surprising that aberrantly folded RNA structures contribute to disease. In this Review, we provide a brief introduction into RNA structural biology and then describe how RNA structures function in cells and cause or contribute to neurological disease. Finally, we highlight successful applications of rational design principles to provide chemical probes and lead compounds targeting structured RNAs. Based on several examples of well-characterized RNA-driven neurological disorders, we demonstrate how designed small molecules can facilitate the study of RNA dysfunction, elucidating previously unknown roles for RNA in disease, and provide lead therapeutics. PMID:26139368

  13. RNA structures as mediators of neurological diseases and as drug targets

    PubMed Central

    Bernat, Viachaslau; Disney, Matthew D.

    2015-01-01

    RNAs adopt diverse folded structures that are essential for function and thus play critical roles in cellular biology. A striking example of this is the ribosome, a complex, three-dimensionally folded macromolecular machine that orchestrates protein synthesis. Advances in RNA biochemistry, structural and molecular biology, and bioinformatics have revealed other non-coding RNAs whose functions are dictated by their structure. It is not surprising that aberrantly folded RNA structures contribute to disease. In this review, we provide a brief introduction into RNA structural biology and then describe how RNA structures function in cells and cause or contribute to neurological disease. Finally, we highlight successful applications of rational design principles to provide chemical probes and lead compounds targeting structured RNAs. Based on several examples of well-characterized RNA-driven neurological disorders, we demonstrate how designed small molecules can facilitate study of RNA dysfunction, elucidating previously unknown roles for RNA in disease, and provide lead therapeutics. PMID:26139368

  14. Label-Free Nanoplasmonic-Based Short Noncoding RNA Sensing at Attomolar Concentrations Allows for Quantitative and Highly Specific Assay of MicroRNA-10b in Biological Fluids and Circulating Exosomes

    PubMed Central

    2015-01-01

    MicroRNAs are short noncoding RNAs consisting of 18–25 nucleotides that target specific mRNA moieties for translational repression or degradation, thereby modulating numerous biological processes. Although microRNAs have the ability to behave like oncogenes or tumor suppressors in a cell-autonomous manner, their exact roles following release into the circulation are only now being unraveled and it is important to establish sensitive assays to measure their levels in different compartments in the circulation. Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility. The sensor was used to specifically detect microRNA-10b at the attomolar (10–18 M) concentration in pancreatic cancer cell lines, derived tissue culture media, human plasma, and media and plasma exosomes. In addition, for the first time, our label-free and nondestructive sensing technique was used to quantify microRNA-10b in highly purified exosomes isolated from patients with pancreatic cancer or chronic pancreatitis, and from normal controls. We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls. Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures. PMID:26444644

  15. The extracellular RNA complement of Escherichia coli

    PubMed Central

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-01

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular