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Sample records for nucleobase analogue fret

  1. Studying Z-DNA and B- to Z-DNA transitions using a cytosine analogue FRET-pair

    PubMed Central

    Dumat, Blaise; Larsen, Anders Foller; Wilhelmsson, L. Marcus

    2016-01-01

    Herein, we report on the use of a tricyclic cytosine FRET pair, incorporated into DNA with different base pair separations, to study Z-DNA and B-Z DNA junctions. With its position inside the DNA structure, the FRET pair responds to a B- to Z-DNA transition with a distinct change in FRET efficiency for each donor/acceptor configuration allowing reliable structural probing. Moreover, we show how fluorescence spectroscopy and our cytosine analogues can be used to determine rate constants for the B- to Z-DNA transition mechanism. The modified cytosines have little influence on the transition and the FRET pair is thus an easily implemented and virtually non-perturbing fluorescence tool to study Z-DNA. This nucleobase analogue FRET pair represents a valuable addition to the limited number of fluorescence methods available to study Z-DNA and we suggest it will facilitate, for example, deciphering the B- to Z-DNA transition mechanism and investigating the interaction of DNA with Z-DNA binding proteins. PMID:26896804

  2. Asymmetric Hydrogenation of α-Purine Nucleobase-Substituted Acrylates with Rhodium Diphosphine Complexes: Access to Tenofovir Analogues.

    PubMed

    Sun, Huan-Li; Chen, Fei; Xie, Ming-Sheng; Guo, Hai-Ming; Qu, Gui-Rong; He, Yan-Mei; Fan, Qing-Hua

    2016-05-01

    The first asymmetric hydrogenation of α-purine nucleobase-substituted α,β-unsaturated esters, catalyzed by a chiral rhodium (R)-Synphos catalyst, has been developed. A wide range of mono- and disubstituted acrylates were successfully hydrogenated under very mild conditions in high yields with good to excellent enantioselectivities (up to 99% ee). This method provides a convenient approach to the synthesis of a new kind of optically pure acyclic nucleoside and Tenofovir analogues. PMID:27112983

  3. A straightforward entry to chiral carbocyclic nucleoside analogues via the enantioselective [3+2] cycloaddition of α-nucleobase substituted acrylates.

    PubMed

    Xie, Ming-Sheng; Wang, Yong; Li, Jian-Ping; Du, Cong; Zhang, Yan-Yan; Hao, Er-Jun; Zhang, Yi-Ming; Qu, Gui-Rong; Guo, Hai-Ming

    2015-08-11

    A straightforward entry to chiral carbocyclic nucleoside analogues has been realized via the enantioselective [3+2] cycloaddition of α-nucleobase substituted acrylates to vinyl cyclopropanes for the first time. With Pd2(dba)3-L5 as the catalyst, carbocyclic purine, uracil, and thymine nucleoside analogues with quaternary stereocenters were obtained in excellent yields (up to 99% yield) and good enantioselectivities (up to 92% ee). PMID:26145719

  4. Ultrafast Dynamics of a Nucleobase Analogue Illuminated by a Short Intense X-ray Free Electron Laser Pulse

    NASA Astrophysics Data System (ADS)

    Nagaya, K.; Motomura, K.; Kukk, E.; Fukuzawa, H.; Wada, S.; Tachibana, T.; Ito, Y.; Mondal, S.; Sakai, T.; Matsunami, K.; Koga, R.; Ohmura, S.; Takahashi, Y.; Kanno, M.; Rudenko, A.; Nicolas, C.; Liu, X.-J.; Zhang, Y.; Chen, J.; Anand, M.; Jiang, Y. H.; Kim, D.-E.; Tono, K.; Yabashi, M.; Kono, H.; Miron, C.; Yao, M.; Ueda, K.

    2016-04-01

    Understanding x-ray radiation damage is a crucial issue for both medical applications of x rays and x-ray free-electron-laser (XFEL) science aimed at molecular imaging. Decrypting the charge and fragmentation dynamics of nucleobases, the smallest units of a macro-biomolecule, contributes to a bottom-up understanding of the damage via cascades of phenomena following x-ray exposure. We investigate experimentally and by numerical simulations the ultrafast radiation damage induced on a nucleobase analogue (5-iodouracil) by an ultrashort (10 fs) high-intensity radiation pulse generated by XFEL at SPring-8 Angstrom Compact free electron Laser (SACLA). The present study elucidates a plausible underlying radiosensitizing mechanism of 5-iodouracil. This mechanism is independent of the exact composition of 5-iodouracil and thus relevant to other such radiosensitizers. Furthermore, we found that despite a rapid increase of the net molecular charge in the presence of iodine, and of the ultrafast release of hydrogen, the other atoms are almost frozen within the 10-fs duration of the exposure. This validates single-shot molecular imaging as a consistent approach, provided the radiation pulse used is brief enough.

  5. Functionalized Solid Electrodes for Electrochemical Biosensing of Purine Nucleobases and Their Analogues: A Review

    PubMed Central

    Sharma, Vimal Kumar; Jelen, Frantisek; Trnkova, Libuse

    2015-01-01

    Interest in electrochemical analysis of purine nucleobases and few other important purine derivatives has been growing rapidly. Over the period of the past decade, the design of electrochemical biosensors has been focused on achieving high sensitivity and efficiency. The range of existing electrochemical methods with carbon electrode displays the highest rate in the development of biosensors. Moreover, modification of electrode surfaces based on nanomaterials is frequently used due to their extraordinary conductivity and surface to volume ratio. Different strategies for modifying electrode surfaces facilitate electron transport between the electrode surface and biomolecules, including DNA, oligonucleotides and their components. This review aims to summarize recent developments in the electrochemical analysis of purine derivatives, as well as discuss different applications. PMID:25594595

  6. Bicyclic and Tricyclic "Expanded" Nucleobase Analogues of Sofosbuvir: New Scaffolds for Hepatitis C Therapies.

    PubMed

    Chen, Zhe; Jochmans, Dirk; Ku, Therese; Paeshuyse, Jan; Neyts, Johan; Seley-Radtke, Katherine L

    2015-08-14

    Given the impressive success of Gilead's Sofosbuvir, many laboratories, including ours, have explored the unique 2'-sugar modification (2'-Me, 2'-F) of nucleoside analogues in the hopes of exploiting the biological activity that this unique modification has imparted to the nucleoside scaffold. In that regard, we have combined our tricyclic "expanded" purine base motif with the 2'-Me, 2'-F sugar modification. Although the synthesis of these complex molecules proved to be nontrivial, with the best results coming from a linear approach, the overall strategy resulted in highly promising biological results for several of the target compounds, including their corresponding McGuigan ProTides. Modest activity against HCV was observed with inhibitory concentrations of as low as 20 μM. PMID:27624884

  7. Cation radii induced structural variation in fluorescent alkaline earth networks constructed from tautomers of a nucleobase analogue.

    PubMed

    Deng, Zhao-Peng; Kang, Wei; Zhu, Zhi-Biao; Huo, Li-Hua; Zhao, Hui; Gao, Shan

    2012-07-21

    Nucleobase tautomers and their metal complexes have attracted considerable attention due to their fascinating architectures along with wide applications. In this paper, 4,6-dihydroxypyrimidine (H(2)DHP), an analogue of uracil and thymine, was employed to react with the vital elements of alkaline earth metals in an aqueous solution and lead to the formation of four novel complexes, [Mg(HDHP)(2) (H(2)O)(4)] (1), [Ca(HDHP)(2)(H(2)O)(3)](n)·nH(2)O (2), [Sr(HDHP)(2)(H(2)O)(3)](n)·nH(2)O (3), and [Ba(HDHP)(2)(H(2)O)(2)](n)·nH(2)O (4), which have been characterized by elemental analysis, IR, TG, UV-Vis, PL, powder and single-crystal X-ray diffraction and progressively evolve from zero-dimensional (0D) mononuclear, one-dimensional (1D) zig-zag double chain, two-dimensional (2D) double layer, to a three-dimensional (3D) porous network along with the increase of cation radii. This tendency in dimensionality follows salient crystal engineering principles and can be explained by considering factors such as hard-soft acid-base principles and cation radii. The deprotonated H(2)DHP ligand exhibits four new coordination modes, namely, O-monodentate (complex 1), N,O-chelating (complexes 2 and 3), O,O-bridging (complexes 2 and 3), and κ(1)O:κ(2)O-bridging mode (complex 4). Interestingly, the structural investigation indicates that the HDHP(-) monoanion shows three unusual types of tautomers, which are essential for the diagnosis of disease and investigation of medicine. Furthermore, the four complexes exhibit strong blue emission compared to free H(2)DHP ligand at room temperature and may be potential candidates for blue fluorescent biological materials used in organisms.

  8. FRETmatrix: a general methodology for the simulation and analysis of FRET in nucleic acids

    PubMed Central

    Preus, Søren; Kilså, Kristine; Miannay, Francois-Alexandre; Albinsson, Bo; Wilhelmsson, L. Marcus

    2013-01-01

    Förster resonance energy transfer (FRET) is a technique commonly used to unravel the structure and conformational changes of biomolecules being vital for all living organisms. Typically, FRET is performed using dyes attached externally to nucleic acids through a linker that complicates quantitative interpretation of experiments because of dye diffusion and reorientation. Here, we report a versatile, general methodology for the simulation and analysis of FRET in nucleic acids, and demonstrate its particular power for modelling FRET between probes possessing limited diffusional and rotational freedom, such as our recently developed nucleobase analogue FRET pairs (base–base FRET). These probes are positioned inside the DNA/RNA structures as a replacement for one of the natural bases, thus, providing unique control of their position and orientation and the advantage of reporting from inside sites of interest. In demonstration studies, not requiring molecular dynamics modelling, we obtain previously inaccessible insight into the orientation and nanosecond dynamics of the bases inside double-stranded DNA, and we reconstruct high resolution 3D structures of kinked DNA. The reported methodology is accompanied by a freely available software package, FRETmatrix, for the design and analysis of FRET in nucleic acid containing systems. PMID:22977181

  9. Microhydration of Deprotonated Nucleobases

    NASA Astrophysics Data System (ADS)

    Wincel, Henryk

    2016-08-01

    Hydration reactions of deprotonated nucleobases (uracil, thymine, 5-fluorouracil,2-thiouracil, cytosine, adenine, and hypoxanthine) produced by electrospray have been experimentally studied in the gas phase at 10 mbar using a pulsed ion-beam high-pressure mass spectrometer. The thermochemical data, ΔH o , ΔS o , and ΔG o , for the monohydrated systems were determined. The hydration enthalpies were found to be similar for all studied systems and varied between 39.4 and 44.8 kJ/mol. A linear correlation was found between water binding energies in the hydrated complexes and the corresponding acidities of the most acidic site of nucleobases. The structural and energetic aspects of the precursors for the hydrated complexes are discussed in conjunction with available literature data.

  10. Fretting in aircraft turbine engines

    NASA Technical Reports Server (NTRS)

    Johnson, R. L.; Bill, R. C.

    1974-01-01

    The problem of fretting in aircraft turbine engines is discussed. Critical fretting can occur on fan, compressor, and turbine blade mountings, as well as on splines, rolling element bearing races, and secondary sealing elements of face type seals. Structural fatigue failures have been shown to occur at fretted areas on component parts. Methods used by designers to reduce the effects of fretting are given.

  11. Extraterrestrial Nucleobases in Carbonaceous Chondrites

    NASA Astrophysics Data System (ADS)

    Martins, Z.; Botta, O.; Fogel, M.; Sephton, M.; Glavin, D.; Watson, J.; Dworkin, J.; Schwartz, A.; Ehrenfreund, P.

    Nucleobases in Carbonaceous Chondrites Z. Martins (1), O. Botta (2), M. L. Fogel (3), M. A. Sephton (4), D. P. Glavin (2), J. S. Watson (5), J. P. Dworkin (2), A. W. Schwartz (6) and P. Ehrenfreund (1,6). (1) Astrobiology Laboratory, Leiden Institute of Chemistry, Leiden, The Netherlands, (2) NASA Goddard Space Flight Center, Goddard Center for Astrobiology, Greenbelt, MD, USA, (3) GL, Carnegie Institution of Washington, Washington DC, USA, (4) Impacts and Astromaterials Research Centre, Department of Earth Science and Engineering, South Kensington Campus, Imperial College, London, UK, (5) Planetary and Space Sciences Research Institute, The Open University, Walton Hall, Milton Keynes, UK, (6) Radboud University Nijmegen, Nijmegen, The Netherlands. E-mail: z.martins@chem.leidenuniv.nl/Phone:+31715274440 Nucleobases are crucial compounds in terrestrial biochemistry, because they are key components of DNA and RNA. Carbonaceous meteorites have been analyzed for nucleobases by different research groups [1-5]. However, significant quantitative and qualitative differences were observed, leading to the controversial about the origin of these nucleobases. In order to establish the origin of these compounds in carbonaceous chondrites and to assess the plausibility of their exogenous delivery to the early Earth, we have performed formic acid extraction of samples of the Murchison meteorite [6], followed by an extensive purification procedure, analysis and quantification by high-performance liquid chromatography with UV absorption detection and gas chromatography-mass spectrometry. Our results were qualitatively consistent with previous results [3, 4], but showed significant quantitative differences. Compound specific carbon isotope values were obtained, using gas chromatography-combustion- isotope ratio mass spectrometry. A soil sample collected in the proximity of the Murchison meteorite fall site was subjected to the same extraction, purification and analysis procedure

  12. Fretting of AISI 9310 and selected fretting resistant surface treatments

    NASA Technical Reports Server (NTRS)

    Bill, R. C.

    1977-01-01

    Fretting wear experiments were conducted with uncoated AISI 9310 mating surfaces, and with combinations incorporating a selected coating to one of the mating surfaces. Wear measurements and SEM observations indicated that surface fatigue, as made evident by spallation and surface crack formation, is an important mechanism in promoting fretting wear to uncoated 9310. Increasing humidity resulted in accelerated fretting, and a very noticeable difference in nature of the fretting debris. Of the coatings evaluated, aluminum bronze with a polyester additive was most effective at reducing wear and minimizing fretting damage to the mating uncoated surface, by means of a selflubricating film that developed on the fretting surfaces. Chromium plate performed as an effective protective coating, itself resisting fretting and not accelerating damage to the uncoated surface.

  13. Pulsed supersonic beams with nucleobases.

    PubMed

    Sarfraz, Adnan; Rademann, Klaus; Christen, Wolfgang

    2012-10-01

    The dissolution of the primary nucleobases in supercritical fluids has been investigated using pulsed molecular beam mass spectrometry. Due to the low critical temperatures of ethylene and carbon dioxide, their adiabatic jet expansion permits transferring thermally sensitive solutes into the gas phase. This feature is particularly attractive for pharmaceutical and biomedical applications. In this study, adenine, guanine, cytosine, thymine, and uracil have been dissolved in supercritical ethylene with a few percent of ethanol as cosolvent. At source temperatures of 313 K, these solutions have been expanded from supercritical pressures into high vacuum using a customized pulsed nozzle. A mass spectrometer was used to monitor the relative amounts of solute, solvent, and cosolvent in the supersonic beam. The results suggest a paramount influence of the cosolvent.

  14. Spectroscopy of Isolated Prebiotic Nucleobases

    NASA Technical Reports Server (NTRS)

    Svadlenak, Nathan; Callahan, Michael P.; Ligare, Marshall; Gulian, Lisa; Gengeliczki, Zsolt; Nachtigallova, Dana; Hobza, Pavel; deVries, Mattanjah

    2011-01-01

    We use multiphoton ionization and double resonance spectroscopy to study the excited state dynamics of biologically relevant molecules as well as prebiotic nucleobases, isolated in the gas phase. Molecules that are biologically relevant to life today tend to exhibit short excited state lifetimes compared to similar but non-biologically relevant analogs. The mechanism is internal conversion, which may help protect the biologically active molecules from UV damage. This process is governed by conical intersections that depend very strongly on molecular structure. Therefore we have studied purines and pyrimidines with systematic variations of structure, including substitutions, tautomeric forms, and cluster structures that represent different base pair binding motifs. These structural variations also include possible alternate base pairs that may shed light on prebiotic chemistry. With this in mind we have begun to probe the ultrafast dynamics of molecules that exhibit very short excited states and search for evidence of internal conversions.

  15. Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.

    PubMed

    Young, J D; Yao, S Y M; Sun, L; Cass, C E; Baldwin, S A

    2008-07-01

    1. The human (h) SLC29 family of integral membrane proteins is represented by four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterized family member, hENT1. They belong to the widely distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporter proteins. 2. A predicted topology of eleven transmembrane helices has been experimentally confirmed for hENT1. The best-characterized members of the family, hENT1 and hENT2, possess similar broad permeant selectivities for purine and pyrimidine nucleosides, but hENT2 also efficiently transports nucleobases. hENT3 has a similar broad permeant selectivity for nucleosides and nucleobases and appears to function in intracellular membranes, including lysosomes. 3. hENT4 is uniquely selective for adenosine, and also transports a variety of organic cations. hENT3 and hENT4 are pH sensitive, and optimally active under acidic conditions. ENTs, including those in parasitic protozoa, function in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis and, in humans, are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. 4. By regulating the concentration of adenosine available to cell surface receptors, mammalian ENTs additionally influence physiological processes ranging from cardiovascular activity to neurotransmission.

  16. Fretted Terrain Valleys

    NASA Technical Reports Server (NTRS)

    2004-01-01

    30 October 2004 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows shallow tributary valleys in the Ismenius Lacus fretted terrain region of northern Arabia Terra. These valleys exhibit a variety of typical fretted terrain valley wall and floor textures, including a lineated, pitted material somewhat reminiscent of the surface of a brain. Origins for these features are still being debated within the Mars science community; there are no clear analogs to these landforms on Earth. This image is located near 39.9oN, 332.1oW. The picture covers an area about 3 km (1.9 mi) wide. Sunlight illuminates the scene from the lower left.

  17. Depolarized FRET (depolFRET) on the cell surface: FRET control by photoselection.

    PubMed

    Bene, László; Gogolák, Péter; Ungvári, Tamás; Bagdány, Miklós; Nagy, István; Damjanovich, László

    2016-02-01

    Sensitivity of FRET in hetero- and homo-FRET systems on the photoselected orientation distribution of donors has been proven by using polarized and depolarized light for excitation. FRET as well as donor and acceptor anisotropies have been simultaneously measured in a dual emission-polarization scheme realized in a conventional flow cytometer by using single laser excitation and applying fluorophore-conjugated mAbs against the MHCI and MHCII cell surface receptors. Depolarization of the originally polarized light have been achieved by using crystal depolarizers based on Cornu's principle, a quarter-wave plate for circular polarization, and a parallel beam splitter acting as a diagonal-polarizer for dual-polarization excitation. Simultaneous analysis of intensity-based FRET efficiency and acceptor depolarization equivocally report that depolarization of light may increase FRET in an amount depending on the acceptor-to-donor concentration ratio. Acceptor depolarization turned to be more sensitive to FRET than donor hyper-polarization and even than intensity-based FRET efficiency. It can be used as a sensitive tool for monitoring changes in the dynamics of the donor-acceptor pairs. The basic observations of FRET enhancement and increased acceptor depolarization obtained for hetero-FRET are paralleled by analog observations of homo-FRET enhancements under depolarized excitation. In terms of the orientation factor for FRET, the FRET enhancements on depolarization in the condition of the macroscopically isotropic orientation distributions such as those of the cell surface bound fluorophores report on the presence of local orientation mismatches of the donor and acceptor preventing the optimal FRET in the polarized case, which may be eliminated by the excitation depolarization. A theory of fluorescence anisotropy for depolarized excitation is also presented. PMID:26657258

  18. Fretted Terrain Valley Floor

    NASA Technical Reports Server (NTRS)

    2004-01-01

    30 December 2003 This December 2003 Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows lineated textures on the floor of a valley in the Deuteronilus region of Mars. Deuteronilus, and neighboring Protonilus and Nilosyrtis, have been known since the Mariner 9 mission as regions of 'fretted terrain.' In this context, 'fretted' does not mean 'worried,' it means 'eroded.' The fretted terrains of Mars are regions along the boundary between cratered highlands and northern lowland plains that have been broken-down into mesas, buttes, and valleys. On the floors of some of these valleys occurs a distinctive lineated and pitted texture--like the example shown here. The cause of the textures is not known, although for decades some scientists have speculated that ice is involved. While this is possible, it is far from a demonstrated fact. This picture is located near 40.1oN, 335.1oW, and covers an area approximately 3 km (1.9 mi) wide; sunlight illuminates the scene from the lower left.

  19. Fretting in aircraft turbine engines

    NASA Technical Reports Server (NTRS)

    Johnson, R. L.; Bill, R. C.

    1974-01-01

    Fretting usually produces both polished and pitted areas. Fretting occurs in connection with the relative motion between contacting surface elements. Particular attention is given to fretting in seals, splines, fans, compressors, and turbine blades. Recommended approaches to mitigate fretting problems include a reduction of the adhesion between two surfaces. Approaches for doing this may include the development of alloys which spontaneously generate a self-healing surface film, different in composition from the bulk alloy. Other fundamental adhesion studies have shown that surface crystal structure and crystallographic orientation significantly affect the adhesion properties of alloys.

  20. Photoelectron spectroscopy of hexachloroplatinate-nucleobase complexes: Nucleobase excited state decay observed via delayed electron emission.

    PubMed

    Sen, Ananya; Matthews, Edward M; Hou, Gao-Lei; Wang, Xue-Bin; Dessent, Caroline E H

    2015-11-14

    We report low-temperature photoelectron spectra of isolated gas-phase complexes of the hexachloroplatinate dianion bound to the nucleobases uracil, thymine, cytosine, and adenine. The spectra display well-resolved, distinct peaks that are consistent with complexes where the hexachloroplatinate dianion is largely intact. Adiabatic electron detachment energies for the hexachloroplatinate-nucleobase complexes are measured as 2.26-2.36 eV. The magnitudes of the repulsive Coulomb barriers (RCBs) of the complexes are all ∼1.7 eV, values that are lower than the RCB of the uncomplexed PtCl6 (2-) dianion as a result of charge solvation by the nucleobases. In addition to the resolved spectral features, broad featureless bands indicative of delayed electron detachment are observed in the 193 nm photoelectron spectra of the four clusters. The 266 nm spectra of the PtCl6 (2-) ⋅ thymine and PtCl6 (2-) ⋅ adenine complexes also display very prominent delayed electron emission bands. These results mirror recent results on the related Pt(CN)4 (2-) ⋅ nucleobase complexes [A. Sen et al., J. Phys. Chem. B 119, 11626 (2015)]. The observation of delayed electron emission bands in the PtCl6 (2-) ⋅ nucleobase spectra obtained in this work, as for the previously studied Pt(CN)4 (2-) ⋅ nucleobase complexes, is attributed to one-photon excitation of nucleobase-centred excited states that can effectively couple to the electron detachment continuum, producing strong electron detachment. Moreover, the selective, strong excitation of the delayed emission bands in the 266 nm spectra is linked to fundamental differences in the individual nucleobase photophysics at this excitation energy. This strongly supports our previous suggestion that the dianion within these clusters can be viewed as a "dynamic tag" which has the propensity to emit electrons when the attached nucleobase decays over a time scale long enough to allow autodetachment.

  1. Photoelectron Spectroscopy of Hexachloroplatinate-Nucleobase Complexes: Nucleobase Excited State Decay Observed via Delayed Electron Emission

    SciTech Connect

    Sen, Ananya; Matthews, Edward M.; Hou, Gao-Lei; Wang, Xue B.; Dessent, Caroline

    2015-11-14

    We report low-temperature photoelectron spectra of isolated gas-phase complexes of the hexachloroplatinate dianion bound to the nucleobases uracil, thymine, cytosine and adenine. The spectra display well-resolved, distinct peaks that are consistent with complexes where the hexachloroplatinate dianion is largely intact. Adiabatic electron detachment energies for the hexachloroplatinate-nucleobase complexes are measured as 2.26-2.36 eV. The magnitudes of the repulsive Coulomb barriers (RCBs) of the complexes are all ~1.7 eV, values that are lower than the RCB of the uncomplexed PtCl6 2- dianion as a result of charge solvation by the nucleobases. In addition to the resolved spectral features, broad featureless bands indicative of delayed electron detachment are observed in the 193 nm photoelectron spectra of the four clusters. The 266 nm spectra of the PtCl6 2-∙thymine and PtCl6 2-∙adenine complexes also display very prominent delayed electron emission bands. These results mirror recent results on the related Pt(CN)4 2-∙nucleobase complexes [Sen et al, J. Phys. Chem. B, 119, 11626, 2015]. The observation of delayed electron emission bands in the PtCl6 2-∙nucleobase spectra obtained in this work, as for the previously studied Pt(CN)4 2-∙nucleobase complexes, is attributed to onephoton excitation of nucleobase-centred excited states that can effectively couple to the electron detachment continuum, producing strong electron detachment. Moreover, the selective, strong excitation of the delayed emission bands in the 266 nm spectra is linked to fundamental differences in the individual nucleobase photophysics at this excitation energy. This strongly supports our previous suggestion that the dianion within these clusters can be viewed as a “dynamic tag” which has the propensity to emit electrons when the attached nucleobase decays over a timescale long enough to allow autodetachment.

  2. Photoelectron spectroscopy of hexachloroplatinate-nucleobase complexes: Nucleobase excited state decay observed via delayed electron emission.

    PubMed

    Sen, Ananya; Matthews, Edward M; Hou, Gao-Lei; Wang, Xue-Bin; Dessent, Caroline E H

    2015-11-14

    We report low-temperature photoelectron spectra of isolated gas-phase complexes of the hexachloroplatinate dianion bound to the nucleobases uracil, thymine, cytosine, and adenine. The spectra display well-resolved, distinct peaks that are consistent with complexes where the hexachloroplatinate dianion is largely intact. Adiabatic electron detachment energies for the hexachloroplatinate-nucleobase complexes are measured as 2.26-2.36 eV. The magnitudes of the repulsive Coulomb barriers (RCBs) of the complexes are all ∼1.7 eV, values that are lower than the RCB of the uncomplexed PtCl6 (2-) dianion as a result of charge solvation by the nucleobases. In addition to the resolved spectral features, broad featureless bands indicative of delayed electron detachment are observed in the 193 nm photoelectron spectra of the four clusters. The 266 nm spectra of the PtCl6 (2-) ⋅ thymine and PtCl6 (2-) ⋅ adenine complexes also display very prominent delayed electron emission bands. These results mirror recent results on the related Pt(CN)4 (2-) ⋅ nucleobase complexes [A. Sen et al., J. Phys. Chem. B 119, 11626 (2015)]. The observation of delayed electron emission bands in the PtCl6 (2-) ⋅ nucleobase spectra obtained in this work, as for the previously studied Pt(CN)4 (2-) ⋅ nucleobase complexes, is attributed to one-photon excitation of nucleobase-centred excited states that can effectively couple to the electron detachment continuum, producing strong electron detachment. Moreover, the selective, strong excitation of the delayed emission bands in the 266 nm spectra is linked to fundamental differences in the individual nucleobase photophysics at this excitation energy. This strongly supports our previous suggestion that the dianion within these clusters can be viewed as a "dynamic tag" which has the propensity to emit electrons when the attached nucleobase decays over a time scale long enough to allow autodetachment. PMID:26567662

  3. Carbonaceous meteorites contain a wide range of extraterrestrial nucleobases.

    PubMed

    Callahan, Michael P; Smith, Karen E; Cleaves, H James; Ruzicka, Josef; Stern, Jennifer C; Glavin, Daniel P; House, Christopher H; Dworkin, Jason P

    2011-08-23

    All terrestrial organisms depend on nucleic acids (RNA and DNA), which use pyrimidine and purine nucleobases to encode genetic information. Carbon-rich meteorites may have been important sources of organic compounds required for the emergence of life on the early Earth; however, the origin and formation of nucleobases in meteorites has been debated for over 50 y. So far, the few nucleobases reported in meteorites are biologically common and lacked the structural diversity typical of other indigenous meteoritic organics. Here, we investigated the abundance and distribution of nucleobases and nucleobase analogs in formic acid extracts of 12 different meteorites by liquid chromatography-mass spectrometry. The Murchison and Lonewolf Nunataks 94102 meteorites contained a diverse suite of nucleobases, which included three unusual and terrestrially rare nucleobase analogs: purine, 2,6-diaminopurine, and 6,8-diaminopurine. In a parallel experiment, we found an identical suite of nucleobases and nucleobase analogs generated in reactions of ammonium cyanide. Additionally, these nucleobase analogs were not detected above our parts-per-billion detection limits in any of the procedural blanks, control samples, a terrestrial soil sample, and an Antarctic ice sample. Our results demonstrate that the purines detected in meteorites are consistent with products of ammonium cyanide chemistry, which provides a plausible mechanism for their synthesis in the asteroid parent bodies, and strongly supports an extraterrestrial origin. The discovery of new nucleobase analogs in meteorites also expands the prebiotic molecular inventory available for constructing the first genetic molecules.

  4. Engineering Genetically Encoded FRET Sensors

    PubMed Central

    Lindenburg, Laurens; Merkx, Maarten

    2014-01-01

    Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening. PMID:24991940

  5. Photoelectron spectroscopy of hexachloroplatinate-nucleobase complexes: Nucleobase excited state decay observed via delayed electron emission

    SciTech Connect

    Sen, Ananya; Matthews, Edward M.; Dessent, Caroline E. H. E-mail: xuebin.wang@pnnl.gov; Hou, Gao-Lei; Wang, Xue-Bin E-mail: xuebin.wang@pnnl.gov

    2015-11-14

    We report low-temperature photoelectron spectra of isolated gas-phase complexes of the hexachloroplatinate dianion bound to the nucleobases uracil, thymine, cytosine, and adenine. The spectra display well-resolved, distinct peaks that are consistent with complexes where the hexachloroplatinate dianion is largely intact. Adiabatic electron detachment energies for the hexachloroplatinate-nucleobase complexes are measured as 2.26-2.36 eV. The magnitudes of the repulsive Coulomb barriers (RCBs) of the complexes are all ∼1.7 eV, values that are lower than the RCB of the uncomplexed PtCl{sub 6}{sup 2−} dianion as a result of charge solvation by the nucleobases. In addition to the resolved spectral features, broad featureless bands indicative of delayed electron detachment are observed in the 193 nm photoelectron spectra of the four clusters. The 266 nm spectra of the PtCl{sub 6}{sup 2−} ⋅ thymine and PtCl{sub 6}{sup 2−} ⋅ adenine complexes also display very prominent delayed electron emission bands. These results mirror recent results on the related Pt(CN){sub 4}{sup 2−} ⋅ nucleobase complexes [A. Sen et al., J. Phys. Chem. B 119, 11626 (2015)]. The observation of delayed electron emission bands in the PtCl{sub 6}{sup 2−} ⋅ nucleobase spectra obtained in this work, as for the previously studied Pt(CN){sub 4}{sup 2−} ⋅ nucleobase complexes, is attributed to one-photon excitation of nucleobase-centred excited states that can effectively couple to the electron detachment continuum, producing strong electron detachment. Moreover, the selective, strong excitation of the delayed emission bands in the 266 nm spectra is linked to fundamental differences in the individual nucleobase photophysics at this excitation energy. This strongly supports our previous suggestion that the dianion within these clusters can be viewed as a “dynamic tag” which has the propensity to emit electrons when the attached nucleobase decays over a time scale long enough to

  6. Communication: Photoactivation of nucleobase bound platinumII metal complexes: Probing the influence of the nucleobase

    NASA Astrophysics Data System (ADS)

    Sen, Ananya; Dessent, Caroline E. H.

    2014-12-01

    We present UV laser action spectra (220-300 nm) of isolated nucleobase-bound PtII(CN)42- complexes, i.e., Pt(CN)42-ṡM, where M = uracil, thymine, cytosine, and adenine. These metal complex-nucleobase clusters represent model systems for identifying the fundamental photophysical and photochemical processes occurring in photodynamic platinum (II) drug therapies that target DNA. This is the first study to explore the specific role of the nucleobase in the photophysics of the aggregate complex. Each of the complexes studied displays a broadly similar absorption spectra, with a strong λmax ˜ 4.7 eV absorption band (nucleobase localized chromophore) and a subsequent increase in the absorption intensity towards higher spectral-energy (Pt(CN)42- localized chromophore). However, strikingly different band widths are observed across the series of complexes, decreasing in the order Pt(CN)42-ṡThymine > Pt(CN)42-ṡUracil > Pt(CN)42-ṡAdenine > Pt(CN)42-ṡCytosine. Changes in the bandwidth of the ˜4.7 eV band are accompanied by distinctive changes in the photofragment product ions observed following photoexcitation, with the narrower-bandwidth complexes showing a greater propensity to decay via electron detachment decay. We discuss these observations in the context of the distinctive nucleobase-dependent excited state lifetimes.

  7. A Guide to Fluorescent Protein FRET Pairs.

    PubMed

    Bajar, Bryce T; Wang, Emily S; Zhang, Shu; Lin, Michael Z; Chu, Jun

    2016-01-01

    Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.

  8. A Guide to Fluorescent Protein FRET Pairs

    PubMed Central

    Bajar, Bryce T.; Wang, Emily S.; Zhang, Shu; Lin, Michael Z.; Chu, Jun

    2016-01-01

    Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies. PMID:27649177

  9. A Guide to Fluorescent Protein FRET Pairs.

    PubMed

    Bajar, Bryce T; Wang, Emily S; Zhang, Shu; Lin, Michael Z; Chu, Jun

    2016-01-01

    Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies. PMID:27649177

  10. Carbonaceous Meteorites Contain a Wide Range of Extraterrestrial Nucleobases

    NASA Technical Reports Server (NTRS)

    Callahan, Michael P.; Smith, Karen E.; Cleaves, H. James, II; Ruzicka, Josef; Stern, Jennifer C.; Glavin, Daniel P.; House, Christopher H.; Dworkin, Jason P.

    2011-01-01

    All terrestrial organisms depend on nucleic acids (RNA and DNA), which use pyrimidine and purine nucleobases to encode genetic information. Carbon-rich meteorites may have been important sources of organic compounds required for the emergence of life on the early Earth; however, the origin and formation of nuc1eobases in meteorites has been debated for over 50 y. So far, the few nuc1eobases reported in meteorites are biologically common and lacked the structural diversity typical of other indigenous meteoritic organics. Here, we investigated the abundance and distribution of nucleobases and nucleobase analogs in formic acid extracts of 12 different meteorites by liquid chromatography-mass spectrometry. The Murchison and Lonewolf Nunataks 94102 meteorites contained a diverse suite of nucleobases, which included three unusual and terrestrially rare nucleobase analogs; purine, 2,6-diminopurine, and 6,8-diaminopurine. In a parallel experiment, we found an identical suite of nucleobases and nucleobase analogs generated in reactions of ammonium cyanide. Additionally, these nucleobase analoge were not detected above our parts-per-billion detection limits in any of the procedural blanks, control samples, a terrestrial soil sample, and an Antarctic ice sample. Our results demonstrate that the purines detected in meteorites are consistent with products of ammonium cyanide chemistry, which provides a plausible mechanism for their synthesis in the asteroid parent bodies, and strongly supports an extraterrestrial origin. The discovery of new nucleobase analogs in meteorites also expands the prebiotic molecular inventory available for constructing the first genetic molecules.

  11. Oxidation of DNA: damage to nucleobases.

    PubMed

    Kanvah, Sriram; Joseph, Joshy; Schuster, Gary B; Barnett, Robert N; Cleveland, Charles L; Landman, Uzi

    2010-02-16

    All organisms store the information necessary to maintain life in their DNA. Any process that damages DNA, causing a loss or corruption of that information, jeopardizes the viability of the organism. One-electron oxidation is such a process. In this Account, we address three of the central features of one-electron oxidation of DNA: (i) the migration of the radical cation away from the site of its formation; (ii) the electronic and structural factors that determine the nucleobases at which irreversible reactions most readily occur; (iii) the mechanism of reaction for nucleobase radical cations. The loss of an electron (ionization) from DNA generates an electron "hole" (a radical cation), located most often on its nucleobases, that migrates reversibly through duplex DNA by hopping until it is trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In aqueous solution, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counterions to the phosphate groups (typically Na(+)) play an important role in facilitating both hopping and the eventual reaction of the radical cation with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA, comprising the four common DNA nucleobases G, C, A, and T, reaction occurs most commonly at a guanine, resulting in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step, primarily by a tandem process. The oxidative damage of DNA is a complex process, influenced by charge transport and reactions that are controlled by a combination of enthalpic, entropic, steric, and

  12. Demonstration of FRET in solutions

    NASA Astrophysics Data System (ADS)

    Shah, Sunil; Gryczynski, Zygmunt; Chib, Rahul; Fudala, Rafal; Baxi, Aatmun; Borejdo, Julian; Synak, Anna; Gryczynski, Ignacy

    2016-03-01

    We measured the Förster resonance energy transfer (FRET) from Uranin (U) donor to Rhodamine 101 (R101) acceptor in propylene glycol. Steady-state fluorescence measurements show a significant difference between mixed and unmixed fluorophore solutions. In the solution with mixed fluorophores, fluorescence intensity of the U donor decreases and intensity of R101 fluorescence increases. This is visualized as a color change from green to orange. Fluorescence anisotropy of the mixture solution increases in the donor emission wavelength region and decreases in the acceptor emission wavelengths; which is consistent with FRET occurrence. Time-resolved (lifetime) measurements show a decrease of the U lifetime in the presence of R101 acceptor. In the intensity decay of R101 acceptor appears a negative component indicating excited state process. All these measurements prove the presence of FRET in U/R101 mixture fluorescence.

  13. SLIM for multispectral FRET imaging

    NASA Astrophysics Data System (ADS)

    Rück, A.; Dolp, F.; Steiner, R.; Steinmetz, C.; von Einem, B.; von Arnim, C. A. F.

    2008-02-01

    Spectral fluorescence lifetime imaging (SLIM) is an advanced imaging technique, which combines spectral with time resolved detection. Real spectral information is achieved by using a grating in front of a PML-array, which allows time-correlated single photon counting (TCSPC). Whereas spectrally resolved fluorescence imaging alone has a reasonable sensitivity, the specificity of fluorescence detection can be improved by considering the fluorescence lifetime. The various possibilities which SLIM offers to improve FRET (resonant energy transfer) will be discussed as well as successfully realized applications. These include FRET measurements for protein interactions, related to Alzheimer's disease. Special attention will be focused on molecules involved in the processing and trafficking of the amyloid precursor protein (APP), as trafficking proteins of the GGA family and β-secretase BACE). Taking into account also the lifetime of the acceptor could enhance reliability of the FRET result.

  14. Evaluation of RSRM case hardware fretting concerns

    NASA Technical Reports Server (NTRS)

    Swauger, Thomas R.

    1990-01-01

    Fretting corrosion was first noted on Shuttle flight STS-26. This flight was the first usage of the Redesigned Solid Rocket Motor (RSRM). The occurrence of fretting has since been observed on both the field and factory joints of the RSRM. Fretting is a form of corrosion that occurs at the interface between contacting, highly loaded, metal surfaces when exposed to slight relative vibratory motions. The engineering effort performed to evaluate the effect of fretting on the RSRM case hardware is summarized. Based on the results of this evaluation, several conclusions were made concerning flight safety. Also, recommendations were made concerning trending the effects of multiple generations of fretting damage.

  15. Silver- and gold-mediated nucleobase bonding.

    PubMed

    Acioli, Paulo H; Srinivas, Sudha

    2014-08-01

    We report the results of a density functional theory investigation of the bonding of nucleobases mediated by silver and gold atoms in the gas phase. Our calculations use the Becke exchange and Perdew-Wang correlation functional (BPW91) combined with the Stuttgart effective core potentials to represent the valence electrons of gold, silver, and platinum, and the all-electron DGTZVP basis set for C, H, N, and O. This combination was chosen based on tests on the metal atoms and tautomers of adenine, cytosine, and guanine. To establish a benchmark to understand the metal-mediated bonding, we calculated the binding energy of each of the base pairs in their canonical forms. Our calculations show rather strong bonds between the Watson-Crick base pairs when compared with typical values for N-H-N and N-H-O hydrogen bonds. The neutral metal atoms tend to bond near the nitrogen atoms. The effect of the metal atoms on the bonding of nucleobases differs depending on whether or not the metal atoms bond to one of the hydrogen-bonding sites. When the silver or gold atoms bond to a non-hydrogen-bonding site, the effect is a slight enhancement of the cytosine-guanine bonding, but there is almost no effect on the adenine-thymine pairing. The metal atoms can block one of the hydrogen-bonding sites, thus preventing the normal cytosine-guanine and adenine-thymine pairings. We also find that both silver and gold can bond to consecutive guanines in a similar fashion to platinum, albeit with a significantly lower binding energy.

  16. Fretting about FRET: Failure of the Ideal Dipole Approximation

    PubMed Central

    Muñoz-Losa, Aurora; Curutchet, Carles; Krueger, Brent P.; Hartsell, Lydia R.; Mennucci, Benedetta

    2009-01-01

    Abstract With recent growth in the use of fluorescence-detected resonance energy transfer (FRET), it is being applied to complex systems in modern and diverse ways where it is not always clear that the common approximations required for analysis are applicable. For instance, the ideal dipole approximation (IDA), which is implicit in the Förster equation, is known to break down when molecules get “too close” to each other. Yet, no clear definition exists of what is meant by “too close”. Here we examine several common fluorescent probe molecules to determine boundaries for use of the IDA. We compare the Coulombic coupling determined essentially exactly with a linear response approach with the IDA coupling to find the distance regimes over which the IDA begins to fail. We find that the IDA performs well down to roughly 20 Å separation, provided the molecules sample an isotropic set of relative orientations. However, if molecular motions are restricted, the IDA performs poorly at separations beyond 50 Å. Thus, isotropic probe motions help mask poor performance of the IDA through cancellation of error. Therefore, if fluorescent probe motions are restricted, FRET practitioners should be concerned with not only the well-known κ2 approximation, but also possible failure of the IDA. PMID:19527638

  17. Fretting about FRET: failure of the ideal dipole approximation.

    PubMed

    Muñoz-Losa, Aurora; Curutchet, Carles; Krueger, Brent P; Hartsell, Lydia R; Mennucci, Benedetta

    2009-06-17

    With recent growth in the use of fluorescence-detected resonance energy transfer (FRET), it is being applied to complex systems in modern and diverse ways where it is not always clear that the common approximations required for analysis are applicable. For instance, the ideal dipole approximation (IDA), which is implicit in the Förster equation, is known to break down when molecules get "too close" to each other. Yet, no clear definition exists of what is meant by "too close". Here we examine several common fluorescent probe molecules to determine boundaries for use of the IDA. We compare the Coulombic coupling determined essentially exactly with a linear response approach with the IDA coupling to find the distance regimes over which the IDA begins to fail. We find that the IDA performs well down to roughly 20 A separation, provided the molecules sample an isotropic set of relative orientations. However, if molecular motions are restricted, the IDA performs poorly at separations beyond 50 A. Thus, isotropic probe motions help mask poor performance of the IDA through cancellation of error. Therefore, if fluorescent probe motions are restricted, FRET practitioners should be concerned with not only the well-known kappa2 approximation, but also possible failure of the IDA. PMID:19527638

  18. Multiple Condensation Reactions Involving Pt(II) /Pd(II) -OH2 , Pt-NH3 , and Cytosine-NH2 Groups: New Twists in Cisplatin-Nucleobase Chemistry.

    PubMed

    Yin-Bandur, Lu; Sanz Miguel, Pablo J; Rodríguez-Santiago, Luis; Sodupe, Mariona; Berghaus, Melanie; Lippert, Bernhard

    2016-09-12

    The coordination chemistry of the antitumor agent cisplatin and related complexes with DNA and its constituents, that is, the nucleobases, appears to be dominated by 1:1 and 1:2 adducts of the types cis-[Pta2 (nucleobase)X] and cis-[Pta2 (nucleobase)2 ] (a=NH3 or amine; a2 =diamine or diimine; X=Cl, OH or OH2 ). Here, we have studied the interactions of the putative 1:1 adducts cis-[Pta2 (1-MeC-N3)(OH2 )](2+) (with a=NH3 , a2 =2,2'-bpy (2,2'-bipyridine), 1-MeC=model nucleobase 1-methylcytosine) with additional cis-[Pt(NH3 )2 (OH2 )2 ](2+) or its kinetically superior analogues [Pd(en)(OH2 )2 ](2+) (en=ethylenediamine) and [Pd(2,2'-bpy)(OH2 )2 ](2+) . Depending upon the conditions applied different compounds of different nuclearity are formed. Without exception they represent condensation products of the components, containing μ-1-MeC-H , μ-OH(-) , as well as μ-NH2 (-) bridges. In the presence of Ag(+) ions, the isolated products in several cases display additionally Pt→Ag dative bonds. On the basis of the cytosine-containing structures established by X-ray crystallography, it is proposed that any of the feasible initial 1:1 nucleobase adducts of cisplatin could form dinuclear Pt complexes upon reaction with additional hydrolyzed cisplatin, thereby generating nucleobase adducts other than the presently established ones. Two findings appear to be of particular significance: First, hydrolyzed cisplatin can have a moderately accelerating effect on the formation of a secondary nucleobase product. Second, NH3 ligands of the cisplatin moiety can be converted into bridging amido ligands following condensation with the diaqua species of cisplatin.

  19. Photobleaching-corrected FRET efficiency imaging of live cells.

    PubMed

    Zal, Tomasz; Gascoigne, Nicholas R J

    2004-06-01

    Fluorescent resonance energy transfer (FRET) imaging techniques can be used to visualize protein-protein interactions in real-time with subcellular resolution. Imaging of sensitized fluorescence of the acceptor, elicited during excitation of the donor, is becoming the most popular method for live FRET (3-cube imaging) because it is fast, nondestructive, and applicable to existing widefield or confocal microscopes. Most sensitized emission-based FRET indices respond nonlinearly to changes in the degree of molecular interaction and depend on the optical parameters of the imaging system. This makes it difficult to evaluate and compare FRET imaging data between laboratories. Furthermore, photobleaching poses a problem for FRET imaging in timelapse experiments and three-dimensional reconstructions. We present a 3-cube FRET imaging method, E-FRET, which overcomes both of these obstacles. E-FRET bridges the gap between the donor recovery after acceptor photobleaching technique (which allows absolute measurements of FRET efficiency, E, but is not suitable for living cells), and the sensitized-emission FRET indices (which reflect FRET in living cells but lack the quantitation and clarity of E). With E-FRET, we visualize FRET in terms of true FRET efficiency images (E), which correlate linearly with the degree of donor interaction. We have defined procedures to incorporate photobleaching correction into E-FRET imaging. We demonstrate the benefits of E-FRET with photobleaching correction for timelapse and three-dimensional imaging of protein-protein interactions in the immunological synapse in living T-cells.

  20. DNA-mediated excitonic upconversion FRET switching

    SciTech Connect

    Kellis, Donald L.; Rehn, Sarah M.; Cannon, Brittany L.; Davis, Paul H.; Graugnard, Elton; Lee, Jeunghoon; Yurke, Bernard; Knowlton, William B.

    2015-11-17

    Excitonics is a rapidly expanding field of nanophotonics in which the harvesting of photons, ensuing creation and transport of excitons via Förster resonant energy transfer (FRET), and subsequent charge separation or photon emission has led to the demonstration of excitonic wires, switches, Boolean logic and light harvesting antennas for many applications. FRET funnels excitons down an energy gradient resulting in energy loss with each step along the pathway. Conversely, excitonic energy up conversion via up conversion nanoparticles (UCNPs), although currently inefficient, serves as an energy ratchet to boost the exciton energy. Although FRET-based up conversion has been demonstrated, it suffers from low FRET efficiency and lacks the ability to modulate the FRET. We have engineered an up conversion FRET-based switch by combining lanthanide-doped UCNPs and fluorophores that demonstrates excitonic energy up conversion by nearly a factor of 2, an excited state donor to acceptor FRET efficiency of nearly 25%, and an acceptor fluorophore quantum efficiency that is close to unity. These findings offer a promising path for energy up conversion in nanophotonic applications including artificial light harvesting, excitonic circuits, photovoltaics, nanomedicine, and optoelectronics.

  1. DNA-mediated excitonic upconversion FRET switching

    DOE PAGES

    Kellis, Donald L.; Rehn, Sarah M.; Cannon, Brittany L.; Davis, Paul H.; Graugnard, Elton; Lee, Jeunghoon; Yurke, Bernard; Knowlton, William B.

    2015-11-17

    Excitonics is a rapidly expanding field of nanophotonics in which the harvesting of photons, ensuing creation and transport of excitons via Förster resonant energy transfer (FRET), and subsequent charge separation or photon emission has led to the demonstration of excitonic wires, switches, Boolean logic and light harvesting antennas for many applications. FRET funnels excitons down an energy gradient resulting in energy loss with each step along the pathway. Conversely, excitonic energy up conversion via up conversion nanoparticles (UCNPs), although currently inefficient, serves as an energy ratchet to boost the exciton energy. Although FRET-based up conversion has been demonstrated, it suffersmore » from low FRET efficiency and lacks the ability to modulate the FRET. We have engineered an up conversion FRET-based switch by combining lanthanide-doped UCNPs and fluorophores that demonstrates excitonic energy up conversion by nearly a factor of 2, an excited state donor to acceptor FRET efficiency of nearly 25%, and an acceptor fluorophore quantum efficiency that is close to unity. These findings offer a promising path for energy up conversion in nanophotonic applications including artificial light harvesting, excitonic circuits, photovoltaics, nanomedicine, and optoelectronics.« less

  2. DNA-mediated excitonic upconversion FRET switching

    NASA Astrophysics Data System (ADS)

    Kellis, Donald L.; Rehn, Sarah M.; Cannon, Brittany L.; Davis, Paul H.; Graugnard, Elton; Lee, Jeunghoon; Yurke, Bernard; Knowlton, William B.

    2015-11-01

    Excitonics is a rapidly expanding field of nanophotonics in which the harvesting of photons, ensuing creation and transport of excitons via Förster resonant energy transfer (FRET), and subsequent charge separation or photon emission has led to the demonstration of excitonic wires, switches, Boolean logic and light harvesting antennas for many applications. FRET funnels excitons down an energy gradient resulting in energy loss with each step along the pathway. Conversely, excitonic energy upconversion via upconversion nanoparticles (UCNPs), although currently inefficient, serves as an energy ratchet to boost the exciton energy. Although FRET-based upconversion has been demonstrated, it suffers from low FRET efficiency and lacks the ability to modulate the FRET. We have engineered an upconversion FRET-based switch by combining lanthanide-doped UCNPs and fluorophores that demonstrates excitonic energy upconversion by nearly a factor of 2, an excited state donor to acceptor FRET efficiency of nearly 25%, and an acceptor fluorophore quantum efficiency that is close to unity. These findings offer a promising path for energy upconversion in nanophotonic applications including artificial light harvesting, excitonic circuits, photovoltaics, nanomedicine, and optoelectronics.

  3. Design of intelligent nucleobases and DNA HOMO mapping.

    PubMed

    Saito, Isao

    2002-01-01

    We have designed a new nucleobase, benzodeazaadenine (BDA) that has a stronger charge transport ability than guanine and is not destroyed during charge transport process. By incorporating this new nucleobase into DNA, we demonstrated a protocol for real DNA nano-wire that is far superior to natural DNA. We also demonstrated that the selectivity for the interaction of Mn(II) ion with guanine N7 in G runs is a HOMO-controlled process, and as a consequence, the selectivity for G-metal ion interactions obtained by 15N-NMR studies would directly reflect the HOMO distribution of G-containing sequences in B-DNA.

  4. Fretting fatigue of 2XXX series aerospace aluminum alloys

    NASA Astrophysics Data System (ADS)

    Giummarra, Cindie

    Fretting is a wear mechanism that occurs at the contact region between two materials subject to minute cyclic relative motion. Fretting causes the initiation of surface cracks within the first few thousand cycles, which in the presence of a fatigue stress, grow to cause material failure approximately 10 to 100 times earlier than expected under standard fatigue conditions. Examples of fretting fatigue have been seen in joints in aircraft, and the aerospace industry acknowledges the possibility of catastrophic failure from this mechanism. Improvements in a material's resistance to fretting would benefit aluminum alloys in aerospace applications. This research investigated the effect of microstructural properties on the fretting response in 2XXX series aerospace aluminum alloys. Fretting wear and fretting fatigue tests were conducted to determine the influence of slip characteristics, alloy purity, grain orientation and yield strength on fretting crack initiation and growth. Crack length measurements and micrographs of the fretting indicated there was no significant difference in the fretting response of these alloys based on their microstructural characteristics. Results showed that fretting initiated cracks in the first 1--8% of the life while standard fatigue initiation took around 90% of the life. This reduction in initiation resulted in a shorter life under fretting conditions. Additionally, fretting normalized the initiation time in all alloys which eliminated any intrinsic initiation resistance. The alloys with the highest stress-life (S-N) fatigue properties exhibiting a greater reduction in fatigue strength under fretting conditions. The fretting stresses appeared to influence the crack growth to a distance below the surface of approximately 17mum under fretting fatigue conditions, after which some cracks changed direction and propagated under the influence of the fatigue stress. Under fretting wear conditions, the cracks tended to arrest at a depth of 8

  5. Isolation of Human Genomic DNA Sequences with Expanded Nucleobase Selectivity.

    PubMed

    Rathi, Preeti; Maurer, Sara; Kubik, Grzegorz; Summerer, Daniel

    2016-08-10

    We report the direct isolation of user-defined DNA sequences from the human genome with programmable selectivity for both canonical and epigenetic nucleobases. This is enabled by the use of engineered transcription-activator-like effectors (TALEs) as DNA major groove-binding probes in affinity enrichment. The approach provides the direct quantification of 5-methylcytosine (5mC) levels at single genomic nucleotide positions in a strand-specific manner. We demonstrate the simple, multiplexed typing of a variety of epigenetic cancer biomarker 5mC with custom TALE mixes. Compared to antibodies as the most widely used affinity probes for 5mC analysis, i.e., employed in the methylated DNA immunoprecipitation (MeDIP) protocol, TALEs provide superior sensitivity, resolution and technical ease. We engineer a range of size-reduced TALE repeats and establish full selectivity profiles for their binding to all five human cytosine nucleobases. These provide insights into their nucleobase recognition mechanisms and reveal the ability of TALEs to isolate genomic target sequences with selectivity for single 5-hydroxymethylcytosine and, in combination with sodium borohydride reduction, single 5-formylcytosine nucleobases. PMID:27429302

  6. The Renaissance of Metal-Pyrimidine Nucleobase Coordination Chemistry.

    PubMed

    Lippert, Bernhard; Sanz Miguel, Pablo J

    2016-08-16

    The significance of metal ions for the function and properties of DNA and RNA, long seen primarily under biological aspects and medicinal uses, has recently gained a renewed momentum. This is a consequence of the advent of novel applications in the fields of materials science, biotechnology, and analytical sensor chemistry that relate to the designed incorporation of transition metal ions into nucleic acid base pairs. Ag(+) and Hg(2+) ions, binding to pyrimidine (pym) nucleobases, represent major players in this development. Interestingly, these metal ions were the ones that some 60 years ago started the field! At the same time, the mentioned metal ions had demonstrated a "special relationship" with the pym nucleobases cytosine, thymine, and uracil! Parallel work conducted with oligonucleotides and model nucleobases fostered numerous significant details of these interactions, in particular when X-ray crystallography was involved, correcting earlier views occasionally. Our own activities during the past three to four decades have focused on, among others, the coordination chemistry of transition and main-group metal ions with pym model nucleobases, with an emphasis on Pt(II) and Pd(II). It has always been our goal to deduce, if possible, the potential relevance of our findings for biological processes. It is interesting to put our data, in particular for trans-a2Pt(II) (a = NH3 or amine), into perspective with those of other metal ions, notably Ag(+) and Hg(2+). Irrespective of major differences in kinetics and lability/inertness between d(8) and d(10) metal ions, there is also a lot of similarity in structural aspects as a result of the preferred linear coordination geometry of these species. Moreover, the apparent clustering of metal ions to the pym nucleobases, which is presumably essential for the formation of nanoclusters on oligonucleotide scaffolds, is impressively reflected in model systems, as are reasons for inter-nucleobase cross-links containing more

  7. The Renaissance of Metal-Pyrimidine Nucleobase Coordination Chemistry.

    PubMed

    Lippert, Bernhard; Sanz Miguel, Pablo J

    2016-08-16

    The significance of metal ions for the function and properties of DNA and RNA, long seen primarily under biological aspects and medicinal uses, has recently gained a renewed momentum. This is a consequence of the advent of novel applications in the fields of materials science, biotechnology, and analytical sensor chemistry that relate to the designed incorporation of transition metal ions into nucleic acid base pairs. Ag(+) and Hg(2+) ions, binding to pyrimidine (pym) nucleobases, represent major players in this development. Interestingly, these metal ions were the ones that some 60 years ago started the field! At the same time, the mentioned metal ions had demonstrated a "special relationship" with the pym nucleobases cytosine, thymine, and uracil! Parallel work conducted with oligonucleotides and model nucleobases fostered numerous significant details of these interactions, in particular when X-ray crystallography was involved, correcting earlier views occasionally. Our own activities during the past three to four decades have focused on, among others, the coordination chemistry of transition and main-group metal ions with pym model nucleobases, with an emphasis on Pt(II) and Pd(II). It has always been our goal to deduce, if possible, the potential relevance of our findings for biological processes. It is interesting to put our data, in particular for trans-a2Pt(II) (a = NH3 or amine), into perspective with those of other metal ions, notably Ag(+) and Hg(2+). Irrespective of major differences in kinetics and lability/inertness between d(8) and d(10) metal ions, there is also a lot of similarity in structural aspects as a result of the preferred linear coordination geometry of these species. Moreover, the apparent clustering of metal ions to the pym nucleobases, which is presumably essential for the formation of nanoclusters on oligonucleotide scaffolds, is impressively reflected in model systems, as are reasons for inter-nucleobase cross-links containing more

  8. Communication: Photoactivation of nucleobase bound platinum{sup II} metal complexes: Probing the influence of the nucleobase

    SciTech Connect

    Sen, Ananya; Dessent, Caroline E. H.

    2014-12-28

    We present UV laser action spectra (220-300 nm) of isolated nucleobase-bound Pt{sup II}(CN){sub 4}{sup 2−} complexes, i.e., Pt(CN){sub 4}{sup 2−}⋅M, where M = uracil, thymine, cytosine, and adenine. These metal complex-nucleobase clusters represent model systems for identifying the fundamental photophysical and photochemical processes occurring in photodynamic platinum (II) drug therapies that target DNA. This is the first study to explore the specific role of the nucleobase in the photophysics of the aggregate complex. Each of the complexes studied displays a broadly similar absorption spectra, with a strong λ{sub max} ∼ 4.7 eV absorption band (nucleobase localized chromophore) and a subsequent increase in the absorption intensity towards higher spectral-energy (Pt(CN){sub 4}{sup 2−} localized chromophore). However, strikingly different band widths are observed across the series of complexes, decreasing in the order Pt(CN){sub 4}{sup 2−}⋅Thymine > Pt(CN){sub 4}{sup 2−}⋅Uracil > Pt(CN){sub 4}{sup 2−}⋅Adenine > Pt(CN){sub 4}{sup 2−}⋅Cytosine. Changes in the bandwidth of the ∼4.7 eV band are accompanied by distinctive changes in the photofragment product ions observed following photoexcitation, with the narrower-bandwidth complexes showing a greater propensity to decay via electron detachment decay. We discuss these observations in the context of the distinctive nucleobase-dependent excited state lifetimes.

  9. Action-FRET of a Gaseous Protein

    NASA Astrophysics Data System (ADS)

    Daly, Steven; Knight, Geoffrey; Halim, Mohamed Abdul; Kulesza, Alexander; Choi, Chang Min; Chirot, Fabien; MacAleese, Luke; Antoine, Rodolphe; Dugourd, Philippe

    2016-08-01

    Mass spectrometry is an extremely powerful technique for analysis of biological molecules, in particular proteins. One aspect that has been contentious is how much native solution-phase structure is preserved upon transposition to the gas phase by soft ionization methods such as electrospray ionization. To address this question—and thus further develop mass spectrometry as a tool for structural biology—structure-sensitive techniques must be developed to probe the gas-phase conformations of proteins. Here, we report Förster resonance energy transfer (FRET) measurements on a ubiquitin mutant using specific photofragmentation as a reporter of the FRET efficiency. The FRET data is interpreted in the context of circular dichroism, molecular dynamics simulation, and ion mobility data. Both the dependence of the FRET efficiency on the charge state—where a systematic decrease is observed—and on methanol concentration are considered. In the latter case, a decrease in FRET efficiency with methanol concentration is taken as evidence that the conformational ensemble of gaseous protein cations retains a memory of the solution phase conformational ensemble upon electrospray ionization.

  10. pH-Insensitive FRET voltage dyes.

    PubMed

    Maher, Michael P; Wu, Nyan-Tsz; Ao, Hong

    2007-08-01

    Many high-throughput ion channel assays require the use of voltage-sensitive dyes to detect channel activity in the presence of test compounds. Dye systems employing Förster resonance energy transfer (FRET) between 2 membrane-bound dyes are advantageous in combining high sensitivity, relatively fast response, and ratiometric output. The most widely used FRET voltage dye system employs a coumarin fluorescence donor whose excitation spectrum is pH dependent. The authors have validated a new class of voltage-sensitive FRET donors based on a pyrene moiety. These dyes are significantly brighter than CC2-DMPE and are not pH sensitive in the physiological range. With the new dye system, the authors demonstrate a new high-throughput assay for the acid-sensing ion channel (ASIC) family. They also introduce a novel method for absolute calibration of voltage-sensitive dyes, simultaneously determining the resting membrane potential of a cell. PMID:17517905

  11. FRET-based Molecular Tension Microscopy.

    PubMed

    Gayrard, Charlène; Borghi, Nicolas

    2016-02-01

    Cells generate and experience mechanical forces that may shape tissues and regulate signaling pathways in a variety of physiological or pathological situations. How forces propagate and transduce signals at the molecular level is poorly understood. The advent of FRET-based Molecular Tension Microscopy now allows to achieve mechanical force measurements at a molecular scale with molecular specificity in situ, and thereby better understand the mechanical architecture of cells and tissues, and mechanotransduction pathways. In this review, we will first expose the basic principles of FRET-based MTM and its various incarnations. We will describe different ways of measuring FRET, their advantages and drawbacks. Then, throughout the range of proteins of interest, cells and organisms to which it has been applied, we will review the tests developed to validate the approach, how molecular tension was related to cell functions, and conclude with possible developments and offshoots.

  12. Excitation Spectra of Nucleobases with Multiconfigurational Density Functional Theory.

    PubMed

    Hubert, Mickaël; Jensen, Hans Jørgen Aa; Hedegård, Erik D

    2016-01-14

    Range-separated hybrid methods between wave function theory and density functional theory (DFT) can provide high-accuracy results, while correcting some of the inherent flaws of both the underlying wave function theory and DFT. We here assess the accuracy for excitation energies of the nucleobases thymine, uracil, cytosine, and adenine, using a hybrid between complete active space self-consistent field (CASSCF) and DFT methods. The method is based on range separation, thereby avoiding all double-counting of electron correlation and is denoted long-range CASSCF short-range DFT (CAS-srDFT). Using a linear response extension of CAS-srDFT, we compare the first 7-8 excited states of the nucleobases with perturbative multireference approaches as well as coupled cluster based methods. Our results show that the CAS-srDFT method can provide accurate excitation energies in good correspondence with the computationally more expensive methods. PMID:26669578

  13. Nucleobase appended viologens: Building blocks for new optoelectronic materials

    NASA Astrophysics Data System (ADS)

    Ciobanu, Marius; Asaftei, Simona

    2015-04-01

    We describe here the fabrication, characterization and possible applications of a new type of optical material - consisting of 4,4‧-bipyridinium core ("viologen") and nucleobases i.e. adenine and/or thymine made by H-bonding. The viologen-nucleobase derivatives were used to construct supramolecular structures in a "biomimetic way" with complementary oligonucleotides (ssDNA) and peptide nucleic acids (ssPNA) as templates. The new nanostructured materials are expected to exhibit enhanced optical and optoelectronic properties with application in the field of supramolecular electronics. Such viologen derivatives could be significant in the design of new 2D and 3D materials with potentially application in optoelectronics, molecular electronics or sensoric.

  14. Local piezoresponse and polarization switching in nucleobase thymine microcrystals

    NASA Astrophysics Data System (ADS)

    Bdikin, Igor; Heredia, Alejandro; Neumayer, Sabine M.; Bystrov, Vladimir S.; Gracio, José; Rodriguez, Brian J.; Kholkin, Andrei L.

    2015-08-01

    Thymine (2-oxy-4-oxy-5 methyl pyrimidine) is one of the four nucleobases of deoxyribonucleic acid (DNA). In the DNA molecule, thymine binds to adenine via two hydrogen bonds, thus stabilizing the nucleic acid structure and is involved in pairing and replication. Here, we show that synthetic thymine microcrystals grown from the solution exhibit local piezoelectricity and apparent ferroelectricity, as evidenced by nanoscale electromechanical measurements via Piezoresponse Force Microscopy. Our experimental results demonstrate significant electromechanical activity and polarization switchability of thymine, thus opening a pathway for piezoelectric and ferroelectric-based applications of thymine and, perhaps, of other DNA nucleobase materials. The results are supported by molecular modeling of polarization switching under an external electric field.

  15. Proton Transfer in Nucleobases is Mediated by Water

    SciTech Connect

    Khistyaev, Kirill; Golan, Amir; Bravaya, Ksenia B.; Orms, Natalie; Krylov, Anna I.; Ahmed, Musahid

    2013-08-08

    Water plays a central role in chemistry and biology by mediating the interactions between molecules, altering energy levels of solvated species, modifying potential energy proles along reaction coordinates, and facilitating ecient proton transport through ion channels and interfaces. This study investigates proton transfer in a model system comprising dry and microhydrated clusters of nucleobases. With mass spectrometry and tunable vacuum ultraviolet synchrotron radiation, we show that water shuts down ionization-induced proton transfer between nucleobases, which is very ecient in dry clusters. Instead, a new pathway opens up in which protonated nucleo bases are generated by proton transfer from the ionized water molecule and elimination of a hydroxyl radical. Electronic structure calculations reveal that the shape of the potential energy prole along the proton transfer coordinate depends strongly on the character of the molecular orbital from which the electron is removed, i.e., the proton transfer from water to nucleobases is barrierless when an ionized state localized on water is accessed. The computed energetics of proton transfer is in excellent agreement with the experimental appearance energies. Possible adiabatic passage on the ground electronic state of the ionized system, while energetically accessible at lower energies, is not ecient. Thus, proton transfer is controlled electronically, by the character of the ionized state, rather than statistically, by simple energy considerations.

  16. Nucleobase and nucleoside transport and integration into plant metabolism

    PubMed Central

    Girke, Christopher; Daumann, Manuel; Niopek-Witz, Sandra; Möhlmann, Torsten

    2014-01-01

    Nucleotide metabolism is an essential process in all living organisms. Besides newly synthesized nucleotides, the recycling (salvage) of partially degraded nucleotides, i.e., nucleosides and nucleobases serves to keep the homeostasis of the nucleotide pool. Both types of metabolites are substrates of at least six families of transport proteins in Arabidopsis thaliana (Arabidopsis) with a total of 49 members. In the last years several members of such transport proteins have been analyzed allowing to present a more detailed picture of nucleoside and nucleobase transport and the physiological function of these processes. Besides functioning in nucleotide metabolism it turned out that individual members of the before named transporters exhibit the capacity to transport a wide range of different substrates including vitamins and phytohormones. The aim of this review is to summarize the current knowledge on nucleobase and nucleoside transport processes in plants and integrate this into nucleotide metabolism in general. Thereby, we will focus on those proteins which have been characterized at the biochemical level. PMID:25250038

  17. CTAB enhancement of FRET in DNA structures.

    PubMed

    Oh, Taeseok; Takahashi, Tsukasa; Kim, Sejung; Heller, Michael J

    2016-01-01

    The effect of cetyl-trimethylammonium bromide (CTAB) on enhancing the fluorescence resonance energy transfer (FRET) between two dye-conjugated DNA strands was studied using fluorescence emission spectroscopy and dynamic light scattering (DLS). For hybridized DNA where one strand is conjugated with a TAMRA donor and the other with a TexasRed acceptor, increasing the concentration of CTAB changes the fluorescence emission properties and improves the FRET transfer efficiency through changes in the polarity of the solvent, neutralization of the DNA backbone and micelle formation. For the DNA FRET system without CTAB, the DNA hybridization leads to contact quenching between TAMRA donor and TexasRed acceptor producing reduced donor emission and only a small increase in acceptor emission. At 50 µM CTAB, however, the sheathing and neutralization of the dye-conjugated dsDNA structure significantly reduces quenching by DNA bases and dye interactions, producing a large increase in FRET efficiency, which is almost four fold higher than without CTAB.

  18. Bioinspired optofluidic FRET lasers via DNA scaffolds

    PubMed Central

    Sun, Yuze; Shopova, Siyka I.; Wu, Chung-Shieh; Arnold, Stephen; Fan, Xudong

    2010-01-01

    Optofluidic dye lasers hold great promise for adaptive photonic devices, compact and wavelength-tunable light sources, and micro total analysis systems. To date, however, nearly all those lasers are directly excited by tuning the pump laser into the gain medium absorption band. Here we demonstrate bioinspired optofluidic dye lasers excited by FRET, in which the donor-acceptor distance, ratio, and spatial configuration can be precisely controlled by DNA scaffolds. The characteristics of the FRET lasers such as spectrum, threshold, and energy conversion efficiency are reported. Through DNA scaffolds, nearly 100% energy transfer can be maintained regardless of the donor and acceptor concentration. As a result, efficient FRET lasing is achieved at an unusually low acceptor concentration of micromolar, over 1,000 times lower than that in conventional optofluidic dye lasers. The lasing threshold is on the order of μJ/mm2. Various DNA scaffold FRET lasers are demonstrated to illustrate vast possibilities in optofluidic laser designs. Our work opens a door to many researches and applications such as intracavity bio/chemical sensing, biocontrolled photonic devices, and biophysics. PMID:20798062

  19. A Transition-State Interaction Shifts Nucleobase Ionization Toward Neutrality to Facilitate Small Ribozyme Catalysis

    PubMed Central

    Liberman, Joseph A.; Guo, Man; Jenkins, Jermaine L.; Krucinska, Jolanta; Chen, Yuanyuan; Carey, Paul R.; Wedekind, Joseph E.

    2012-01-01

    One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pKa values for the Ade38 N1-imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pKa value of 6.27 ± 0.05, which agrees strikingly well with values measured by pH-rate analyses. To identify the chemical attributes that contribute to the shifted pKa we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pKa values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pKa >0.8 log units relative to the precatalytic state. The agreement of the microscopic and macroscopic pKa values and the accompanying structural analysis support a mechanism in which Ade38 N1(H)+ functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity. PMID:22989273

  20. Determination of pKa values for deprotonable nucleobases in short model oligonucleotides.

    PubMed

    González-Olvera, Julio C; Martínez-Reyes, José; González-Jasso, Eva; Pless, Reynaldo C

    2015-11-01

    The deprotonation of ionizable nucleobases centrally placed in short model oligonucleotides was examined under different physical conditions, using UV absorption spectroscopy. The oligonucleotide sequences were designed so that only the central base would be ionized over the pH range examined. pKa values of 9.90±0.01 and 9.34±0.04 were determined for the guanine group in the oligomer d-ACAGCAC and 2'-deoxyguanosine, respectively, both at 25°C and 0.1M NaCl. Lengthening the oligonucleotide up to the tridecamer stage further increases the pKa of the central guanine moiety. Electrolyte concentration, temperature, and mixed water-ethanol solvents affect the acidity of the central base. Changes in the sequence surrounding the central guanine can also have a significant effect, especially in the case of strongly stacking sequences. The pKa values were also determined for the hepta(2'-O-methyl)ribonucleotide and the heptamer PNA of identical sequence, as well as for oligodeoxyribonucleotides with different deprotonable bases, viz. thymine, uracil, or hypoxanthine, in the central position. The results are interpreted in terms of the electric-field effect exerted on the departing proton by the negative electric charges located on the internucleotide phosphate groups, and calculations show this effect to approximately explain the magnitude of the pKa difference observed between the deoxyriboheptanucleotide and its electroneutral PNA analogue. PMID:26188860

  1. 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

  2. Nonenzymatic template-directed reactions on altritol oligomers, preorganized analogues of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Zielinski, M.; Allart, B.; Kerremans, L.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    2000-01-01

    Altritol nucleic acids (ANAs) are RNA analogues with a phosphorylated D-altritol backbone. The nucleobase is attached at the 2-(S)-position of the carbohydrate moiety. We report that ANA oligomers are superior to the corresponding DNA, RNA, and HNA (hexitol nucleic acid) in supporting efficient nonenzymatic template-directed synthesis of complementary RNAs from nucleoside-5'-phosphoro-2-methyl imidazolides. Activated ANA and HNA monomers do not oligomerize efficiently on DNA, RNA, HNA, or ANA templates.

  3. A Re-Examination of Nucleobases in Carbonaceous Chondrites

    NASA Astrophysics Data System (ADS)

    Martins, Z.; Botta, O.; de Vries, M.; Becker, L.; Ehrenfreund, E.

    The biomolecular building blocks of life, as we know it, are amino acids, purines and pyrimidines. The latter two form the bases of DNA and RNA, molecules that are used in the storage, transcription and translation of genetic information in all terrestrial organisms. A dedicated search for these compounds in meteorites can shed light on the origins of life in two ways: (i) Results can help assess the plausibility of extraterrestrial formation of prebiotic molecules followed by their meteoritic delivery to the early Earth. (ii) Such studies can also provide insights into possible prebiotic synthetic routes. We will search for these compounds in selected carbonaceous chondrites using formic acid extraction and reverse phase high performance liquid chromatography (HPLC) to isolate specific nucleobases from the bulk meteorite material as previously reported [1,2,3]. We will also use a new technique, resonant two-photon ionization mass spectrometry (R2PI) that can, not only identify organic compounds by their mass, but at the same time by their vibronic spectroscopy [4]. R2PI dramatically enhances the specificity for certain compounds (e.g. amino acids, nucleobases) and allows for distinction of structural isomers, tautomers and enantiomers as well as providing additional information due to isotope shifts. The optical spectroscopy can thus help us to further discriminate between terrestrial and extraterrestrial nucleobases. References: [1] Van Der Velden, W. and Schwarts, A. W. (1977) Geochim. Cosmochim. Acta, 41, 961-968. [2] Stoks, P. G. and Schwartz, A. W. (1979a) Nature, 282, 709-10. [3] Glavin, D. P. and Bada, J. L. (2004) In Lunar and Planetary Science XXXV, Abstract # 1022, Houston. [4] Nir, E., Grace, L. I., Brauer, B. and de Vries, M. S. (1999) Journal of the American Chemical Society, 121, 4896-4897.

  4. Oxidative stress modulates nucleobase transport in microvascular endothelial cells.

    PubMed

    Bone, Derek B J; Antic, Milica; Vilas, Gonzalo; Hammond, James R

    2014-09-01

    Purine nucleosides and nucleobases play key roles in the physiological response to vascular ischemia/reperfusion events. The intra- and extracellular concentrations of these compounds are controlled, in part, by equilibrative nucleoside transporter subtype 1 (ENT1; SLC29A1) and by equilibrative nucleobase transporter subtype 1 (ENBT1). These transporters are expressed at the membranes of numerous cell types including microvascular endothelial cells. We studied the impact of reactive oxygen species on the function of ENT1 and ENBT1 in primary (CMVEC) and immortalized (HMEC-1) human microvascular endothelial cells. Both cell types displayed similar transporter expression profiles, with the majority (>90%) of 2-chloro[(3)H]adenosine (nucleoside) uptake mediated by ENT1 and [(3)H]hypoxanthine (nucleobase) uptake mediated by ENBT1. An in vitro mineral oil-overlay model of ischemia/reperfusion had no effect on ENT1 function, but significantly reduced ENBT1 Vmax in both cell types. This decrease in transport function was mimicked by the intracellular superoxide generator menadione and could be reversed by the superoxide dismutase mimetic MnTMPyP. In contrast, neither the extracellular peroxide donor TBHP nor the extracellular peroxynitrite donor 3-morpholinosydnonimine (SIN-1) affected ENBT1-mediated [(3)H]hypoxanthine uptake. SIN-1 did, however, enhance ENT1-mediated 2-chloro[(3)H]adenosine uptake. Our data establish HMEC-1 as an appropriate model for study of purine transport in CMVEC. Additionally, these data suggest that the generation of intracellular superoxide in ischemia/reperfusion leads to the down-regulation of ENBT1 function. Modification of purine transport by oxidant stress may contribute to ischemia/reperfusion induced vascular damage and should be considered in the development of therapeutic strategies.

  5. A Versatile Approach Towards Nucleobase-Modified Aptamers.

    PubMed

    Tolle, Fabian; Brändle, Gerhard M; Matzner, Daniel; Mayer, Günter

    2015-09-01

    A novel and versatile method has been developed for modular expansion of the chemical space of nucleic acid libraries, thus enabling the generation of nucleobase-modified aptamers with unprecedented recognition properties. Reintroduction of the modification after enzymatic replication gives broad access to many chemical modifications. This wide applicability, which is not limited to a single modification, will rapidly advance the application of in vitro selection approaches beyond what is currently feasible and enable the generation of aptamers to many targets that have so far not been addressable. PMID:26224087

  6. Adeninealkylresorcinol, the first alkylresorcinol tethered with nucleobase from Lasiodiplodia sp.

    PubMed

    Gao, Yu-Meng; Sun, Tian-Yu; Ma, Min; Chen, Guo-Dong; Zhou, Zheng-Qun; Wang, Chuan-Xi; Hu, Dan; Chen, Li-Guo; Yao, Xin-Sheng; Gao, Hao

    2016-07-01

    Adeninealkylresorcinol (1), an unusual alkylresorcinol with adenine-alkylresorcinol conjoined skeleton, was isolated from an endophytic fungus Lasiodiplodia sp. obtained from a traditional Chinese medicine Houttuynia cordata Thunb., together with three new biogenetically related compounds (2-4). Their structures were elucidated by comprehensive spectroscopic analysis, and the absolute configuration of 4 was determined by the modified Mosher's method and quantum chemical calculation. Among them, adeninealkylresorcinol (1) is the first alkylresorcinol tethered with nucleobase. In addition, the antioxidant, cytotoxic, and antimicrobial activities of 1-3 were evaluated. PMID:27343368

  7. Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy.

    PubMed

    Hirata, Eishu; Kiyokawa, Etsuko

    2016-09-20

    Förster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena.

  8. Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy.

    PubMed

    Hirata, Eishu; Kiyokawa, Etsuko

    2016-09-20

    Förster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena. PMID:27475975

  9. Investigation of Fretting by Microscopic Observation

    NASA Technical Reports Server (NTRS)

    Godfrey, Douglas

    1951-01-01

    An experimental investigation, using microscopic observation and color motion photomicrographs of the action, was conducted to determine the cause of fretting. Glass and other noncorrosive materials, as well as metals, were used as specimens. A very simple apparatus vibrated convex surfaces in contact with stationary flat surfaces at frequencies of 120 cycles or less than l cycle per second, an amplitude of 0.0001 inch, and load of 0.2 pound.

  10. An optofluidic FRET laser using quantum dots as donor

    NASA Astrophysics Data System (ADS)

    Chen, Qiushu; Kiraz, Alper; Fan, Xudong

    2016-03-01

    The integration of optofluidic laser and FRET mechanism provides novel research frontiers, including sensitive biochemical analysis and novel photonic devices, such as on-chip coherent light sources and bio-tunable lasers. Here we investigated an optofluidic FRET laser using quantum dots (QDs) as FRET donors. We achieved lasing from Cy5 as the acceptor in the QD-Cy5 pair with excitation at 450 nm where Cy5 has negligible absorption by itself. The threshold was approximately 14 μJ/mm2. The demonstrated capability of QDs as the donor in a FRET laser greatly improves the versatility of optofluidic laser operation due to the broad and large absorption cross section of QDs in the blue and UV spectral region. The excitation efficiency of the acceptor molecules through FRET channel was also analyzed, showing that the energy transfer rate and the non-radiative Auger recombination rate of QDs plays a significant role in FRET laser performance.

  11. The role of oxidation in the fretting wear process

    NASA Technical Reports Server (NTRS)

    Bill, R. C.

    1980-01-01

    Fretting experiments were conducted on titanium, a series of Ni-Cr-Al alloys and on some high temperature turbine alloys at room temperature and at elevated temperatures in air and in various inert environments. It was found that, depending on temperature and environment, the fretting behavior of the materials examined could be classified according to four general types of behavior. Briefly, these types of behavior were: (1) the complete absence of oxidation, as in inert environments, generally leading to low rates of fretting wear but high fretting friction; (2) gradual attrition of surface oxide with each fretting stroke, found in these experiments to operate in concert with other dominating mechanisms; (3) rapid oxidation at surface fatigue damage sites, resulting in undermining and rapid disintegration of the load bearing surface; and (4) the formation of coherent, protective oxide film, resulting in low rates of fretting wear. An analytical model predicting conditions favorable to the fourth type of behavior was outlined.

  12. Fretting of titanium at temperatures to 650 C in air

    NASA Technical Reports Server (NTRS)

    Bill, R. C.

    1975-01-01

    Fretting wear experiments were conducted on high-purity titanium at temperatures up to 650 C. Results indicate that up to about 500 C, the fretting wear increases with temperature. A further increase in the temperature up to 650 C results in decreasing fretting wear. This change in trend of fretting wear with temperature is shown to be associated with a change in oxidation rate. Additional experiments at 650 C showed a transmission from a low rate of fretting wear to a higher rate occurred after exposure to a number of fretting cycles; the number of cycles required to cause this transition was dependent on the normal load. Scanning electron microscopy studies revealed that this transition was marked by cracking and disruption of the surface oxide film. A model was proposed that coupled the oxidation rate kinetics of titanium at 650 C with the occurrence of wear at the surface of the oxide film.

  13. High-Frequency, High-Temperature Fretting Experiments

    NASA Technical Reports Server (NTRS)

    Matlik, J. F.; Farris, T. N.; Haake, F. K.; Swanson, G. R.; Duke, G. C.

    2005-01-01

    Fretting is a structural damage mechanism observed when two nominally clamped surfaces are subjected to an oscillatory loading. A critical location for fretting induced damage has been identified at the blade/disk and blade/damper interfaces of gas turbine engine turbomachinery and space propulsion components. The high-temperature, high-frequency loading environment seen by these components lead to severe stress gradients at the edge-of-contact. These contact stresses drive crack nucleation and propagation in fretting and are very sensitive to the geometry of the contacting bodies, the contact loads, materials, temperature, and contact surface tribology (friction). To diagnose the threat that small and relatively undetectable fretting cracks pose to damage tolerance and structural integrity of in-service components, the objective of this work is to develop a well-characterized experimental fretting rig capable of investigating fretting behavior of advanced aerospace alloys subjected to load and temperature conditions representative of such turbomachinery components.

  14. FRET-based glucose monitoring for bioprocessing

    NASA Astrophysics Data System (ADS)

    Bartolome, Amelita; Smalls-Mantey, Lauren; Lin, Debora; Rao, Govind; Tolosa, Leah

    2006-02-01

    The glucose-mediated conformational changes in the glucose binding protein (GBP) have been exploited in the development of fluorescence based glucose sensors. The fluorescence response is generated by a polarity sensitive dye attached to a specific site. Such fluorescent sensors respond to submicromolar glucose at diffusion-controlled rates mimicking the wild type. However, such sensors have been limited to in vitro glucose sensing because of the preliminary dye-labeling step. In the study described here, the dye-labeling step is omitted by genetically encoding the GBP with two green fluorescent mutants namely, the green fluorescent protein (GFP) and the yellow fluorescent protein (YFP) in the N- and C-terminal ends, respectively. These two GFP mutants comprise a fluorescence resonance energy transfer (FRET) donor and acceptor pair. Thus, when glucose binds with GBP, the conformational changes affect the FRET efficiency yielding a dose-dependent response. A potential application for this FRET-based glucose biosensor is online glucose sensing in bioprocessing and cell culture. This was demonstrated by the measurement of glucose consumption in yeast fermentation. Further development of this system should yield in vivo measurement of glucose in bioprocesses.

  15. Two-Photon-Induced Fluorescence of Isomorphic Nucleobase Analogs

    PubMed Central

    Lane, Richard S. K.; Jones, Rosemary; Sinkeldam, Renatus W.

    2014-01-01

    Five isomorphic fluorescent uridine mimics have been subjected to two-photon (2P) excitation analysis to investigate their potential applicability as non-perturbing probes for the single-molecule detection of nucleic acids. We find that small structural differences can cause major changes in the two-photon excitation probability, with the 2P cross sections varying by over one order of magnitude. Two of the probes, both furan-modified uridine analogs, have the highest 2P cross sections (3.8 GM and 7.6 GM) reported for nucleobase analogs, using a conventional Ti:sapphire laser for excitation at 690 nm; they also have the lowest emission quantum yields. In contrast, the analogs with the highest reported quantum yields have the lowest 2P cross sections. The structure-photophysical property relationship presented here is a first step towards the rational design of emissive nucleobase analogs with controlled 2P characteristics. The results demonstrate the potential for major improvements through judicious structural modifications. PMID:24604669

  16. RNA fragment modeling with a nucleobase discrete-state model

    NASA Astrophysics Data System (ADS)

    Zhang, Jian; Bian, Yunqiang; Lin, Hui; Wang, Wei

    2012-02-01

    In this work we develop an approach for predicting the tertiary structures of RNA fragments by combining an RNA nucleobase discrete state (RNAnbds) model, a sequential Monte Carlo method, and a statistical potential. The RNAnbds model is designed for optimizing the configuration of nucleobases with respect to their preceding ones along the sequence and their spatial neighbors, in contrast to previous works that focus on RNA backbones. The tests of our approach with the fragments taken from a small RNA pseudoknot and a 23S ribosome RNA show that for short fragments (<10 nucleotides), the root mean square deviations (RMSDs) between the predicted and the experimental ones are generally smaller than 3 Å; for slightly longer fragments (10-15 nucleotides), most RMSDs are smaller than 4 Å. The comparison of our method with another physics-based predictor with a testing set containing nine loops shows that ours is superior in both accuracy and efficiency. Our approach is useful in facilitating RNA three-dimensional structure prediction as well as loop modeling. It also holds the promise of providing insight into the structural ensembles of RNA loops.

  17. Microstructural characterization of titanium alloys with fretting damage

    NASA Astrophysics Data System (ADS)

    Swalla, Dana Ray

    The primary focus of this work is to understand the role of microstructure in the fretting damage process and develop quantifying measures in fretting damage accumulation in a dual phase Ti-6Al-4V as well as two single phase materials: commercially pure titanium (CP-Ti), which consists of pure alpha-phase titanium, and a near alpha Ti-5Al-2.5Sn. The size and distribution of crystallographic orientation of the alpha-phase, which has an HCP crystalline structure, is significant in fretting crack formation. In particular, the effect of slip displacement amplitude and number of fretting cycles on the evolution of grain morphology, grain orientation, misorientation distribution, composition, and microhardness is investigated. The fretting behavior is also related to the macroscopic monotonic and cyclic deformation response. The research goals are accomplished using state-of-the-art surface characterization tools such as orientation image microscopy (OIM) using electron backscatter diffraction (EBSD), energy dispersive X-ray analysis (EDX) and nanoindentation. This study is the first of its kind to use OIM to characterize fretting damage and also makes contributions to the body of knowledge about deformation mechanisms in titanium alloys. The results provide a foundation for developing and validating computational crystal plasticity models and their application to fretting and sliding contact problems. New fretting assessment measures have also been identified and have application for components that suffer from fretting wear and/or fatigue related failures.

  18. Effect of humidity on fretting wear of several pure metals

    NASA Technical Reports Server (NTRS)

    Goto, H.; Buckley, D. H.

    1984-01-01

    Fretting wear experiments with several pure metals were conducted in air at various relative humidity levels. The materials used were iron, aluminum, copper, silver, chromium, titanium, and nickel. Each pure metal had a maximum fretting wear volume at a specific humidity level RH sub max that was not dependent on mechanical factors such as contact load, fretting amplitude, and frequency in the ranges studied. The weight loss due to fretting wear at RH sub max for each pure metal decreased with increasing heat of oxygen adsorption on the metal, indicating that adhesive wear dominated at RH sub max.

  19. In silico FRET from simulated dye dynamics

    NASA Astrophysics Data System (ADS)

    Hoefling, Martin; Grubmüller, Helmut

    2013-03-01

    Single molecule fluorescence resonance energy transfer (smFRET) experiments probe molecular distances on the nanometer scale. In such experiments, distances are recorded from FRET transfer efficiencies via the Förster formula, E=1/(1+(). The energy transfer however also depends on the mutual orientation of the two dyes used as distance reporter. Since this information is typically inaccessible in FRET experiments, one has to rely on approximations, which reduce the accuracy of these distance measurements. A common approximation is an isotropic and uncorrelated dye orientation distribution. To assess the impact of such approximations, we present the algorithms and implementation of a computational toolkit for the simulation of smFRET on the basis of molecular dynamics (MD) trajectory ensembles. In this study, the dye orientation dynamics, which are used to determine dynamic FRET efficiencies, are extracted from MD simulations. In a subsequent step, photons and bursts are generated using a Monte Carlo algorithm. The application of the developed toolkit on a poly-proline system demonstrated good agreement between smFRET simulations and experimental results and therefore confirms our computational method. Furthermore, it enabled the identification of the structural basis of measured heterogeneity. The presented computational toolkit is written in Python, available as open-source, applicable to arbitrary systems and can easily be extended and adapted to further problems. Catalogue identifier: AENV_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENV_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GPLv3, the bundled SIMD friendly Mersenne twister implementation [1] is provided under the SFMT-License. No. of lines in distributed program, including test data, etc.: 317880 No. of bytes in distributed program, including test data, etc.: 54774217 Distribution format: tar.gz Programming language

  20. Click Reaction on Solid Phase Enables High Fidelity Synthesis of Nucleobase-Modified DNA.

    PubMed

    Tolle, Fabian; Rosenthal, Malte; Pfeiffer, Franziska; Mayer, Günter

    2016-03-16

    The post-synthetic functionalization of nucleic acids via click chemistry (CuAAC) has seen tremendous implementation, extending the applicability of nucleobase-modified nucleic acids in fields like fluorescent labeling, nanotechnology, and in vitro selection. However, the production of large quantities of high-density functionalized material via solid phase synthesis has been hampered by oxidative by-product formation associated with the alkaline workup conditions. Herein, we describe a rapid and cost-effective protocol for the high fidelity large-scale production of nucleobase-modified nucleic acids, exemplified with a recently described nucleobase-modified aptamer.

  1. Click Reaction on Solid Phase Enables High Fidelity Synthesis of Nucleobase-Modified DNA.

    PubMed

    Tolle, Fabian; Rosenthal, Malte; Pfeiffer, Franziska; Mayer, Günter

    2016-03-16

    The post-synthetic functionalization of nucleic acids via click chemistry (CuAAC) has seen tremendous implementation, extending the applicability of nucleobase-modified nucleic acids in fields like fluorescent labeling, nanotechnology, and in vitro selection. However, the production of large quantities of high-density functionalized material via solid phase synthesis has been hampered by oxidative by-product formation associated with the alkaline workup conditions. Herein, we describe a rapid and cost-effective protocol for the high fidelity large-scale production of nucleobase-modified nucleic acids, exemplified with a recently described nucleobase-modified aptamer. PMID:26850226

  2. BOBA FRET: bootstrap-based analysis of single-molecule FRET data.

    PubMed

    König, Sebastian L B; Hadzic, Mélodie; Fiorini, Erica; Börner, Richard; Kowerko, Danny; Blanckenhorn, Wolf U; Sigel, Roland K O

    2013-01-01

    Time-binned single-molecule Förster resonance energy transfer (smFRET) experiments with surface-tethered nucleic acids or proteins permit to follow folding and catalysis of single molecules in real-time. Due to the intrinsically low signal-to-noise ratio (SNR) in smFRET time traces, research over the past years has focused on the development of new methods to extract discrete states (conformations) from noisy data. However, limited observation time typically leads to pronounced cross-sample variability, i.e., single molecules display differences in the relative population of states and the corresponding conversion rates. Quantification of cross-sample variability is necessary to perform statistical testing in order to assess whether changes observed in response to an experimental parameter (metal ion concentration, the presence of a ligand, etc.) are significant. However, such hypothesis testing has been disregarded to date, precluding robust biological interpretation. Here, we address this problem by a bootstrap-based approach to estimate the experimental variability. Simulated time traces are presented to assess the robustness of the algorithm in conjunction with approaches commonly used in thermodynamic and kinetic analysis of time-binned smFRET data. Furthermore, a pair of functionally important sequences derived from the self-cleaving group II intron Sc.ai5γ (d3'EBS1/IBS1) is used as a model system. Through statistical hypothesis testing, divalent metal ions are shown to have a statistically significant effect on both thermodynamic and kinetic aspects of their interaction. The Matlab source code used for analysis (bootstrap-based analysis of smFRET data, BOBA FRET), as well as a graphical user interface, is available via http://www.aci.uzh.ch/rna/. PMID:24386343

  3. BOBA FRET: Bootstrap-Based Analysis of Single-Molecule FRET Data

    PubMed Central

    König, Sebastian L. B.; Hadzic, Mélodie; Fiorini, Erica; Börner, Richard; Kowerko, Danny; Blanckenhorn, Wolf U.; Sigel, Roland K. O.

    2013-01-01

    Time-binned single-molecule Förster resonance energy transfer (smFRET) experiments with surface-tethered nucleic acids or proteins permit to follow folding and catalysis of single molecules in real-time. Due to the intrinsically low signal-to-noise ratio (SNR) in smFRET time traces, research over the past years has focused on the development of new methods to extract discrete states (conformations) from noisy data. However, limited observation time typically leads to pronounced cross-sample variability, i.e., single molecules display differences in the relative population of states and the corresponding conversion rates. Quantification of cross-sample variability is necessary to perform statistical testing in order to assess whether changes observed in response to an experimental parameter (metal ion concentration, the presence of a ligand, etc.) are significant. However, such hypothesis testing has been disregarded to date, precluding robust biological interpretation. Here, we address this problem by a bootstrap-based approach to estimate the experimental variability. Simulated time traces are presented to assess the robustness of the algorithm in conjunction with approaches commonly used in thermodynamic and kinetic analysis of time-binned smFRET data. Furthermore, a pair of functionally important sequences derived from the self-cleaving group II intron Sc.ai5γ (d3'EBS1*/IBS1*) is used as a model system. Through statistical hypothesis testing, divalent metal ions are shown to have a statistically significant effect on both thermodynamic and kinetic aspects of their interaction. The Matlab source code used for analysis (bootstrap-based analysis of smFRET data, BOBA FRET), as well as a graphical user interface, is available via http://www.aci.uzh.ch/rna/. PMID:24386343

  4. Optofluidic FRET Lasers Using Aqueous Quantum Dots as Donors

    PubMed Central

    Chen, Qiushu; Kiraz, Alper; Fan, Xudong

    2015-01-01

    An optofluidic FRET (fluorescence resonance energy transfer) laser is formed by putting FRET pairs inside a microcavity acting as gain medium. This integration of optofluidic laser and FRET mechanism provides novel research frontiers, including sensitive biochemical analysis and novel photonic devices, such as on-chip coherent light sources and bio-tunable lasers. Here we investigated an optofluidic FRET laser using quantum dots (QDs) as FRET donors. We achieved lasing from Cy5 as the acceptor in the QD-Cy5 pair when excited at 450 nm where Cy5 has negligible absorption by itself. The threshold was approximately 14 µJ/mm2. The demonstrated capability of QDs as the donor in a FRET laser greatly improves the versatility of optofluidic laser operation due to the broad and large absorption cross section of QDs in the blue and UV spectral region. The excitation efficiency of the acceptor molecules through FRET channel was also analyzed, showing that the energy transfer rate and the non-radiative Auger recombination rate of QDs plays a significant role in FRET laser performance. PMID:26659274

  5. Optofluidic FRET lasers using aqueous quantum dots as donors.

    PubMed

    Chen, Qiushu; Kiraz, Alper; Fan, Xudong

    2016-01-21

    An optofluidic FRET (fluorescence resonance energy transfer) laser is formed by putting FRET pairs inside a microcavity acting as a gain medium. This integration of an optofluidic laser and the FRET mechanism provides novel research frontiers, including sensitive biochemical analysis and novel photonic devices, such as on-chip coherent light sources and bio-tunable lasers. Here, we investigated an optofluidic FRET laser using quantum dots (QDs) as FRET donors. We achieved lasing from Cy5 as the acceptor in a QD-Cy5 pair upon excitation at 450 nm, where Cy5 has negligible absorption by itself. The threshold was approximately 14 μJ mm(-2). The demonstrated capability of QDs as donors in the FRET laser greatly improves the versatility of optofluidic laser operation due to the broad and large absorption cross section of the QDs in the blue and UV spectral regions. The excitation efficiency of the acceptor molecules through a FRET channel was also analyzed, showing that the energy transfer rate and the non-radiative Auger recombination rate of QDs play a significant role in FRET laser performance.

  6. Metal ion mediated nucleobase recognition by the ZTP riboswitch

    PubMed Central

    Trausch, Jeremiah J.; Marcano-Velázquez, Joan G.; Matyjasik, Michal M.; Batey, Robert T.

    2015-01-01

    SUMMARY The ZTP riboswitch is a widespread family of regulatory RNAs that upregulate de novo purine synthesis in response to increased intracellular levels of ZTP or ZMP (AICAR). As an important intermediate in purine biosynthesis, ZMP also serves as a proxy for the concentration of 10-formyltetrahydrofolate, a key component of one carbon metabolism. Here we report the structure of the ZTP riboswitch bound to ZMP at a resolution of 1.80 Å. The RNA contains two subdomains brought together through a long-range pseudoknot further stabilized through helix-helix packing. ZMP is bound at the subdomain interface of the RNA through a set of interactions with the ligand's base, ribose sugar and phosphate moieties. Unique to nucleobase recognition by RNAs, the Z base is inner sphere coordinated to a magnesium cation bound by two backbone phosphates. This interaction, along with steric hindrance by the backbone, imparts specificity over related analogs such as ATP/AMP. PMID:26144884

  7. Does stacking restrain the photodynamics of individual nucleobases?

    PubMed

    Nachtigallová, Dana; Zelený, Tomás; Ruckenbauer, Matthias; Müller, Thomas; Barbatti, Mario; Hobza, Pavel; Lischka, Hans

    2010-06-23

    Nonadiabatic photodynamical simulations of 4-aminopyrimidine (4-APy) used as a model for adenine were performed by embedding it between two stacking methyl-guanine (mGua) molecules to determine the effect of spatial restrictions on the ultrafast photodeactivation mechanism of this nucleobase. A hybrid multiconfigurational ab initio/molecular mechanical approach in combination with surface hopping was used. During the dynamics the formation of a significant fraction of intrastrand hydrogen bonding from 4-APy to mGua above and below is observed. These findings show that this type of hydrogen bond may play an important role for the photodynamics within one DNA strand and that it should be of interest even in irregular segments of double stranded nucleic acids structures. The relaxation mechanism of internal conversion to the ground state is dominated by ring puckering, and an overall elongation of the lifetime of the embedded system by approximately 20% as compared to the isolated 4-APy is computed. PMID:20513159

  8. Sensitive cylindrical SERS substrate array for rapid microanalysis of nucleobases.

    PubMed

    Rajapandiyan, Panneerselvam; Yang, Jyisy

    2012-12-01

    In this work, a cylindrical-substrate array for surface-enhanced Raman scattering (SERS) measurements was developed to enable analysis of nucleobases in a few microliters of liquid. To eliminate uncertainties associated with SERS detection of aqueous samples, a new type of cylindrical SERS substrate was designed to confine the aqueous sample at the tip of the SERS probe. Poly(methyl methacrylate) (PMMA) optical fibers in a series of different diameters were used as the basic substrate. A solution of poly(vinylidene fluoride)/dimethylformamide (PVDF/DMF) was used to coat the tip of each fiber to increase the surface roughness and facilitate adsorption of silver nanoparticles (AgNPs) for enhancing Raman signals. A chemical reduction method was used to form AgNPs in and on the PVDF coating layer. The reagents and reaction conditions were systematically examined with the aim of estimating the optimum parameters. Unlike the spreading of aqueous sample on most SERS substrates, particularly flat ones, the new SERS substrates showed enough hydrophobicity to restrict aqueous sample to the tip area, thus enabling quantitative analysis. The required volume of sample could be as low as 1 μL with no need for a drying step in the procedure. By aligning the cylindrical SERS substrates into a solid holder, an array of cylindrical substrates was produced for mass analysis of aqueous samples. This new substrate improves both reproducibility and sensitivity for detection in aqueous samples. The enhancement factor approaches 7 orders in magnitude with a relative standard error close to 8%. Using the optimized conditions, nucleobases of adenine, cytosine, thymine, and uracil could be detected with limits approaching a few hundreds nanomolar in only a few microliters of solution. PMID:23140099

  9. Anchoring of FRET Sensors—A Requirement for Spatiotemporal Resolution

    PubMed Central

    Ivanova, Elena V.; Figueroa, Ricardo A.; Gatsinzi, Tom; Hallberg, Einar; Iverfeldt, Kerstin

    2016-01-01

    FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling. PMID:27196902

  10. Large Scale Bacterial Colony Screening of Diversified FRET Biosensors

    PubMed Central

    Litzlbauer, Julia; Schifferer, Martina; Ng, David; Fabritius, Arne; Thestrup, Thomas; Griesbeck, Oliver

    2015-01-01

    Biosensors based on Förster Resonance Energy Transfer (FRET) between fluorescent protein mutants have started to revolutionize physiology and biochemistry. However, many types of FRET biosensors show relatively small FRET changes, making measurements with these probes challenging when used under sub-optimal experimental conditions. Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors. Here we describe procedures for optimizing FRET changes by large scale screening of mutant biosensor libraries in bacterial colonies. We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions. The procedures reported here may help in improving FRET changes in multiple suitable classes of biosensors. PMID:26061878

  11. Probing protein-lipid interactions by FRET between membrane fluorophores

    NASA Astrophysics Data System (ADS)

    Trusova, Valeriya M.; Gorbenko, Galyna P.; Deligeorgiev, Todor; Gadjev, Nikolai

    2016-09-01

    Förster resonance energy transfer (FRET) is a powerful fluorescence technique that has found numerous applications in medicine and biology. One area where FRET proved to be especially informative involves the intermolecular interactions in biological membranes. The present study was focused on developing and verifying a Monte-Carlo approach to analyzing the results of FRET between the membrane-bound fluorophores. This approach was employed to quantify FRET from benzanthrone dye ABM to squaraine dye SQ-1 in the model protein-lipid system containing a polycationic globular protein lysozyme and negatively charged lipid vesicles composed of phosphatidylcholine and phosphatidylglycerol. It was found that acceptor redistribution between the lipid bilayer and protein binding sites resulted in the decrease of FRET efficiency. Quantification of this effect in terms of the proposed methodology yielded both structural and binding parameters of lysozyme-lipid complexes.

  12. First Principles Study of Nuclear Quadrupole Interactions in Single and Double Chain DNA and Solid Nucleobases

    NASA Astrophysics Data System (ADS)

    Das, T. P.; Pink, R. H.; Badu, S. R.; Dubey, Archana; Scheicher, R. H.; Saha, H. P.; Chow, Lee; Huang, M. B.

    2009-03-01

    Nuclear Quadrupole Interactions (NQI) of ^17O, ^14N and ^2H nuclei have been studied for free nucleobases and nucleobases in single strand and double strand DNA and in solid state. Our first-principles investigations were carried out using the Gaussian 2003 set of programs to implement the Hartree-Fock procedure combined with many-body effects included using many-body perturbation theory. As expected for NQI in general, many-body effects are found to be small. Results will be presented for the quadrupole coupling constants (e^2qQ) and asymmetry parameters (η) for the nucleobases in the various environments. Trends in e^2qQ and η in the different environments will be discussed. In the case of the solid nucleobases, comparisons will be made with available experimental data [1] for ^17O nuclei.[3pt] [1] Gang Wu et al., J. Am. Chem. Soc. 124, 1768 (2002)

  13. Binding Strength of Nucleobases and Nucleosides on Silver Nanoparticles Probed by a Colorimetric Method.

    PubMed

    Yu, Lu; Li, Na

    2016-06-01

    Because of their unique and tunable properties, oligonucleotide-functionalized noble metal nanoparticles have provided a versatile platform for various engineering and biomedical applications. The vast majority of such applications were demonstrated with gold nanoparticles (AuNPs) while only a few were demonstrated with sliver nanoparticles (AgNPs). This is largely due to the lack of robust protocols to functionalize AgNPs with thiol-modified oligonucleotides. Previous studies have revealed strong interactions between nucleobases and AgNPs. This could enable an alternative way to functionalize AgNPs with non-thiolated oligonucleotides. However, there is no quantitative study on the interaction strengths between AgNPs and oligonucleotides. Several methods have been used for quantitative evaluation of the interaction strengths between AuNPs and oligonucleotides. These methods often require specialized equipment that might not be widely accessible or rely on labor-intensive procedures to obtain the adsorption isotherms. Herein, we developed a colorimetric method, as a simple and high-throughput alternative of existing methods, to quantify the binding strength between AgNPs and nucleobases/nucleosides. In this colorimetric method, concentration-dependent destabilizing effects of nucleobase/nucleoside adsorption on AgNPs are utilized to indirectly quantify the amount of nucleobases/nucleosides adsorbed on AgNPs, thus deriving the binding strength between AgNPs and nucleobases/nucleosides. First, the concentration-dependent AgNP aggregation kinetics in the presence of nucleobases/nucleosides were systematically investigated. Then, this colorimetric method was used to determine the binding strengths between AgNPs and various DNA/RNA nucleobases/nucleosides. It was found that the ranking of interaction strengths between AgNPs and DNA/RNA nucleosides (dC < dT < dA, rC < rU < rA) is generally agreed with that between AgNPs and corresponding nucleobases (C < T < U < A). This

  14. FLIM-FRET for Cancer Applications

    PubMed Central

    Rajoria, Shilpi; Zhao, Lingling; Intes, Xavier; Barroso, Margarida

    2015-01-01

    Optical imaging assays, especially fluorescence molecular assays, are minimally invasive if not completely noninvasive, and thus an ideal technique to be applied to live specimens. These fluorescence imaging assays are a powerful tool in biomedical sciences as they allow the study of a wide range of molecular and physiological events occurring in biological systems. Furthermore, optical imaging assays bridge the gap between the in vitro cell-based analysis of subcellular processes and in vivo study of disease mechanisms in small animal models. In particular, the application of Förster resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM), well-known techniques widely used in microscopy, to the optical imaging assay toolbox, will have a significant impact in the molecular study of protein-protein interactions during cancer progression. This review article describes the application of FLIM-FRET to the field of optical imaging and addresses their various applications, both current and potential, to anti-cancer drug delivery and cancer research. PMID:26023359

  15. Quantum mechanical treatment of binding energy between DNA nucleobases and carbon nanotube: A DFT analysis

    NASA Astrophysics Data System (ADS)

    Chehel Amirani, Morteza; Tang, Tian; Cuervo, Javier

    2013-12-01

    The interactions between DNA and carbon nanotubes (CNTs) have been widely studied in recent years. The binding process of DNA with CNT as well as the electronic properties of DNA/CNT hybrids constitutes an interesting yet complicated problem. The binding energy (BE) of the hybridization is one of the most extensively studied parameters for the problem. In this work, density functional theory (DFT) was used to perform geometry optimization of neutral nucleobases including adenine, cytosine, guanine and thymine absorbed on a zigzag (7,0) single-walled CNT and to evaluate the basis set superposition error corrected BE of the optimized configuration. All DFT calculations were performed using the M05-2X functional. The 6-31G(d) basis set was used for the optimization step and single point energy calculations were done using the 6-31G(d,p) basis set. For each nucleobase, we examined the influence of the initial configuration (IC) on the BE value. In particular, we considered 24 different ICs for each nucleobase, and each IC was subjected to an independent optimization and BE calculation. Our results showed that different ICs result in very different BE values and can even change the order of the BE corresponding to different nucleobases. The difference in the BE for a particular nucleobase caused by changes in its IC can be comparable to the difference in the BE between different nucleobases at the same initial position relative to the CNT. This provides an explanation for the discrepancies that exist in the literature on the nucleobase/CNT BE, and suggests that the potential energy surface between the nucleobases and the CNT can have many local minima and care should be exercised in the calculation and interpretation of the BE.

  16. Which Electronic and Structural Factors Control the Photostability of DNA and RNA Purine Nucleobases?

    NASA Astrophysics Data System (ADS)

    Pollum, Marvin; Reichardt, Christian; Crespo-Hernández, Carlos E.; Martínez-Fernández, Lara; Corral, Inés; Rauer, Clemens; Mai, Sebastian; Marquetand, Philipp; González, Leticia

    2015-06-01

    Following ultraviolet excitation, the canonical purine nucleobases, guanine and adenine, are able to efficiently dissipate the absorbed energy within hundreds of femtoseconds. This property affords these nucleobases with great photostability. Conversely, non-canonical purine nucleobases exhibit high fluorescence quantum yields or efficiently populate long-lived triplet excited states from which chemistry can occur. Using femtosecond broadband transient absorption spectroscopy in combination with ab initio static and surface hopping dynamics simulations we have determined the electronic and structural factors that regulate the excited state dynamics of the purine nucleobase derivatives. Importantly, we have uncovered that the photostability of the guanine and adenine nucleobases is not due to the structure of the purine core itself and that the substituent at the C6 position of the purine nucleobase plays a more important role than that at the C2 position in the ultrafast relaxation of deleterious electronic energy. [The authors acknowledge the CAREER program of the National Science Foundation (Grant No. CHE-1255084) for financial support.

  17. Adsorption of nucleobase pairs on hexagonal boron nitride sheet: hydrogen bonding versus stacking.

    PubMed

    Ding, Ning; Chen, Xiangfeng; Wu, Chi-Man Lawrence; Li, Hui

    2013-07-14

    The adsorption of hydrogen-bonded and stacked nucleobase pairs on the hexagonal boron nitride (h-BN) surface was studied by density functional theory and molecular dynamics methods. Eight types of nucleobase pairs (i.e., GG, AA, TT, CC, UU, AT, GC, and AU) were chosen as the adsorbates. The adsorption configurations, interaction energies, and electronic properties of the nucleobase pair on the h-BN surface were obtained and compared. The density of states analysis result shows that both the hydrogen-bonded and stacked nucleobase pairs were physisorbed on h-BN with minimal charge transfer. The hydrogen-bonded base pairs lying on the h-BN surface are significantly more stable than the stacked forms in both the gas and water phase. The molecular dynamics simulation result indicates that h-BN possessed high sensitivity for the nucleobases and the h-BN surface adsorption could revert the base pair interaction from stacking back to hydrogen bonding in aqueous environment. The h-BN surface could immobilize the nucleobases on its surface, which suggests the use of h-BN has good potential in DNA/RNA detection biosensors and self-assembly nanodevices. PMID:23689542

  18. Adsorption of DNA/RNA nucleobases on hexagonal boron nitride sheet: an ab initio study.

    PubMed

    Lin, Qing; Zou, Xiaolong; Zhou, Gang; Liu, Rui; Wu, Jian; Li, Jia; Duan, Wenhui

    2011-07-14

    Our ab initio calculations indicate that the interaction of deoxyribonucleic/ribonucleic acid (DNA/RNA) nucleobases [guanine (G), adenine (A), thymine (T), cytosine (C), and uracil (U)] with the hexagonal boron nitride (h-BN) sheet, a polar but chemically inert surface, is governed by mutual polarization. Unlike the case of graphene, all nucleobases exhibit the same stacking arrangement on the h-BN sheet due to polarization effects: the anions (N and O atoms) of nucleobases prefer to stay on top of cations (B) of the substrate as far as possible, regardless of the biological properties of nucleobases. The adsorption energies, ranging from 0.5 eV to 0.69 eV, increase in the order of U, C, T, A and G, which can be attributed to different side groups or atoms of nucleobases. The fundamental nature of DNA/RNA nucleobases and h-BN sheet remains unchanged upon adsorption, suggesting that the h-BN sheet is a promising template for DNA/RNA-related research, such as self-assembly. PMID:21637870

  19. Synthesis of alanyl nucleobase amino acids and their incorporation into proteins.

    PubMed

    Talukder, Poulami; Dedkova, Larisa M; Ellington, Andrew D; Yakovchuk, Petro; Lim, Jaebum; Anslyn, Eric V; Hecht, Sidney M

    2016-09-15

    Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail. PMID:27452282

  20. Synthesis of alanyl nucleobase amino acids and their incorporation into proteins.

    PubMed

    Talukder, Poulami; Dedkova, Larisa M; Ellington, Andrew D; Yakovchuk, Petro; Lim, Jaebum; Anslyn, Eric V; Hecht, Sidney M

    2016-09-15

    Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.

  1. Quantitative tomographic imaging of intermolecular FRET in small animals

    PubMed Central

    Venugopal, Vivek; Chen, Jin; Barroso, Margarida; Intes, Xavier

    2012-01-01

    Forster resonance energy transfer (FRET) is a nonradiative transfer of energy between two fluorescent molecules (a donor and an acceptor) in nanometer range proximity. FRET imaging methods have been applied to proteomic studies and drug discovery applications based on intermolecular FRET efficiency measurements and stoichiometric measurements of FRET interaction as quantitative parameters of interest. Importantly, FRET provides information about biomolecular interactions at a molecular level, well beyond the diffraction limits of standard microscopy techniques. The application of FRET to small animal imaging will allow biomedical researchers to investigate physiological processes occurring at nanometer range in vivo as well as in situ. In this work a new method for the quantitative reconstruction of FRET measurements in small animals, incorporating a full-field tomographic acquisition system with a Monte Carlo based hierarchical reconstruction scheme, is described and validated in murine models. Our main objective is to estimate the relative concentration of two forms of donor species, i.e., a donor molecule involved in FRETing to an acceptor close by and a nonFRETing donor molecule. PMID:23243567

  2. Fret wear mediation of NIRCam filter wheel assembly

    NASA Astrophysics Data System (ADS)

    Privári, Béla I.

    2011-10-01

    We will discuss a fret wear solution developed for the James Webb Space Telescope NIRCam filter wheel assembly by implementation of a hard coating. With mechanisms and structures designed for space flight application, titanium is often selected as the choice material of construction. Titanium offers a low-density high strength material that is good for use with many optical instruments due to its' favorable thermal properties. An important factor to consider with titanium mechanisms and structures are component fits and the vibration environment that must be survived during launch. In many instances, small (slip) fits between titanium components can cause fret wear during launch induced vibration. Titanium is particularly susceptible to fret wear, although other materials also demonstrate the fret wear. Fretting is adhesive failure of a material that experiences impact and micro-slip with an adjacent part. The mechanism of fret wear involves small particles that are pulled from the surface of parts that turn into hard oxides that further accelerate the wear between the parts. To mitigate fret wear, the mechanism or structure can be designed to eliminate all slip fits altogether, lubricants may be added to the wear surfaces or hard coatings can be applied to the wear surfaces when the other approaches are not feasible. For the NIRCam filter wheel assembly, which must operate at 35K and remain optically clean, only hard coatings are feasible. A discussion of several coating alternatives and associated wear testing will be presented along with the selection of an optimal solution.

  3. Understanding FRET as a Research Tool for Cellular Studies

    PubMed Central

    Shrestha, Dilip; Jenei, Attila; Nagy, Péter; Vereb, György; Szöllősi, János

    2015-01-01

    Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1–10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types. PMID:25815593

  4. Small-molecule FRET probes for protein kinase activity monitoring in living cells

    SciTech Connect

    Vaasa, Angela; Lust, Marje; Terrin, Anna; Uri, Asko; Zaccolo, Manuela

    2010-07-09

    In this study, the applicability of fluorescently labeled adenosine analogue-oligoarginine conjugates (ARC-Photo probes) for monitoring of protein kinase A (PKA) activity in living cells was demonstrated. ARC-Photo probes possessing subnanomolar affinity towards the catalytic subunit of PKA (PKAc) and competitive with the regulatory subunit (PKAr), penetrate cell plasma membrane and associate with PKAc fused with yellow fluorescent protein (PKAc-YFP). Detection of inter-molecular Foerster resonance energy transfer (FRET) efficiency between the fluorophores of the fusion protein and ARC-Photo probe can be used for both the evaluation of non-labeled inhibitors of PKAc and for monitoring of cAMP signaling via detection of changes in the activity of PKA as a cAMP downstream effector.

  5. Intonation and compensation of fretted string instruments

    NASA Astrophysics Data System (ADS)

    Varieschi, Gabriele U.; Gower, Christina M.

    2010-01-01

    We discuss theoretical and physical models that are useful for analyzing the intonation of musical instruments such as guitars and mandolins and can be used to improve the tuning on these instruments. The placement of frets on the fingerboard is designed according to mathematical rules and the assumption of an ideal string. The analysis becomes more complicated when we include the effects of deformation of the string and inharmonicity due to other string characteristics. As a consequence, perfect intonation of all the notes on the instrument cannot be achieved, but complex compensation procedures can be introduced to minimize the problem. To test the validity of these procedures, we performed extensive measurements using standard monochord sonometers and other acoustical devices, confirming the correctness of our theoretical models. These experimental activities can be integrated into acoustics courses and laboratories and can become a more advanced version of basic experiments with monochords and sonometers.

  6. Elevated temperature fretting fatigue of nickel based alloys

    NASA Astrophysics Data System (ADS)

    Gean, Matthew C.

    This document details the high temperature fretting fatigue of high temperature nickel based alloys common to turbine disk and blade applications. The research consists of three area of focus: Experiments are conducted to determine quantitatively the fretting fatigue lives of advanced nickel based alloys; Analytical tools are developed and used to investigate the fretting fatigue response of the material; Fractographic analysis of the experimental results is used to improve the analytical models employed in the analysis of the experiments. Sixty three fretting fatigue experiments were conducted at 649 °C using a polycrystalline Nickel specimen in contact with directionally solidified and single crystal Nickel pads. Various influences on the fretting fatigue life are investigated. Shot peened Rene' 95 had better fretting fatigue life compared to shot peened Rene' 88. Shot peening produced a 2x increase in life for Rene' 95, but only a marginal improvement in the fretting fatigue life for Rene' 88. Minor cycles in variable amplitude loading produces significant damage to the specimen. Addition of occasional overpeaks in load produces improvements in fretting fatigue life. Contact tractions and stresses are obtained through a variety of available tools. The contact tractions can be efficiently obtained for limited geometries, while FEM can provide the contact tractions for a broader class of problems, but with the cost of increased CPU requirements. Similarly, the subsurface contact stresses can be obtained using the contact tractions as a boundary condition with either a semi-analytical FFT method or FEM. It is found that to calculate contact stresses the FFT was only marginally faster than FEM. The experimental results are combined with the analysis to produce tools that are used to design against fretting fatigue. Fractographic analysis of the fracture surface indicates the nature of the fretting fatigue crack behavior. Interrupted tests were performed to analyze

  7. Seeding the Pregenetic Earth: Meteoritic Abundances of Nucleobases and Potential Reaction Pathways

    NASA Astrophysics Data System (ADS)

    Pearce, Ben K. D.; Pudritz, Ralph E.

    2015-07-01

    Carbonaceous chondrites are a class of meteorite known for having high contents of water and organics. In this study, the abundances of the nucleobases, i.e., the building blocks of RNA and DNA, found in carbonaceous chondrites are collated from a variety of published data and compared across various meteorite classes. An extensive review of abiotic chemical reactions producing nucleobases is then performed. These reactions are then reduced to a list of 15 individual reaction pathways that could potentially occur within meteorite parent bodies. The nucleobases guanine, adenine, and uracil are found in carbonaceous chondrites in amounts of 1–500 ppb. It is currently unknown which reaction is responsible for their synthesis within the meteorite parent bodies. One class of carbonaceous meteorite dominates the abundances of both amino acids and nucleobases—the so-called CM2 (e.g., Murchison meteorite). CR2 meteorites (e.g., Graves Nunataks) also dominate the abundances of amino acids, but are the least abundant in nucleobases. The abundances of total nucleobases in these two classes are 330 ± 250 and 16 ± 13 ppb, respectively. Guanine most often has the greatest abundances in carbonaceous chondrites with respect to the other nucleobases, but is 1–2 orders of magnitude less abundant in CM2 meteorites than glycine (the most abundant amino acid). Our survey of the reaction mechanisms for nucleobase formation suggests that Fischer–Tropsch synthesis (i.e., CO, H2, and NH3 gases reacting in the presence of a catalyst such as alumina or silica) is the most likely candidate for conditions that characterize the early states of planetesimals.

  8. Unified reaction pathways for the prebiotic formation of RNA and DNA nucleobases.

    PubMed

    Jeilani, Yassin Aweis; Williams, Phoenix N; Walton, Sofia; Nguyen, Minh Tho

    2016-07-27

    The reaction pathways for the prebiotic formation of nucleobases are complex and lead to the formation of a mixture of products. In the past 50 years, there has been a concerted effort for identifying a unified mechanism for the abiotic origin of the biomolecules but with little success. In the present theoretical study, we identified two prominent precursors for the building up of RNA and DNA nucleobases under prebiotic conditions: (a) 1,2-diaminomaleonitrile (DAMN), which is a tetramer of hydrogen cyanide (HCN), and (b) formamide, a hydrolysis product of HCN; it is important to emphasize that HCN is the source of both precursors. We find that free radical pathways are potentially appropriate to account for the origin of nucleobases from HCN. The current study unites the formamide pathways with the DAMN pathways. The mechanisms for the formation of the RNA and DNA nucleobases (uracil, adenine, purine, cytosine) were studied by quantum chemical computations using density functional theory at the B3LYP/6-311G(d,p) level. All the routes involved proceed with relatively low energy barriers (within the error margin of DFT methods). We showed that the radical mechanisms for the formation of nucleobases could be unified through common precursors. The results demonstrated that 4-aminoimidazole-5-carbonitrile (AICN), which is a known precursor for nucleobases, is a product of DAMN. The overall mechanisms are internally consistent with the abiotic formation of the nucleobases, namely (a) under a meteoritic impact scenario on the early Earth's surface that generated high internal energy, and/or (b) in the (gas phase) interstellar regions without the presence of catalysts.

  9. Seeding the Pregenetic Earth: Meteoritic Abundances of Nucleobases and Potential Reaction Pathways

    NASA Astrophysics Data System (ADS)

    Pearce, Ben K. D.; Pudritz, Ralph E.

    2015-07-01

    Carbonaceous chondrites are a class of meteorite known for having high contents of water and organics. In this study, the abundances of the nucleobases, i.e., the building blocks of RNA and DNA, found in carbonaceous chondrites are collated from a variety of published data and compared across various meteorite classes. An extensive review of abiotic chemical reactions producing nucleobases is then performed. These reactions are then reduced to a list of 15 individual reaction pathways that could potentially occur within meteorite parent bodies. The nucleobases guanine, adenine, and uracil are found in carbonaceous chondrites in amounts of 1-500 ppb. It is currently unknown which reaction is responsible for their synthesis within the meteorite parent bodies. One class of carbonaceous meteorite dominates the abundances of both amino acids and nucleobases—the so-called CM2 (e.g., Murchison meteorite). CR2 meteorites (e.g., Graves Nunataks) also dominate the abundances of amino acids, but are the least abundant in nucleobases. The abundances of total nucleobases in these two classes are 330 ± 250 and 16 ± 13 ppb, respectively. Guanine most often has the greatest abundances in carbonaceous chondrites with respect to the other nucleobases, but is 1-2 orders of magnitude less abundant in CM2 meteorites than glycine (the most abundant amino acid). Our survey of the reaction mechanisms for nucleobase formation suggests that Fischer-Tropsch synthesis (i.e., CO, H2, and NH3 gases reacting in the presence of a catalyst such as alumina or silica) is the most likely candidate for conditions that characterize the early states of planetesimals.

  10. Synthesis and characterization of nucleobase-carbon nanotube hybrids.

    PubMed

    Singh, Prabhpreet; Kumar, Jitendra; Toma, Francesca Maria; Raya, Jesus; Prato, Maurizio; Fabre, Bruno; Verma, Sandeep; Bianco, Alberto

    2009-09-23

    We report the synthesis and characterization of adenine-single-walled carbon nanotube (SWCNT) hybrid materials, where for the first time nucleobases are covalently attached to the exosurface of SWCNTs. The structural properties of all hybrids have been characterized using usual spectroscopic and microscopic techniques. The degree of functional groups for functionalized SWCNTs (f-SWCNTs) 2a and 2b is one adenine group for each 26 and 37 carbon atoms, respectively. Solid-state magic angle spinning (13)C NMR spectroscopy (MAS NMR) and electrochemistry have been also applied for the characterization of these f-SWCNTs. AFM images of f-SWCNT 2b showed an interesting feature of horizontally aligned nanotubes along the surface when deposited on highly oriented pyrolytic graphite surface. Furthermore, we evaluated the coordinating ability of these hybrid materials toward silver ions, and interestingly, we found a pattern of silver nanoparticles localized over the surface of the carbon nanotube network. The presence of aligned and randomly oriented CNTs and their ability to coordinate with metal ions make this class of materials very interesting for applications in the development of novel electronic devices and as new supports for different catalytic transformations. PMID:19673527

  11. DNA photoreacts by nucleobase ring cleavage to form labile isocyanates.

    PubMed

    Buschhaus, Laura; Rolf, Josefin; Kleinermanns, Karl

    2013-11-14

    Differential infrared absorption spectroscopy was used to study the formation of isocyanates and further photo-products in the oligonucleotides dG10, dC10 and dT10 and in their mononucleosides by ultraviolet light at 266 nm. We find that α-cleavage takes place in oligonucleotides and mononucleosides both in films and in solution. The very intense and spectrally isolated isocyanate (N=C=O) asymmetric stretch vibration at 2277 cm(-1) is used as a spectroscopic marker for detection of the photo-product. The band disappears upon reaction with small amounts of water vapour as expected for isocyanates. Quantum yields for isocyanate formation by nucleobase ring cleavage in the α-position to the carbonyl group are ∼5 × 10(-5) in the mononucleosides and up to 5 × 10(-4) in the oligonucleotides. In the mixed oligonucleotides dG10/dC10 and dA10/dT10 the quantum yield of α-cleavage drops by a factor of 10 compared to the single oligonucleotides. Implications for DNA repair and photo-induced DNA-protein cross-linking via isocyanate reaction with NH2 groups of amino acids are discussed.

  12. Formation of the nucleobases around the star forming region

    NASA Astrophysics Data System (ADS)

    Saha, Rajdeep; Majumdar, Liton; Das, Ankan; Chakrabarti, Sandip K.; Chakrabarti, Sonali

    2013-06-01

    Chemistry of the dense and cold interstellar clouds are mainly dominated by the ion-molecular and radical-radical interactions though some neutral-neutral reactions are also barrierless and feasible at this condition. The base pairs of RNA are guanine (G) & uracil (U) (G-U pair) and adenine (A) & cytosine (C) (A-C pair). We perform quantum chemical calculations to predict the energetically most economical as well as favorable root for the formation of major bases of the nucleic acids. The outcome of this quantum chemical calculations could be used into our hydro-chemical model to obtain the abundances of some of the important bases of RNA during the formation of a proto star. It is well known that the thymine (T) and uracil (U) are the two nucleobases which are not common in DNA and RNA. Our quantum chemical calculation suggests that uracil could be produced prior to thymine in our chemical network. These findings could be used to support the RNA world hypothesis.

  13. Accumulation of formamide in hydrothermal pores to form prebiotic nucleobases.

    PubMed

    Niether, Doreen; Afanasenkau, Dzmitry; Dhont, Jan K G; Wiegand, Simone

    2016-04-19

    Formamide is one of the important compounds from which prebiotic molecules can be synthesized, provided that its concentration is sufficiently high. For nucleotides and short DNA strands, it has been shown that a high degree of accumulation in hydrothermal pores occurs, so that temperature gradients might play a role in the origin of life [Baaske P, et al. (2007)Proc Natl Acad Sci USA104(22):9346-9351]. We show that the same combination of thermophoresis and convection in hydrothermal pores leads to accumulation of formamide up to concentrations where nucleobases are formed. The thermophoretic properties of aqueous formamide solutions are studied by means of Infrared Thermal Diffusion Forced Rayleigh Scattering. These data are used in numerical finite element calculations in hydrothermal pores for various initial concentrations, ambient temperatures, and pore sizes. The high degree of formamide accumulation is due to an unusual temperature and concentration dependence of the thermophoretic behavior of formamide. The accumulation fold in part of the pores increases strongly with increasing aspect ratio of the pores, and saturates to highly concentrated aqueous formamide solutions of ∼85 wt% at large aspect ratios. Time-dependent studies show that these high concentrations are reached after 45-90 d, starting with an initial formamide weight fraction of[Formula: see text]wt % that is typical for concentrations in shallow lakes on early Earth. PMID:27044100

  14. The role of nucleobase interactions in RNA structure and dynamics

    PubMed Central

    Bottaro, Sandro; Di Palma, Francesco; Bussi, Giovanni

    2014-01-01

    The intricate network of interactions observed in RNA three-dimensional structures is often described in terms of a multitude of geometrical properties, including helical parameters, base pairing/stacking, hydrogen bonding and backbone conformation. We show that a simple molecular representation consisting in one oriented bead per nucleotide can account for the fundamental structural properties of RNA. In this framework, canonical Watson-Crick, non-Watson-Crick base-pairing and base-stacking interactions can be unambiguously identified within a well-defined interaction shell. We validate this representation by performing two independent, complementary tests. First, we use it to construct a sequence-independent, knowledge-based scoring function for RNA structural prediction, which compares favorably to fully atomistic, state-of-the-art techniques. Second, we define a metric to measure deviation between RNA structures that directly reports on the differences in the base–base interaction network. The effectiveness of this metric is tested with respect to the ability to discriminate between structurally and kinetically distant RNA conformations, performing better compared to standard techniques. Taken together, our results suggest that this minimalist, nucleobase-centric representation captures the main interactions that are relevant for describing RNA structure and dynamics. PMID:25355509

  15. Accumulation of formamide in hydrothermal pores to form prebiotic nucleobases.

    PubMed

    Niether, Doreen; Afanasenkau, Dzmitry; Dhont, Jan K G; Wiegand, Simone

    2016-04-19

    Formamide is one of the important compounds from which prebiotic molecules can be synthesized, provided that its concentration is sufficiently high. For nucleotides and short DNA strands, it has been shown that a high degree of accumulation in hydrothermal pores occurs, so that temperature gradients might play a role in the origin of life [Baaske P, et al. (2007)Proc Natl Acad Sci USA104(22):9346-9351]. We show that the same combination of thermophoresis and convection in hydrothermal pores leads to accumulation of formamide up to concentrations where nucleobases are formed. The thermophoretic properties of aqueous formamide solutions are studied by means of Infrared Thermal Diffusion Forced Rayleigh Scattering. These data are used in numerical finite element calculations in hydrothermal pores for various initial concentrations, ambient temperatures, and pore sizes. The high degree of formamide accumulation is due to an unusual temperature and concentration dependence of the thermophoretic behavior of formamide. The accumulation fold in part of the pores increases strongly with increasing aspect ratio of the pores, and saturates to highly concentrated aqueous formamide solutions of ∼85 wt% at large aspect ratios. Time-dependent studies show that these high concentrations are reached after 45-90 d, starting with an initial formamide weight fraction of[Formula: see text]wt % that is typical for concentrations in shallow lakes on early Earth.

  16. Accumulation of formamide in hydrothermal pores to form prebiotic nucleobases

    PubMed Central

    Niether, Doreen; Afanasenkau, Dzmitry; Dhont, Jan K. G.

    2016-01-01

    Formamide is one of the important compounds from which prebiotic molecules can be synthesized, provided that its concentration is sufficiently high. For nucleotides and short DNA strands, it has been shown that a high degree of accumulation in hydrothermal pores occurs, so that temperature gradients might play a role in the origin of life [Baaske P, et al. (2007) Proc Natl Acad Sci USA 104(22):9346−9351]. We show that the same combination of thermophoresis and convection in hydrothermal pores leads to accumulation of formamide up to concentrations where nucleobases are formed. The thermophoretic properties of aqueous formamide solutions are studied by means of Infrared Thermal Diffusion Forced Rayleigh Scattering. These data are used in numerical finite element calculations in hydrothermal pores for various initial concentrations, ambient temperatures, and pore sizes. The high degree of formamide accumulation is due to an unusual temperature and concentration dependence of the thermophoretic behavior of formamide. The accumulation fold in part of the pores increases strongly with increasing aspect ratio of the pores, and saturates to highly concentrated aqueous formamide solutions of ∼85 wt% at large aspect ratios. Time-dependent studies show that these high concentrations are reached after 45–90 d, starting with an initial formamide weight fraction of 10−3 wt % that is typical for concentrations in shallow lakes on early Earth. PMID:27044100

  17. Anharmonic IR Spectra of Biomolecules: Nucleobases and Their Oligomers

    NASA Astrophysics Data System (ADS)

    Barone, Vincenzo; Biczysko, Malgorzata; Bloino, Julien; Carnimeo, Ivan; Fornaro, Teresa

    2014-06-01

    Computational spectroscopy techniques have become in the last years effective means to predict and characterize spectra, such as infrared, for molecular systems of increasing dimensions with account for different environments. We are actively developing a comprehensive and robust computational protocol, set within a perturbative vibrational framework [1], aimed at a quantitative reproduction of the spectra of biomolecules. In order to model the vibrational spectra of weakly bound molecular complexes, dispersion interactions should be taken into proper account. In this work, we present critical assessment of dispersion-corrected DFT approaches for anharmonic vibrational frequency calculations. It is shown that fully anharmonic IR spectra, simulated through full and reduced-dimensionality generalized second-order vibrational perturbation theory (GVPT2)[1] with the potential energy surfaces computed with the B3LYP-D3 approach, may be used to interpret experimental data of nucleobases and their complexes[2] by the direct comparison of experimental IR spectra with their theoretical anharmonic counterpart, taking into account also overtones and combination bands. [1] V. Barone, M. Biczysko, J. Bloino, Phys. Chem. Chem. Phys., 2014,16, 1759-1787 [2] T. Fornaro, M. Biczysko, S. Monti, V. Barone, Phys. Chem. Chem. Phys., 2014, DOI: 10.1039/C3CP54724H

  18. QD-Based FRET Probes at a Glance

    PubMed Central

    Shamirian, Armen; Ghai, Aashima; Snee, Preston T.

    2015-01-01

    The unique optoelectronic properties of quantum dots (QDs) give them significant advantages over traditional organic dyes, not only as fluorescent labels for bioimaging, but also as emissive sensing probes. QD sensors that function via manipulation of fluorescent resonance energy transfer (FRET) are of special interest due to the multiple response mechanisms that may be utilized, which in turn imparts enhanced flexibility in their design. They may also function as ratiometric, or “color-changing” probes. In this review, we describe the fundamentals of FRET and provide examples of QD-FRET sensors as grouped by their response mechanisms such as link cleavage and structural rearrangement. An overview of early works, recent advances, and various models of QD-FRET sensors for the measurement of pH and oxygen, as well as the presence of metal ions and proteins such as enzymes, are also provided. PMID:26053750

  19. Wide and high resolution tension measurement using FRET in embryo.

    PubMed

    Yamashita, Satoshi; Tsuboi, Takashi; Ishinabe, Nanako; Kitaguchi, Tetsuya; Michiue, Tatsuo

    2016-06-23

    During embryonic development, physical force plays an important role in morphogenesis and differentiation. Stretch sensitive fluorescence resonance energy transfer (FRET) has the potential to provide non-invasive tension measurements inside living tissue. In this study, we introduced a FRET-based actinin tension sensor into Xenopus laevis embryos and demonstrated that this sensor captures variation of tension across differentiating ectoderm. The actinin tension sensor, containing mCherry and EGFP connected by spider silk protein, was validated in human embryonic kidney (HEK) cells and embryos. It co-localized with actin filaments and changed FRET efficiencies in response to actin filament destruction, myosin deactivation, and osmotic perturbation. Time-lapse FRET analysis showed that the prospective neural ectoderm bears higher tension than the epidermal ectoderm during gastrulation and neurulation, and cells morphogenetic behavior correlated with the tension difference. These data confirmed that the sensor enables us to measure tension across tissues concurrently and with high resolution.

  20. Pulse-shaping based two-photon FRET stoichiometry.

    PubMed

    Flynn, Daniel C; Bhagwat, Amar R; Brenner, Meredith H; Núñez, Marcos F; Mork, Briana E; Cai, Dawen; Swanson, Joel A; Ogilvie, Jennifer P

    2015-02-01

    Förster Resonance Energy Transfer (FRET) based measurements that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under two-photon excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.

  1. Wide and high resolution tension measurement using FRET in embryo

    PubMed Central

    Yamashita, Satoshi; Tsuboi, Takashi; Ishinabe, Nanako; Kitaguchi, Tetsuya; Michiue, Tatsuo

    2016-01-01

    During embryonic development, physical force plays an important role in morphogenesis and differentiation. Stretch sensitive fluorescence resonance energy transfer (FRET) has the potential to provide non-invasive tension measurements inside living tissue. In this study, we introduced a FRET-based actinin tension sensor into Xenopus laevis embryos and demonstrated that this sensor captures variation of tension across differentiating ectoderm. The actinin tension sensor, containing mCherry and EGFP connected by spider silk protein, was validated in human embryonic kidney (HEK) cells and embryos. It co-localized with actin filaments and changed FRET efficiencies in response to actin filament destruction, myosin deactivation, and osmotic perturbation. Time-lapse FRET analysis showed that the prospective neural ectoderm bears higher tension than the epidermal ectoderm during gastrulation and neurulation, and cells morphogenetic behavior correlated with the tension difference. These data confirmed that the sensor enables us to measure tension across tissues concurrently and with high resolution. PMID:27335157

  2. Wide and high resolution tension measurement using FRET in embryo.

    PubMed

    Yamashita, Satoshi; Tsuboi, Takashi; Ishinabe, Nanako; Kitaguchi, Tetsuya; Michiue, Tatsuo

    2016-01-01

    During embryonic development, physical force plays an important role in morphogenesis and differentiation. Stretch sensitive fluorescence resonance energy transfer (FRET) has the potential to provide non-invasive tension measurements inside living tissue. In this study, we introduced a FRET-based actinin tension sensor into Xenopus laevis embryos and demonstrated that this sensor captures variation of tension across differentiating ectoderm. The actinin tension sensor, containing mCherry and EGFP connected by spider silk protein, was validated in human embryonic kidney (HEK) cells and embryos. It co-localized with actin filaments and changed FRET efficiencies in response to actin filament destruction, myosin deactivation, and osmotic perturbation. Time-lapse FRET analysis showed that the prospective neural ectoderm bears higher tension than the epidermal ectoderm during gastrulation and neurulation, and cells morphogenetic behavior correlated with the tension difference. These data confirmed that the sensor enables us to measure tension across tissues concurrently and with high resolution. PMID:27335157

  3. An Experimental Study of Fretting of Gear Teeth

    NASA Technical Reports Server (NTRS)

    Krantz, Timothy L.

    2008-01-01

    Experiments were conducted to study fretting of gears. The gears were made from case-carburized AISI 9310 alloy to match the material of a flight actuator gearbox of interest. The objective of the testing was to produce damage representative of that observed on flight hardware. The following correlations and observations were noted. The amplitude of dithering motion very strongly influenced the type and magnitude of damage. Sliding amounts on the order of 30% of the width of the line contact were judged to most readily produce fretting damage. There was observed an incubation period on the order of tens-of-thousands of cycles, and the incubation period was influenced by surface roughness, torque, and the motion extent. Fretting damage could be produced for any of the torques tested, and the severity of damage increased slightly with torque. Gear teeth having surface roughness of 0.7-0.8 micrometer were somewhat more resistant to fretting than were smoother surfaces.

  4. Detecting Pyrolysis Products from Bacteria in a Mars Soil Analogue

    NASA Technical Reports Server (NTRS)

    Glavin, D. P.; Cleaves, H. J.; Schubert, M.; Aubrey, A.; Buch, A.; Mahaffy, P. R.; Bada, J. L.

    2004-01-01

    One of the primary objectives of the 1976 Viking missions was to determine whether organic compounds, possibly of biological origin, were present in the Martian surface soils. The Viking gas chromatography mass spectrometry (GCMS) instruments found no evidence for any organic compounds of Martian origin above a few parts per billion in the upper 10 cm of surface soil, suggesting the absence of a widely distributed Martian biota. However, it is now known that key organic compounds important to biology, such as amino acids, carboxylic acids and nucleobases, would likely have been missed by the Viking GCMS instruments. In this study, a Mars soil analogue that was inoculated with approx. 10 billion Escherichia coli cells was heated at 500 C under Martian ambient pressure to release volatile organic compounds from the sample. The pyrolysis products were then analyzed for amino acids and nucleobases using high performance liquid chromatography (HPLC) and GCMS. Our experimental results indicate that at the part per billion level, the degradation products generated from several million bacterial cells per gram of Martian soil would not have been detected by the Viking GCMS instruments. Upcoming strategies for Mars exploration will require in-situ analyses by instruments that can assess whether any organic compounds, especially those that might be associated with life, are present in Martian surface samples.

  5. Fretting maps of glass fiber-reinforced composites

    SciTech Connect

    Turki, C.; Salvia, M.; Vincent, L.

    1993-12-31

    Industrial development of new materials are often limited due to an insufficient knowledge in their functional properties. The paper deals with fretting behavior of glass fiber reinforced epoxy/metal contacts. Fretting is a plague for all industries, especially in the case of quasi-static loadings. Furthermore friction testing under small displacements appeared well fitted to understand the effect of fiber orientations and to relate results to microstructure (fiber, matrix and interface).

  6. N-h and N-C bond activation of pyrimidinic nucleobases and nucleosides promoted by an osmium polyhydride.

    PubMed

    Esteruelas, Miguel A; García-Raboso, Jorge; Oliván, Montserrat; Oñate, Enrique

    2012-05-21

    Complex OsH(6)(P(i)Pr(3))(2) (1) reacts with 1-methylthymine and 1-methyluracil to give OsH(3)(P(i)Pr(3))(2)(nucleobase') (2, 3) containing the deprotonated nucleobases (nucleobase') κ(2)-N,O coordinated by the nitrogen atom at position 3 and the oxygen bonded to the carbon atom of the ring at position 4. Similarly, the reactions of 1 with thymidine, 5-methyluridine, deoxyuridine, and uridine lead to OsH(3)(P(i)Pr(3))(2)(nucleoside') (4-7) with the deprotonated nucleoside (nucleoside') κ(2)-N,O coordinated by the nitrogen atom at position 3 and the oxygen bonded to the carbon atom at position 4 of the nucleobases. Treatment of complexes 5 and 7, containing nucleosides derived from ribose, with OsH(2)Cl(2)(P(i)Pr(3))(2) (8) in the presence of Et(3)N affords dinuclear species OsH(3)(P(i)Pr(3))(2)(nucleobase')-(ribose)(P(i)Pr(3))(2)H(2)Os (9, 10) formed by two different metal fragments. Complex 1 also promotes the cleavage of the N-C bond of 2-7 to give the dinuclear species {OsH(3)(P(i)Pr(3))(2)}(2)(nucleobase'') (11, 12) with the nucleobase skeleton (nucleobase'') κ(2)-N,O coordinated to both metal fragments. These compounds can be also prepared by reaction of 1 with 0.5 equiv of thymine and uracil. The use of 1:1 hexahydride:nucleobase molar ratios gives rise to the preferred formation of the mononuclear complexes OsH(3)(P(i)Pr(3))(2)(nucleobase''') (13, 14; nucleobase''' = monodeprotonated thymine or uracil). The X-ray structures of complexes 6, 11, and 14 are also reported.

  7. Two-step FRET as a structural tool.

    PubMed

    Watrob, Heather M; Pan, Chia-Pin; Barkley, Mary D

    2003-06-18

    The power of FRET to study molecular complexes is expanded by the use of two or more donor/acceptor pairs. A general theoretical framework for distance measurements in three-chromophore systems is presented. Three energy transfer schemes applicable to many diverse situations are considered: (I) two-step FRET relay with FRET between the first and second chromophores and between the second and third, (II) FRET from a single donor to two different acceptors, and (III) two-step FRET relay with FRET also between the first and third chromophores. Equations for the efficiencies involving multiple energy transfer steps are derived for both donor quenching and sensitized emission measurements. The theory is supported by experimental data on model systems of known structure using steady-state donor quenching, lifetime quenching, and sensitized emission. The distances measured in the three-chromophore systems agree with those in two-chromophore systems and molecular models. Finally, labeling requirements for diagnosis of the energy transfer scheme and subsequent distance measurements are discussed.

  8. FRET-based biosensors to detect infectious agents

    NASA Astrophysics Data System (ADS)

    Xu, Juntao; Grant, Sheila A.

    2002-02-01

    We report herein on the development of a FRET-based method to detect changes caused by viral protein-receptor binding. FRET fluorophore pairs (donor and acceptor fluorophores) were tagged to two specific receptors, both which bind to a viral protein. When the binding event occurs, the distance between the donor and acceptor FRET fluorophores is decreased, thus initiating the fluorescence resonance energy transfer (FRET). Since the binding event is unique to the viral protein, fluorescent change indicates the present of the virus. In this paper, the viral protein gp120, which is the featured protein on the surface of HIV-1, was detected. The receptors, CD4 and gp120-antibody which specifically bind to gp120, were conjugated to the FRET fluorophore pair, AMCA-NHS (succinimidyl-7-amino-4-methylcoumarin-3-acetic acid) and FITC (fluorescein isothiocyanate) respectively. Spectrofluorimetry was used to detect the fluorescent change between AMCA-NHS and FITC peak intensities when the receptors bind to the gp120. Specific binding gp120 and non-specific binding gp120 were used to test the selectivity of the sensor. The results indicated that FRET-conjugated receptors can efficiently detect the presence of gp120.

  9. a Study on the Fretting Fatigue Life of Zircaloy Alloys

    NASA Astrophysics Data System (ADS)

    Kwon, Jae-Do; Park, Dae-Kyu; Woo, Seung-Wan; Chai, Young-Suck

    Studies on the strength and fatigue life of machines and structures have been conducted in accordance with the development of modern industries. In particular, fine and repetitive cyclic damage occurring in contact regions has been known to have an impact on fretting fatigue fractures. The main component of zircaloy alloy is Zr, and it possesses good mechanical characteristics at high temperatures. This alloy is used in the fuel rod material of nuclear power plants because of its excellent resistance. In this paper, the effect of the fretting damage on the fatigue behavior of the zircaloy alloy is studied. Further, various types of mechanical tests such as tension and plain fatigue tests are performed. Fretting fatigue tests are performed with a flat-flat contact configuration using a bridge-type contact pad and plate-type specimen. Through these experiments, it is found that the fretting fatigue strength decreases by about 80% as compared to the plain fatigue strength. Oblique cracks are observed in the initial stage of the fretting fatigue, in which damaged areas are found. These results can be used as the basic data for the structural integrity evaluation of corrosion-resisting alloys considering the fretting damages.

  10. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    PubMed Central

    Hochreiter, Bernhard; Pardo Garcia, Alan; Schmid, Johannes A.

    2015-01-01

    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them. PMID:26501285

  11. Formation of Nucleobases and Other Prebiotic Species from the UV Irradiation of Pyrimidine in Astrophysical Ices

    NASA Astrophysics Data System (ADS)

    Nuevo, M.; Sandford, S. A.; Milam, S. N.; Materese, C. K.; Elsila, J. E.; Dworkin, J. P.

    2011-05-01

    Nucleobases are N-heterocycles which are the informational subunits of DNA and RNA. Biological nucleobases are divided in two types: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites and their extraterrestrial origin has been confirmed by isotope measurements, but no N-heterocycle has ever been observed in the ISM. Experiments showed that the UV irradiation of pyrimidine mixed in astrophysical ices such as H_2O, NH_3, CH_3OH, or any combination of these at low temperature (20-30 K) leads to the formation of multiple photo-products derived from pyrimidine including the nucleobases uracil and cytosine. Theoretical studies on the formation of uracil confirmed its experimental formation pathway and demonstrated that the H_2O matrix plays a key role in the chemistry [9]. Thymine, however, was not found in any of the samples, though other pyrimidine derivatives, as well as other species of prebiotic interest such as urea and the amino acid glycine, could be identified [8]. We will extend this study to the formation of nucleobases and other prebiotic species from the UV irradiation of pyrimidine in astrophysically relevant ice mixtures containing H_2O, NH_3, CH_3OH, CO, and CO_2.

  12. The Formation of Nucleobases from the Irradiation of Purine in Astophysical Ices and Comparisons with Meteorites.

    NASA Technical Reports Server (NTRS)

    Sandford, S. A.; Materese, C. K.; Nuevo, M.

    2016-01-01

    N-heterocycles have been identified in meteorites and their extraterrestrial origins are suggested by isotopic ratio measurements. Although small N- heterocycles have not been detected in the interstellar medium (ISM), recent experiments in our lab have shown that the irradiation of the aromatic molecules like benzene (C6H6) and naphthalene (C10H8) in mixed molecular ices leads to the formation of O- and N-heterocyclic molecules. Among the class of N-heterocycles are the nucleobases, which are of astrobiological interest because they are the information bearing units of DNA and RNA. Nucleobases have been detected in meteorites [3-5], with isotopic signatures that are also consistent with an extraterrestrial origin. Three of the biologically relevant nucleobases (uracil, cytosine, and guanine) have a pyrimidine core structure while the remaining two (adenine and guanine) possess a purine core. Previous experiments in our lab have demonstrated that all of the bio-logical nucleobases (and numerous other molecules) with a pyrimidine core structure can be produced by irradiating pyrimidine in mixed molecular ices of several compositions [6-8]. In this work, we study the formation of purine-based molecules, including the nucleobases adenine, and guanine, from the ultraviolet (UV) irradiation of purine in ices consisting mixtures of H2O and NH3 at low temperature. The experiments are designed to simulate the astrophysical conditions under which these species may be formed in dense molecular clouds, protoplanetary disks, or on the surfaces of icy bodies in planetary systems.

  13. Characterization of poly(N-isopropylacrylamide)-nucleobase supramolecular complexes featuring bio-multiple hydrogen bonds.

    PubMed

    Yang, Hsiu-Wen; Lee, Ai-Wei; Huang, Chi-Hsien; Chen, Jem-Kun

    2014-11-01

    In this study we employed poly(N-isopropylacrylamide) (PNIPAAm) as a matrix that we hybridized with five different nucleobase units (adenine, thymine, uracil, guanine, cytosine) to generate PNIPAAm-nucleobase supramolecular complexes (PNSCs) stabilized through bio-multiple hydrogen bonds (BMHBs). These nucleobase units interacted with PNIPAAm through BMHBs of various strengths, leading to competition between the BMHBs and the intramolecular hydrogen bonds (HBs) of PNIPAAm. The changes in morphology, crystalline structure, and thermoresponsive behavior of PNIPAAm were related to the strength of its BMHBs with the nucleobases. The strengths of the BMHBs followed the order guanine > adenine > thymine > cytosine > uracil, as verified through analyses of Fourier transform infrared spectra, lower critical solution temperatures, and inter-association equilibrium constants. The PNSCs also exhibited remarkable improvements in conductivity upon the formation of BMHBs, which facilitated proton transport. The neat PNIPAAm film was an insulator, but it transformed into a semiconductor after hybridizing with the nucleobases. In particular, the resistivity of the PNIPAAm-guanine supramolecular complex decreased to 1.35 × 10(5) ohm cm. The resistivity of the PNIPAAm-cytosine supramolecular complex increased significantly from 5.83 × 10(6) to 3 × 10(8) ohm cm upon increasing the temperature from 40 to 50 °C, suggesting that this material might have applicability in thermo-sensing. The ability to significantly improve the conductivity of hydrogels through such a simple approach involving BMHBs might facilitate their use as novel materials in bioelectronics. PMID:25196131

  14. A new trend to determine biochemical parameters by quantitative FRET assays

    PubMed Central

    Liao, Jia-yu; Song, Yang; Liu, Yan

    2015-01-01

    Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1–10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (Kd), enzymatic velocity (kcat) and Km. We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution. PMID:26567729

  15. Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements.

    PubMed

    George Abraham, Bobin; Sarkisyan, Karen S; Mishin, Alexander S; Santala, Ville; Tkachenko, Nikolai V; Karp, Matti

    2015-01-01

    Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM). PMID:26237400

  16. In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair

    SciTech Connect

    Hsu, Y.-Y.; Liu, Y.-N.; Wang Wenyen; Kao, Fu-Jen; Kung, S.-H. . E-mail: szkung@ym.edu.tw

    2007-02-23

    An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP{sup 2})-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2A{sup pro}) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2A{sup pro} from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research.

  17. Photoelectron Spectroscopy of Rare-Gas Solvated Nucleobase Anions

    NASA Astrophysics Data System (ADS)

    Buonaugurio, Angela M.; Chen, Jing; Bowen, Kit H.

    2012-06-01

    Gas-phase polar molecular anions [uracil (U^-), thymine (T^-), 1-3 dimethyluracil (DMU^-)] solvated by rare gas atoms were studied by means of negative ion photoelectron spectroscopy. The photoelectron spectrum (PES) of U^-, T^-, and DMU^- each exhibit a distinctive dipole-bound (DB) spectral signature. The spectra of U^-, U^- (Ar)_1,2 and U^- (Kr)_1 also only displayed the DB anion feature. Upon the solvation of more rare gas atoms, the spectra of U^- (Ar)_3, U^- (Kr)_2, and U^- (Xe)1-3 not only retained the DB signature but also exhibited the valence anion features. Moreover, the DB and the valence features shifted together to higher electron binding energies (EBEs) with increasing numbers of rare gas solvent atoms. Therefore, the co-existing DB and the valence anions appeared to be strongly coupled with each other, i.e. they effectively form a single state that is a superposition of both DB and valence anion states. For both U^- and T^- series, the ``onset size" of the Xe, Kr, and Ar solvents for the co-existing of the two anionic states was 1, 2, and 3 respectively. In addition, a minimum of 2 methane (CH_4) molecules or 1 ethane (C_2H_6) molecule were required to induce the coupling between the two states in the T^- series. Thus, the nucleobase anion interaction with non-polar solvent atoms tracks as the sum of the solvent polarizabilities. However for the DMU- series, the DB and the valence anions of DMU^-(Xe)_1, DMU^-(Kr)_2, and DMU^-(Ar)_3 were completely absent in both the mass spectra and the PES. Beyond these ``holes", their PES displayed the similar behaviors to the U^- and T^- series. Extrapolated EA values for these missing species were at or very close to zero, which may explain why they were not seen. However, why this was the case is not clear. With better Franck-Condon overlap between the origins of the NB^- (Rg)_n valence anion and the neutral NB(Rg)n than between those of the NB^- (H2O)n valence anion and the neutral NB(H2O)n, extrapolation of

  18. Evaluation of Ti-48Al-2Nb Under Fretting Conditions

    NASA Technical Reports Server (NTRS)

    Miyoshi, Kazuhisa; Lerch, Bradley A.; Draper, Susan L.; Raj, Sai V.

    2001-01-01

    An investigation was conducted to examine the fretting behavior of lambda-TiAl (Ti-48Al-2Cr-2Nb) in contact with a nickel-base superalloy (Inconel 718) in air at temperatures from 23 to 550 C. Fretting wear experiments were conducted with 9.4-mm-diameter hemispherical Inconel (IN) 718 pins in contact with Ti-48Al-2Cr-2Nb flats (and the reverse) at loads from 1 to 40 N and fretting frequencies from 50 to 160 Hz with slip amplitudes from 50 to 200 microns for 1 to 20 million fretting cycles. The results were similar for both combinations of pin and flat. Reference fretting wear experiments were also conducted with 9.4-mm-diameter hemispherical Ti-6Al-4V pins in contact with IN718 flats. The interfacial adhesive bonds between Ti-48Al-2Cr-2Nb and IN718 in contact were generally stronger than the cohesive bonds in the cohesively weaker Ti-48Al-2Cr-2Nb. The failed Ti-48Al-2Cr-2Nb subsequently transferred to the IN718 surface at any fretting condition. The wear scars produced on Ti-48Al-2Cr-2Nb contained metallic and oxide wear debris, scratches, plastically deformed asperities, cracks, and fracture pits. Oxide layers readily formed on the Ti-48Al-2Cr-2Nb surface at 550 C, but cracks easily occurred in the oxide layers. Factors including fretting frequency, temperature, slip amplitude, and load influenced the fretting behavior of Ti-48Al-2Cr-2Nb in contact with IN718. The wear volume loss of Ti-48Al-2Cr-2Nb generally decreased with increasing fretting frequency. The increasing rate of oxidation at elevated temperatures up to 200 C led to a drop in wear volume loss at 200 C. However, the fretting wear increased as the temperature was increased from 200 to 550 C. The highest temperatures of 450 and 550 C resulted in oxide film disruption with generation of cracks, loose wear debris, and pits on the Ti-48Al-2Cr-2Nb wear surface. The wear volume loss generally increased as the slip amplitude increased. The wear volume loss also generally increased as the load increased

  19. Accumulation of formamide in hydrothermal pores to form prebiotic nucleobases

    NASA Astrophysics Data System (ADS)

    Niether, Doreen; Afanasenkau, Dzmitry; Dhont, Jan K. G.

    2016-04-01

    Formamide is one of the important compounds from which prebiotic molecules can be synthesized, provided that its concentration is sufficiently high. For nucleotides and short DNA strands, it has been shown that a high degree of accumulation in hydrothermal pores occurs, so that temperature gradients might play a role in the origin of life [Baaske P, et al. (2007) Proc Natl Acad Sci USA 104(22):9346-9351]. We show that the same combination of thermophoresis and convection in hydrothermal pores leads to accumulation of formamide up to concentrations where nucleobases are formed. The thermophoretic properties of aqueous formamide solutions are studied by means of Infrared Thermal Diffusion Forced Rayleigh Scattering. These data are used in numerical finite element calculations in hydrothermal pores for various initial concentrations, ambient temperatures, and pore sizes. The high degree of formamide accumulation is due to an unusual temperature and concentration dependence of the thermophoretic behavior of formamide. The accumulation fold in part of the pores increases strongly with increasing aspect ratio of the pores, and saturates to highly concentrated aqueous formamide solutions of ˜85 wt% at large aspect ratios. Time-dependent studies show that these high concentrations are reached after 45-90 d, starting with an initial formamide weight fraction of 10-310-3 wt % that is typical for concentrations in shallow lakes on early Earth.

  20. Dissociative electron attachment to the gas-phase nucleobase hypoxanthine

    NASA Astrophysics Data System (ADS)

    Dawley, M. Michele; Tanzer, Katrin; Carmichael, Ian; Denifl, Stephan; Ptasińska, Sylwia

    2015-06-01

    We present high-resolution measurements of the dissociative electron attachment (DEA) to isolated gas-phase hypoxanthine (C5H4N4O, Hyp), a tRNA purine base. The anion mass spectra and individual ion efficiency curves from Hyp were measured as a function of electron energy below 9 eV. The mass spectra at 1 and 6 eV exhibit the highest anion yields, indicating possible common precursor ions that decay into the detectable anionic fragments. The (Hyp - H) anion (C5H3N4O-) exhibits a sharp resonant peak at 1 eV, which we tentatively assign to a dipole-bound state of the keto-N1H,N9H tautomer in which dehydrogenation occurs at either the N1 or N9 position based upon our quantum chemical computations (B3LYP/6-311+G(d,p) and U(MP2-aug-cc-pVDZ+)) and prior studies with adenine. This closed-shell dehydrogenated anion is the dominant fragment formed upon electron attachment, as with other nucleobases. Seven other anions were also observed including (Hyp - NH)-, C4H3N4-/C4HN3O-, C4H2N3-, C3NO-/HC(HCN)CN-, OCN-, CN-, and O-. Most of these anions exhibit broad but weak resonances between 4 and 8 eV similar to many analogous anions from adenine. The DEA to Hyp involves significant fragmentation, which is relevant to understanding radiation damage of biomolecules.

  1. Dissociative electron attachment to the gas-phase nucleobase hypoxanthine

    SciTech Connect

    Dawley, M. Michele; Tanzer, Katrin; Denifl, Stephan E-mail: Sylwia.Ptasinska.1@nd.edu; Carmichael, Ian; Ptasińska, Sylwia E-mail: Sylwia.Ptasinska.1@nd.edu

    2015-06-07

    We present high-resolution measurements of the dissociative electron attachment (DEA) to isolated gas-phase hypoxanthine (C{sub 5}H{sub 4}N{sub 4}O, Hyp), a tRNA purine base. The anion mass spectra and individual ion efficiency curves from Hyp were measured as a function of electron energy below 9 eV. The mass spectra at 1 and 6 eV exhibit the highest anion yields, indicating possible common precursor ions that decay into the detectable anionic fragments. The (Hyp − H) anion (C{sub 5}H{sub 3}N{sub 4}O{sup −}) exhibits a sharp resonant peak at 1 eV, which we tentatively assign to a dipole-bound state of the keto-N1H,N9H tautomer in which dehydrogenation occurs at either the N1 or N9 position based upon our quantum chemical computations (B3LYP/6-311+G(d,p) and U(MP2-aug-cc-pVDZ+)) and prior studies with adenine. This closed-shell dehydrogenated anion is the dominant fragment formed upon electron attachment, as with other nucleobases. Seven other anions were also observed including (Hyp − NH){sup −}, C{sub 4}H{sub 3}N{sub 4}{sup −}/C{sub 4}HN{sub 3}O{sup −}, C{sub 4}H{sub 2}N{sub 3}{sup −}, C{sub 3}NO{sup −}/HC(HCN)CN{sup −}, OCN{sup −}, CN{sup −}, and O{sup −}. Most of these anions exhibit broad but weak resonances between 4 and 8 eV similar to many analogous anions from adenine. The DEA to Hyp involves significant fragmentation, which is relevant to understanding radiation damage of biomolecules.

  2. The origin of efficient triplet state population in sulfur-substituted nucleobases

    NASA Astrophysics Data System (ADS)

    Mai, Sebastian; Pollum, Marvin; Martínez-Fernández, Lara; Dunn, Nicholas; Marquetand, Philipp; Corral, Inés; Crespo-Hernández, Carlos E.; González, Leticia

    2016-10-01

    Elucidating the photophysical mechanisms in sulfur-substituted nucleobases (thiobases) is essential for designing prospective drugs for photo- and chemotherapeutic applications. Although it has long been established that the phototherapeutic activity of thiobases is intimately linked to efficient intersystem crossing into reactive triplet states, the molecular factors underlying this efficiency are poorly understood. Herein we combine femtosecond transient absorption experiments with quantum chemistry and nonadiabatic dynamics simulations to investigate 2-thiocytosine as a necessary step to unravel the electronic and structural elements that lead to ultrafast and near-unity triplet-state population in thiobases in general. We show that different parts of the potential energy surfaces are stabilized to different extents via thionation, quenching the intrinsic photostability of canonical DNA and RNA nucleobases. These findings satisfactorily explain why thiobases exhibit the fastest intersystem crossing lifetimes measured to date among bio-organic molecules and have near-unity triplet yields, whereas the triplet yields of canonical nucleobases are nearly zero.

  3. High-energy chemistry of formamide: a simpler way for nucleobase formation.

    PubMed

    Ferus, Martin; Michalčíková, Regina; Shestivská, Violetta; Šponer, Jiří; Šponer, Judit E; Civiš, Svatopluk

    2014-01-30

    The formation of nucleobases from formamide during a high-energy density event, i.e., the impact of an extraterrestrial body into the planetary atmosphere, was studied by irradiation of formamide ice and liquid samples with a high-power laser in the presence of potential catalysts. FTIR spectroscopy, time-resolved emission spectroscopy, and GC-MS were subsequently used to monitor the dissociation of this molecule into stable molecular fragments (HCN, H2O, HNCO, H2, CO, and NH3) and unstable species (HNC, •CN, and •NH). The kinetic and thermodynamic models of the high-energy density event molecular dynamics have been suggested together with the reaction routes leading from the dissociation products to the nucleobases. In addition, using theoretical calculations, we propose a simple new reaction pathway for the formation of both pyrimidine and purine nucleobases involving •CN radical chemistry. PMID:24437678

  4. Nucleobases and Other Prebiotic Species from the UV Irradiation of Pyrimidine in Astrophysical Ices

    NASA Technical Reports Server (NTRS)

    Sandford, Scott; Materese, Christopher; Nuevo, Michel

    2012-01-01

    Nucleobases are aromatic N-heterocycles that constitute the informational subunits of DNA and RNA and are divided into two families: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites and their extraterrestrial origin confirmed by isotope measurement. Although no N-heterocycles have been individually identified in the ISM, the 6.2-micron interstellar emission feature seen towards many astronomical objects suggests a population of such molecules is likely present. We report on a study of the formation of pyrimidine-based molecules, including nucleobases and other species of prebiotic interest, from the ultraviolet (UV) irradiation of pyrimidine in low temperature ices containing H2O, NH3, C3OH, and CH4, to simulate the astrophysical conditions under which prebiotic species may be formed in the Solar System.

  5. Intonation and compensation of fretted string instruments

    NASA Astrophysics Data System (ADS)

    Varieschi, Gabriele; Gower, Christina

    2011-04-01

    We discuss theoretical and physical models that are useful for analyzing the intonation of musical instruments such as guitars and mandolins and can be used to improve the tuning on these instruments. The placement of frets on the fingerboard is designed according to mathematical rules and the assumption of an ideal string. The analysis becomes more complicated when we include the effects of deformation of the string and inharmonicity due to other string characteristics. As a consequence, perfect intonation of all the notes on the instrument cannot be achieved, but complex compensation procedures can be introduced to minimize the problem. To test the validity of these procedures, we performed extensive measurements using standard monochord sonometers and other acoustical devices, confirming the correctness of our theoretical models. These experimental activities can be integrated into acoustics courses and laboratories and can become a more advanced version of basic experiments with monochords and sonometers. This work was supported by a grant from the Frank R. Seaver College of Science and Engineering, Loyola Marymount University.

  6. Nucleobase recognition in ssDNA at the central constriction of the αhemolysin pore

    PubMed Central

    Stoddart, David; Heron, Andrew J.; Klingelhoefer, Jochen; Mikhailova, Ellina; Maglia, Giovanni; Bayley, Hagan

    2010-01-01

    Nanopores are under investigation for single-molecule DNA sequencing. The α-hemolysin (αHL) protein nanopore contains three recognition points capable of nucleobase discrimination in individual immobilized ssDNA molecules. We have modified the recognition point R1 by extensive mutagenesis of residue 113. Amino acids that provide an energy barrier to ion flow (e.g. bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier weaken it. Amino acids with related side chains produce similar patterns of nucleobase recognition providing a rationale for the redesign of recognition points. PMID:20704324

  7. The development of FRET-based dual receptor optical biosensor

    NASA Astrophysics Data System (ADS)

    Xu, Juntao

    The focus of the research presented in this dissertation is the development of a new FRET-based dual receptor sensing method for detecting the human immunodeficiency virus (HIV). The new detection method presented in this dissertation imitates the way HIV infects cells. It utilizes the two receptor-binding event and integrates a chemical transducer system with two unique protein receptors, CD4 and mAb (HIV-1 gp120 monoclonal antibody), which both bind to gp120. The chemical transduction system is based on the distance-dependant principle of fluorescence resonance energy transfer (FRET). The work presented in this dissertation attempts to demonstrate the feasibility of this new sensing method both in solution and on an optical fiber. Appropriate FRET pairs which have high energy transfer efficiency as well as good conjugation properties with receptors were selected and optimized. The two receptors, CD4 and mAb which specifically bind to gp120, were conjugated to one of the optimized FRET fluorophore pairs, AMCA-NHS (succinimidyl-7-amino-4-methylcoumarin-3-acetic acid) and FITC (fluorescein isothiocyanate), respectively. For the solution test, the viral protein gp120, which is the featured protein on the surface of HIV-1, was detected by the mixed solution of the two FRET pair tagged receptors. A spectrofluorometer was used to detect the fluorescent change between AMCA-NHS and FITC peak intensities when the receptors bind to the gp120. Specific binding and non-specific binding gp120 were used to test the selectivity of this method. The results of the solution test indicated that FRET-conjugated receptors can efficiently distinguish the presence of specific and non-specific binding gp120 and proved the feasibility of the FRET-based dual receptor method in detecting the presence of gp120 with a limit of detection of 5ng/ml (0.5nM) in solution. For the optical fiber test, two FRET-conjugated receptors were immobilized onto an optical fiber silica core tip to detect the

  8. Heterogeneity and restricted state selection in FRET with fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Blacker, T. S.; Duchen, M. R.; Bain, A. J.

    2016-02-01

    Most fluorescent proteins exhibit multi-exponential fluorescence decays, indicating the presence of a heterogeneous excited state population. In the analysis of FRET to and between fluorescent proteins, it is often convenient to assume that a single interaction pathway is involved. However, in recent work we have shown that this assumption does not hold. Moreover, certain pathways can be highly constrained, leading to the potential misinterpretation of experimental data concerning protein-protein interactions. FRET and single-photon absorption both obey the same global electric dipole selection rules but differ greatly in the mechanism of the acceptor photoselection. In an isotropic medium, single-photon excitation accesses all acceptor transition dipole moment orientations with an equal probability. However, the FRET rate depends on the relative orientation of the donor and acceptor through the κ2 orientation parameter. We show how time- and spectrally- resolved fluorescence intensity and anisotropy decay measurements following direct acceptor excitation, combined with those of the interacting FRET pair, can be used to identify restricted FRET state selection and thus provide accurate measurements of protein-protein interaction dynamics.

  9. Dual-laser homo-FRET on the cell surface.

    PubMed

    Bene, László; Ungvári, Tamás; Fedor, Roland; Nagy, István; Damjanovich, László

    2015-05-01

    Inhomogeneous broadening and red-edge effects have been detected on a highly mobile system of fluorescently conjugated mAbs targeted to cell surface receptors. By exploiting site-selective spectroscopy and the characteristic loss of homo-FRET on increasing excitation and decreasing emission wavelengths, contributions of physical rotation and homo-FRET to the depolarization of fluorescence anisotropy have been separated. Absolute homo-FRET efficiency has been determined by ratioing two anisotropies: a homo-FRET-sensitive one, which is excited at the absorption main band and detected at the long wavelength region of emission, and a homo-FRET-insensitive one, which is excited at the long wavelength region of absorption and detected at the short wavelength region of emission. Because the anisotropies are simultaneously detected in a unified detection scheme of a dual T-format arrangement, the method is applicable for the real-time tracking of dynamical changes of physical rotations and proximities. The utility of the method is demonstrated in the context of the MHCII molecule and the heavy and light chains of the MHCI molecule, a system of three receptors with well-characterized close mutual proximities. Although the method is presented for a flow cytometer, it can also be realized in a fluorescence microscope capable for dual-laser excitation and dual-anisotropy detection.

  10. High-throughput FRET assay yields allosteric SERCA activators.

    PubMed

    Cornea, Razvan L; Gruber, Simon J; Lockamy, Elizabeth L; Muretta, Joseph M; Jin, Dongzhu; Chen, Jiqiu; Dahl, Russell; Bartfai, Tamas; Zsebo, Krisztina M; Gillispie, Gregory D; Thomas, David D

    2013-01-01

    Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) by its cardiac regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca(2+) regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20 000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 hits (0.2%), 31 (72%) were found to be false-positives upon more thorough FRET testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and preclinical testing. We were concerned about the high rate of false-positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HTS.

  11. Influence of fretting on flexural fatigue of 304 stainless steel and mild steel

    NASA Technical Reports Server (NTRS)

    Bill, R. C.; Rohn, D. A.

    1978-01-01

    Fretting fatigue experiments conducted on 304 stainless steel using a flexural fatigue test arrangement with bolted on fretting pads demonstrated that fatigue life is reduced by at least a factor of 10 in the 265 to 334 MPa (38,500 - to 48,500 psi) nominal flexural fatigue stress range. In addition, experiments in which the fretting pads were removed after selected numbers of cycles, followed by continued flexural fatigue without fretting show that continued fretting beyond 50,000 cycles does not significantly further reduce fatigue life of 304 stainless steel at 317 MPa (46,000 psi). Microscopic examination of the fretted contact areas revealed fracture initiation sites as well as numerous cracks that did not propagate to failure. Flexural fretting fatigue experiments performed on mild steel showed an insensitivity of fatigue life to the incidence of fretting under flexural stress conditions of from 162 to 217 MPa (23,500 to 31,500 psi).

  12. Role of the heat capacity change in understanding and modeling melting thermodynamics of complementary duplexes containing standard and nucleobase-modified LNA.

    PubMed

    Hughesman, Curtis B; Turner, Robin F B; Haynes, Charles A

    2011-06-14

    Melting thermodynamic data obtained by differential scanning calorimetry (DSC) are reported for 43 duplexed oligonucleotides containing one or more locked nucleic acid (LNA) substitutions. The measured heat capacity change (ΔC(p)) for the helix-to-coil transition is used to compute the changes in enthalpy and entropy for melting of an LNA-bearing duplex at the T(m) of its corresponding isosequential unmodified DNA duplex to allow rigorous thermodynamic analysis of the stability enhancements provided by LNA substitutions. Contrary to previous studies, our analysis shows that the origin of the improved stability is almost exclusively a net reduction (ΔΔS° < 0) in the entropy gain accompanying the helix-to-coil transition, with the magnitude of the reduction dependent on the type of nucleobase and its base pairing properties. This knowledge and our average measured value for ΔC(p) of 42 ± 11 cal mol(-1) K(-1) bp(-1) are then used to derive a new model that accurately predicts melting thermodynamics and the increased melting temperature (ΔT(m)) of heteroduplexes formed between an unmodified DNA strand and a complementary strand containing any number and configuration of standard LNA nucleotides A, T, C, and G. This single-base thermodynamic (SBT) model requires only four entropy-related parameters in addition to ΔC(p). Finally, DSC data for 20 duplexes containing the nucleobase-modified LNAs 2-aminoadenine (D) and 2-thiothymine (H) are reported and used to determine SBT model parameters for D and H. The data and model suggest that along with the greater stability enhancement provided by D and H bases relative to their corresponding A and T analogues, the unique pseudocomplementary properties of D-H base pairs may make their use appealing for in vitro and in vivo applications.

  13. ICE AND DEBRIS IN THE FRETTED TERRAIN, MARS.

    USGS Publications Warehouse

    Lucchitta, Baerbel K.

    1984-01-01

    Viking moderate- and high-resolution images along the northern highland margin were studied monoscopically and stereoscopically to contribute to an understanding of the development of fretted terrain. Results support the hypothesis that the fretting process involved flow facilitated by interstitial ice. The process apparently continued for a long period of time, and debris-apron formation shaped the fretted terrain in the past as well as the present. Interstitial ice in debris aprons is most likely derived from ground ice obtained by sapping or scarp collapse. Debris aprons could have been removed by sublimation if they consisted mostly of ice, or by deflation if they consisted mostly of debris. To remove the debris, wind erosion was either very intense early in martian history, or was intermittent, perhaps owing to climatic cycles.

  14. Borromean three-body FRET in frozen Rydberg gases

    PubMed Central

    Faoro, R.; Pelle, B.; Zuliani, A.; Cheinet, P.; Arimondo, E.; Pillet, P.

    2015-01-01

    Controlling the interactions between ultracold atoms is crucial for quantum simulation and computation purposes. Highly excited Rydberg atoms are considered in this prospect for their strong and controllable interactions known in the dipole-dipole case to induce non-radiative energy transfers between atom pairs, similarly to fluorescence resonance energy transfer (FRET) in biological systems. Here we predict few-body FRET processes in Rydberg atoms and observe the first three-body resonance energy transfer in cold Rydberg atoms using cold caesium atoms. In these resonances, additional relay atoms carry away an energy excess preventing the two-body resonance, leading thus to a Borromean type of energy transfer. These few-body processes present strong similarities with multistep FRET between chromophores sometimes called donor-bridge-acceptor or superexchange. Most importantly, they generalize to any Rydberg atom and could lead to new implementations of few-body quantum gates or entanglement. PMID:26348821

  15. A portable FRET analyzer for rapid detection of sugar content.

    PubMed

    Kim, Haseong; Kim, Hyo Sang; Ha, Jae-Seok; Lee, Seung-Goo

    2015-05-21

    Fluorescence resonance energy transfer (FRET) is widely used as a core process in biometric sensors to detect small molecules such as sugars, calcium ions, or amino acids. However, FRET based biosensors with innate weak signal intensity require the use of expensive, high-sensitive equipment. In the present study, these shortcomings were overcome with the fabrication of a sensitive, inexpensive, and portable analyzer which provides quantitative detection of small molecules in a liquid sample. The usability of the developed analyzer was successfully tested by measuring sucrose and maltose contents in commercially available beverage samples, with better performance than the conventional monochromator-type spectrofluorometer. It is anticipated that miniaturization of the equipment and improving the FRET based biosensors will contribute to the practical use of this hand-held analyzer in conditions where high-end equipment is not available. PMID:25857344

  16. The Photochemistry of Pyrimidine in Realistic Astrophysical Ices and the Production of Nucleobases

    NASA Astrophysics Data System (ADS)

    Nuevo, Michel; Materese, Christopher K.; Sandford, Scott A.

    2014-10-01

    Nucleobases, together with deoxyribose/ribose and phosphoric acid, are the building blocks of DNA and RNA for all known life. The presence of nucleobase-like compounds in carbonaceous chondrites delivered to the Earth raises the question of an extraterrestrial origin for the molecules that triggered life on our planet. Whether these molecules are formed in interstellar/protostellar environments, in small parent bodies in the solar system, or both, is currently unclear. Recent experiments show that the UV irradiation of pyrimidine (C4H4N2) in H2O-rich ice mixtures that contain NH3, CH3OH, or CH4 leads to the formation of the pyrimidine-based nucleobases uracil, cytosine, and thymine. In this work, we discuss the low-temperature UV irradiation of pyrimidine in realistic astrophysical ice mixtures containing H2O, CH3OH, and NH3, with or without CH4, to search for the production of nucleobases and other prebiotic compounds. These experiments show the presence of uracil, urea, glycerol, hexamethylenetetramine, small amino acids, and small carboxylic acids in all samples. Cytosine was only found in one sample produced from ices irradiated with a higher UV dose, while thymine was not found in any sample, even after irradiation with a higher UV dose. Results are discussed to evaluate the role of the photochemistry of pyrimidine in the inventory of organic molecules detected in meteorites and their astrophysical/astrobiological implications.

  17. Hartree-Fock Cluster Study of Electronic Structures and Nuclear Quadrupole Interactions in Solid Nucleobases.

    NASA Astrophysics Data System (ADS)

    Scheicher, R. H.; Dubey, Archana; Badu, S. R.; Saha, H. P.; Pink, R. H.; Nagamine, K.; Torikai, E.; Chow, Lee; Das, T. P.

    2008-03-01

    In recent work [1] we have studied nucleobases attached to a CH3 group to simulate the influence of their binding to the sugar rings and the phosphate groups in DNA and RNA and the effect of this binding on the nuclear quadrupole interactions of ^14N, ^17O and ^2H nuclei. Our results from this work have indicated that for ^17O, the binding to the CH3 group moves our results from the free nucleobases closer to the experimentally observed data [2] in the solid nucleobases. We are now investigating the solid nucleobases by the first --principles Hartree-Fock cluster procedure that we have employed earlier for the halogen molecular solids [3]. Our results for the binding energy of an imidazole molecule in the molecular solid system and the ^14N, ^17O and ^2H nuclear quadrupole interaction parameters will be presented. [1] T.P. Das et al (at this APS meeting), [2] Gang Wu et al, J. Am.Chem. Soc. 124, 1768(2002). [3] M.M. Aryal et al Hyperfine Interactions (to be published).

  18. 6-Pyrazolylpurine as an Artificial Nucleobase for Metal-Mediated Base Pairing in DNA Duplexes

    PubMed Central

    Léon, J. Christian; Sinha, Indranil; Müller, Jens

    2016-01-01

    The artificial nucleobase 6-pyrazol-1-yl-purine (6PP) has been investigated with respect to its usability in metal-mediated base pairing. As was shown by temperature-dependent UV spectroscopy, 6PP may form weakly stabilizing 6PP–Ag(I)–6PP homo base pairs. Interestingly, 6PP can be used to selectively recognize a complementary pyrimidine nucleobase. The addition of Ag(I) to a DNA duplex comprising a central 6PP:C mispair (C = cytosine) leads to a slight destabilization of the duplex. In contrast, a stabilizing 6PP–Ag(I)–T base pair is formed with a complementary thymine (T) residue. It is interesting to note that 6PP is capable of differentiating between the pyrimidine moieties despite the fact that it is not as sterically crowded as 6-(3,5-dimethylpyrazol-1-yl)purine, an artificial nucleobase that had previously been suggested for the recognition of nucleic acid sequences via the formation of a metal-mediated base pair. Hence, the additional methyl groups of 6-(3,5-dimethylpyrazol-1-yl)purine may not be required for the specific recognition of the complementary nucleobase. PMID:27089326

  19. Characterization of nucleobases and nucleosides in the fruit of Alpinia oxyphylla collected from different cultivation regions.

    PubMed

    Song, Wenjing; Li, Yonghui; Wang, Jianguo; Li, Zeyou; Zhang, Junqing

    2014-03-01

    The fruit of Alpinia oxyphylla, known as Yizhi, Yakuchi and Ikji in Chinese, Japanese, and Korean, respectively, has been utilized as an important drug for the treatment of diarrhea, dyspepsia, spermatorrhea, kidney asthenia and abdominal pain in East Asian traditional medicine for thousands of years. Since the therapeutic effects of A. oxyphylla are attributed to multiple components and nucleobases and nucleosides exhibit various bioactivities, it is necessary to explore the chemical characterization of nucleobases and nucleosides in this herb. Herein, 10 common nucleobases and nucleosides, including cytidine, adenosine, thymidine, inosine, guanosine, 2'-deoxyinosine, guanine, adenine, cytosine, and hypoxanthine, were quantified simultaneously in the fruit of A. oxyphylla collected from different geographical regions. Changes in their contents were discussed, and hierarchical cluster analysis (HCA) was performed to classify all samples on the basis of the contents of the investigated analytes. The results indicated that there was a large variation in the contents of nucleobases and nucleosides among the herbs from different regions, and the samples collected from the same cultivation region were mostly classified in one cluster. The method can be used for comprehensive quality evaluation of A. oxyphylla.

  20. Survey of Analogue Spacetimes

    NASA Astrophysics Data System (ADS)

    Visser, Matt

    Analogue spacetimes (and more boldly, analogue models both of and for gravity), have attracted significant and increasing attention over the last decade and a half. Perhaps the most straightforward physical example, which serves as a template for most of the others, is Bill Unruh's model for a dumb hole,(mute black hole, acoustic black hole), wherein sound is dragged along by a moving fluid—and can even be trapped behind an acoustic horizon. This and related analogue models for curved spacetimes are useful in many ways: analogue spacetimes provide general relativists with extremely concrete physical models to help focus their thinking, and conversely the techniques of curved spacetime can sometimes help improve our understanding of condensed matter and/or optical systems by providing an unexpected and countervailing viewpoint. In this chapter, I shall provide a few simple examples of analogue spacetimes as general background for the rest of the contributions.

  1. 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.

  2. Fretting Stresses in Single Crystal Superalloy Turbine Blade Attachments

    NASA Technical Reports Server (NTRS)

    Arakere, Nagaraj K.; Swanson, Gregory

    2000-01-01

    Single crystal nickel base superalloy turbine blades are being utilized in rocket engine turbopumps and turbine engines because of their superior creep, stress rupture, melt resistance and thermomechanical fatigue capabilities over polycrystalline alloys. Currently the most widely used single crystal nickel base turbine blade superalloys are PWA 1480/1493 and PWA 1484. These alloys play an important role in commercial, military and space propulsion systems. High Cycle Fatigue (HCF) induced failures in aircraft gas turbine and rocket engine turbopump blades is a pervasive problem. Blade attachment regions are prone to fretting fatigue failures. Single crystal nickel base superalloy turbine blades are especially prone to fretting damage because the subsurface shear stresses induced by fretting action at the attachment regions can result in crystallographic initiation and crack growth along octahedral planes. Furthermore, crystallographic crack growth on octahedral planes under fretting induced mixed mode loading can be an order of magnitude faster than under pure mode I loading. This paper presents contact stress evaluation in the attachment region for single crystal turbine blades used in the NASA alternate Advanced High Pressure Fuel Turbo Pump (HPFTP/AT) for the Space Shuttle Main Engine (SSME). Single crystal materials have highly orthotropic properties making the position of the crystal lattice relative to the part geometry a significant factor in the overall analysis. Blades and the attachment region are modeled using a large-scale 3D finite element (FE) model capable of accounting for contact friction, material orthotrophy, and variation in primary and secondary crystal orientation. Contact stress analysis in the blade attachment regions is presented as a function of coefficient of friction and primary and secondary crystal orientation, Stress results are used to discuss fretting fatigue failure analysis of SSME blades. Attachment stresses are seen to reach

  3. Photochemistry of Pyrimidine in Astrophysical Ices: Formation of Nucleobases and Other Prebiotic Species

    NASA Technical Reports Server (NTRS)

    Nuevo, Michel; Sandford, Scott A.; Materese, Christopher K.; Milam, Stefanie N.

    2012-01-01

    Nucleobases are N-heterocycles that are the informational subunits of DNA and RNA. They are divided into two molecular groups: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites, and their extraterrestrial origin confirmed by isotopic measurements. Although no N-heterocycles have ever been observed in the ISM, the positions of the 6.2- m interstellar emission features suggest a population of such molecules is likely to be present. However, laboratory experiments have shown that the ultraviolet (UV) irradiation of pyrimidine in ices of astrophysical relevance such as H2O, NH3, CH3OH, CH4, CO, or combinations of these at low temperature (less than or equal to 20 K) leads to the formation of several pyrimidine derivatives including the nucleobases uracil and cytosine, as well as precursors such as 4(3H)-pyrimidone and 4-aminopyrimidine. Quantum calculations on the formation of 4(3H)-pyrimidone and uracil from the irradiation of pyrimidine in pure H2O ices are in agreement with their experimental formation pathways.10 In those residues, other species of prebiotic interest such as urea as well as the amino acids glycine and alanine could also be identified. However, only very small amounts of pyrimidine derivatives containing CH3 groups could be detected, suggesting that the addition of methyl groups to pyrimidine is not an efficient process. For this reason, the nucleobase thymine was not observed in any of the samples. In this work, we study the formation of nucleobases and other photo-products of prebiotic interest from the UV irradiation of pyrimidine in ices containing H2O, NH3, CH3OH, and CO, mixed in astrophysical proportions.

  4. Nonstationary analogue black holes

    NASA Astrophysics Data System (ADS)

    Eskin, Gregory

    2014-12-01

    We study the existence of analogue nonstationary spherically symmetric black holes. The prime example is the acoustic model see Unruh (1981 Phys. Rev. Lett. 46 1351). We consider also a more general class of metrics that could be useful in other physical models of analogue black and white holes. We give examples of the appearance of black holes and of disappearance of white holes. We also discuss the relation between the apparent and the event horizons for the case of analogue black holes. In the end we study the inverse problem of determination of black or white holes by boundary measurements for the spherically symmetric nonstationary metrics.

  5. Fretting of Nickel-Chromium-Aluminum Alloys at Temperatures to 816 C

    NASA Technical Reports Server (NTRS)

    Bill, R. C.

    1974-01-01

    A series of four nickel-based alloys containing 10 percent and 20 percent chromium in combination with 2 percent and 5 percent aluminum were fretted in dry air at temperatures to 816 C. At all temperatures, the alloys showed far less fretting wear than did high-purity nickel. This was attributed to the formation of protective oxide films on the alloys, the result of the selective oxidation of the alloy constituents. Increasing the aluminum concentration reduced fretting wear at all temperatures. Increasing the chromium concentration from 10 percent to 20 percent resulted in decreased fretting wear at 23 and 540 C, but increased fretting wear at 650 and 816 C.

  6. Plant-based FRET biosensor discriminates enviornmental zinc levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heavy metal accumulation in the environment poses great risks to flora and fauna. However, monitoring sites prone to accumulation poses scale and economic challenges. In this study, we present and test a method for monitoring these sites using fluorescent resonance energy transfer (FRET) change in r...

  7. Fretting Fatigue of Gamma TiAl Studied

    NASA Technical Reports Server (NTRS)

    Miyoshi, Kazuhisa; Lerch, Bradley A.; Draper, Susan L.

    2003-01-01

    Gamma titanium-aluminum alloy (g-TiAl) is an attractive new material for aerospace applications because of its low density and high specific strength in comparison to currently used titanium and nickel-base alloys. Potential applications for this material are compressor and low-pressure turbine blades. These blades are fitted into either the compressor or turbine disks via a dovetail connection. The dovetail region experiences a complex stress state due to the alternating centrifugal force and the natural high-frequency vibration of the blade. Because of the dovetail configuration and the complex stress state, fretting is often a problem in this area. Furthermore, the local stress state becomes more complex when the influence of the metal-metal contact and the edge of the contact is evaluated. Titanium and titanium-based alloys in the clean state exhibit strong adhesive bonds when in contact with themselves and other materials (refs. 1 and 2). This adhesion causes heavy surface damage and high friction in practical cases. Although the wear produced by fretting may be mild, the reduction in fatigue life can be substantial. Thus, there is the potential for fretting problems with these TiAl applications. Since TiAl is an emerging material, there has been limited information about its fretting behavior.

  8. Uncovering aberrant mutant PKA function with flow cytometric FRET

    PubMed Central

    Lee, Shin-Rong; Sang, Lingjie; Yue, David T.

    2016-01-01

    SUMMARY Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPI). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell, FRET-based binding curves using a commercially-available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validate our binding assay against the gold-standard isothermal calorimetry (ITC), and use flow cytometric FRET to uncover the structural and functional effects of the Cushing syndrome-causing mutation (L206R) on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners, but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability and power of flow cytometric FRET. PMID:26997269

  9. Application of FRET probes in the analysis of neuronal plasticity

    PubMed Central

    Ueda, Yoshibumi; Kwok, Showming; Hayashi, Yasunori

    2013-01-01

    Breakthroughs in imaging techniques and optical probes in recent years have revolutionized the field of life sciences in ways that traditional methods could never match. The spatial and temporal regulation of molecular events can now be studied with great precision. There have been several key discoveries that have made this possible. Since green fluorescent protein (GFP) was cloned in 1992, it has become the dominant tracer of proteins in living cells. Then the evolution of color variants of GFP opened the door to the application of Förster resonance energy transfer (FRET), which is now widely recognized as a powerful tool to study complicated signal transduction events and interactions between molecules. Employment of fluorescent lifetime imaging microscopy (FLIM) allows the precise detection of FRET in small subcellular structures such as dendritic spines. In this review, we provide an overview of the basic and practical aspects of FRET imaging and discuss how different FRET probes have revealed insights into the molecular mechanisms of synaptic plasticity and enabled visualization of neuronal network activity both in vitro and in vivo. PMID:24133415

  10. High-Throughput FRET Assay Yields Allosteric SERCA Activators

    PubMed Central

    Cornea, Razvan L.; Lockamy, Elizabeth L.; Gruber, Simon J.; Muretta, Joseph M.; Jin, Dongzhu; Chen, Jiqiu; Dahl, Russell; Bartfai, Tamas; Zsebo, Krisztina M.; Gillispie, Gregory D.; Thomas, David D.

    2013-01-01

    Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT. PMID:22923787

  11. Structural kinetics of myosin by transient time-resolved FRET

    PubMed Central

    Nesmelov, Yuri E.; Agafonov, Roman V.; Negrashov, Igor V.; Blakely, Sarah E.; Titus, Margaret A.; Thomas, David D.

    2011-01-01

    For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)2FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)2FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)2FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism. PMID:21245357

  12. Triazolo-β-aza-ε-amino acid and its aromatic analogue as novel scaffolds for β-turn peptidomimetics.

    PubMed

    Bag, Subhendu Sekhar; Jana, Subhashis; Yashmeen, Afsana; De, Suranjan

    2015-03-28

    Triazolo-β-aza-ε-amino acid and its aromatic analogue ((Al)TAA/(Ar)TAA) in the peptide backbone mark a novel class of conformationally constrained molecular scaffolds to induce β-turn conformations. This was demonstrated for (Al)TAA in a Leu-enkephalin analogue and in a designed pentapeptide wherein the FRET process was established. Restricted rotation induced chirality and turn conformation into the achiral aromatic amino acid scaffold, (Ar)TAA, which in a short tripeptide backbone acted as a β-turn mimic as a β-sheet folding nucleator.

  13. Molecular signaling in live cells studied by FRET

    NASA Astrophysics Data System (ADS)

    Chien, Shu; Wang, Yingxiao

    2012-03-01

    Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) enables visualization of signaling events in live cells with high spatiotemporal resolution. We have used FRET to assess temporal and spatial characteristics for signaling molecules, including tyrosine kinases Src and FAK, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. Activations of Src and Rac by platelet derived growth factor (PDGF) led to distinct subcellular patterns during cell migration on micropatterned surface, and these two enzymes interact with each other to form a feedback loop with differential regulations at different subcellular locations. We have developed FRET biosensors to monitor FAK activities at rafts vs. non-raft regions of plasma membrane in live cells. In response to cell adhesion on matrix proteins or stimulation by PDGF, the raft-targeting FAK biosensor showed a stronger FRET response than that at non-rafts. The FAK activation at rafts induced by PDGF is mediated by Src. In contrast, the FAK activation at rafts induced by adhesion is independent of Src activity, but rather is essential for Src activation. Thus, Src is upstream to FAK in response to chemical stimulation (PDGF), but FAK is upstream to Src in response to mechanical stimulation (adhesion). A novel biosensor has been developed to dynamically visualize the activity of membrane type-1-matrix metalloproteinase (MT1-MMP), which proteolytically remodels the extracellular matrix. Epidermal growth factor (EGF) directed active MT1-MMP to the leading edge of migrating live cancer cells with local accumulation of EGF receptor via a process dependent on an intact cytoskeletal network. In summary, FRET-based biosensors enable the elucidation of molecular processes and hierarchies underlying spatiotemporal regulation of biological and pathological processes, thus advancing our knowledge on how cells perceive mechanical/chemical cues in space and time to coordinate

  14. Molecular signaling in live cells studied by FRET

    NASA Astrophysics Data System (ADS)

    Chien, Shu; Wang, Yingxiao

    2011-11-01

    Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) enables visualization of signaling events in live cells with high spatiotemporal resolution. We have used FRET to assess temporal and spatial characteristics for signaling molecules, including tyrosine kinases Src and FAK, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. Activations of Src and Rac by platelet derived growth factor (PDGF) led to distinct subcellular patterns during cell migration on micropatterned surface, and these two enzymes interact with each other to form a feedback loop with differential regulations at different subcellular locations. We have developed FRET biosensors to monitor FAK activities at rafts vs. non-raft regions of plasma membrane in live cells. In response to cell adhesion on matrix proteins or stimulation by PDGF, the raft-targeting FAK biosensor showed a stronger FRET response than that at non-rafts. The FAK activation at rafts induced by PDGF is mediated by Src. In contrast, the FAK activation at rafts induced by adhesion is independent of Src activity, but rather is essential for Src activation. Thus, Src is upstream to FAK in response to chemical stimulation (PDGF), but FAK is upstream to Src in response to mechanical stimulation (adhesion). A novel biosensor has been developed to dynamically visualize the activity of membrane type-1-matrix metalloproteinase (MT1-MMP), which proteolytically remodels the extracellular matrix. Epidermal growth factor (EGF) directed active MT1-MMP to the leading edge of migrating live cancer cells with local accumulation of EGF receptor via a process dependent on an intact cytoskeletal network. In summary, FRET-based biosensors enable the elucidation of molecular processes and hierarchies underlying spatiotemporal regulation of biological and pathological processes, thus advancing our knowledge on how cells perceive mechanical/chemical cues in space and time to coordinate

  15. Single-Molecule Pull-down FRET (SiMPull-FRET) to dissect the mechanisms of biomolecular machines

    PubMed Central

    Kahlscheuer, Matthew L.; Widom, Julia; Walter, Nils G.

    2016-01-01

    Spliceosomes are multi-megadalton RNA-protein complexes responsible for the faithful removal of non-coding segments (introns) from pre-messenger RNAs (pre-mRNAs), a process critical for the maturation of eukaryotic mRNAs for subsequent translation by the ribosome. Both the spliceosome and ribosome, as well as many other RNA and DNA processing machineries, contain central RNA components that endow biomolecular complexes with precise, sequence-specific nucleic acid recognition and versatile structural dynamics. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) microscopy is a powerful tool for the study of local and global conformational changes of both simple and complex biomolecular systems involving RNA. The integration of biochemical tools such as immunoprecipitation with advanced methods in smFRET microscopy and data analysis has opened up entirely new avenues towards studying the mechanisms of biomolecular machines isolated directly from complex biological specimens such as cell extracts. Here we detail the general steps for using prism-based total internal reflection fluorescence (TIRF) microscopy in exemplary single molecule pull-down FRET (SiMPull-FRET) studies of the yeast spliceosome and discuss the broad application potential of this technique. PMID:26068753

  16. Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals

    PubMed Central

    Furukawa, Junji; Inoue, Katsuhisa; Maeda, Junya; Yasujima, Tomoya; Ohta, Kinya; Kanai, Yoshikatsu; Takada, Tappei; Matsuo, Hirotaka; Yuasa, Hiroaki

    2015-01-01

    The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, those which are specifically responsible for utilization of extracellular nucleobases remain unknown. Here we present the molecular and functional characterization of SLC43A3, an orphan transporter belonging to an amino acid transporter family, as a purine-selective nucleobase transporter. SLC43A3 was highly expressed in the liver, where it was localized to the sinusoidal membrane of hepatocytes, and the lung. In addition, SLC43A3 expressed in MDCKII cells mediated the uptake of purine nucleobases such as adenine, guanine, and hypoxanthine without requiring typical driving ions such as Na+ and H+, but it did not mediate the uptake of nucleosides. When SLC43A3 was expressed in APRT/HPRT1-deficient A9 cells, adenine uptake was found to be low. However, it was markedly enhanced by the introduction of SLC43A3 with APRT. In HeLa cells, knock-down of SLC43A3 markedly decreased adenine uptake. These data suggest that SLC43A3 is a facilitative and purine-selective nucleobase transporter that mediates the cellular uptake of extracellular purine nucleobases in cooperation with salvage enzymes. PMID:26455426

  17. Exciton energy transfer-based quantum dot fluorescence sensing array: "chemical noses" for discrimination of different nucleobases.

    PubMed

    Liu, Jianbo; Li, Gui; Yang, Xiaohai; Wang, Kemin; Li, Li; Liu, Wei; Shi, Xing; Guo, Yali

    2015-01-20

    A novel exciton energy transfer-based fluorescence sensing array for the discrimination of different nucleobases was developed through target nucleobase-triggered self-assembly of quantum dots (QDs). Four QD nanoprobes with different ligand receptors, including mercaptoethylamine, N-acetyl-l-cysteine, 2-dimethyl-aminethanethiol, and thioglycolic acid, were created to detect and identify nucleobase targets. These QDs served as both selective recognition scaffolds and signal transduction elements for a biomolecule target. The extent of particle assembly, induced by the analyte-triggered self-assembly of QDs, led to an exciton energy transfer effect between interparticles that gave a readily detectable fluorescence quenching and distinct fluorescence response patterns. These patterns are characteristic for each nucleobase and can be quantitatively differentiated by linear discriminate analysis. Furthermore, a fingerprint-based barcode was established to conveniently discriminate the nucleobases. This pattern sensing was successfully used to identify nucleobase samples at unknown concentrations and five rare bases. In this "chemical noses" strategy, the robust characteristics of QD nanoprobes, coupled with the diversity of surface functionality that can be readily obtained using nanoparticles, provides a simple and label-free biosensing approach that shows great promise for biomedical applications. PMID:25495103

  18. rFRET: A comprehensive, Matlab-based program for analyzing intensity-based ratiometric microscopic FRET experiments.

    PubMed

    Nagy, Peter; Szabó, Ágnes; Váradi, Tímea; Kovács, Tamás; Batta, Gyula; Szöllősi, János

    2016-04-01

    Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry.

  19. Binding of nucleobases with graphene and carbon nanotube: a review of computational studies.

    PubMed

    Chehel Amirani, Morteza; Tang, Tian

    2015-01-01

    Functionalized carbon nanotubes (CNTs) constitute a new class of nanostructured materials that have vast applications in CNT purification and separation, biosensing, drug delivery, etc. Hybrids formed from the functionalization of CNT with biological molecules have shown interesting properties and have attracted great attention in recent years. Of particular interest is the hybridization of single- or double-stranded nucleic acid (NA) with CNT. Nucleobases, as the building blocks of NA, interact with CNT and contribute strongly to the stability of the NA-CNT hybrids and their properties. In this work, we present a thorough review of previous studies on the binding of nucleobases with graphene and CNT, with a focus on the simulation works that attempted to evaluate the structure and strength of binding. Discrepancies among these works are identified, and factors that might contribute to such discrepancies are discussed.

  20. One-pot microbial synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase.

    PubMed

    Horinouchi, Nobuyuki; Ogawa, Jun; Kawano, Takako; Sakai, Takafumi; Saito, Kyota; Matsumoto, Seiichiro; Sasaki, Mie; Mikami, Yoichi; Shimizu, Sakayu

    2006-06-01

    A one-pot enzymatic synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker's yeast (Saccharomyces cerevisiae) generated ATP which was used to produce D: -glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The D: -glyceraldehyde 3-phosphate produced was transformed to 2'-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2'-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2'-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.

  1. Detecting Organic Compounds in Martian Soil Analogues Using Gas Chromatography Mass Spectrometry

    NASA Technical Reports Server (NTRS)

    Glavin, D. P.; Buch, A.; Mahaffy, P. R.

    2004-01-01

    One of the primary objectives of the 1976 Viking missions was to determine whether organic compounds, possibly of biological origin, were present in the Martian surface soils. The Viking gas chromatography mass spectrometry (GCMS) instruments found no evidence for any organic compounds of Martian origin above a few parts per billion in the upper 10 cm of surface soil [l], suggesting the absence of a widely distributed Martian biota. However, Benner et d. have suggested that significant amounts of non-volatile organic compounds, possibly including oxidation products of bioorganic molecules (e.g. carboxylic acids) would not have been detected by the Viking GCMS [2]. Moreover, other key organic compounds important to biology, such as amino acids and nucleobases, would also likely have been missed by the Viking GCMS as these compounds require chemical derivatization to be stable in a GC column [3]. Recent pyrolysis experiments with a Mars soil analogue that had been innoculated with Escherichia coli bacteria have shown that amino acid decomposition products (amines) and nucleobases are among the most abundant products generated after pyrolysis of the bacterial cells [4,5]. At the part per billion level (Viking GCMS detection limit), these pyrolysis products generated from several million bacterial cells per gram of Martian soil would not have been detected by the Viking GCMS instruments [4]. Analytical protocols are under development for upcoming in situ lander opportunities to target several important biological compounds including amino acids and nucleobases. For example, extraction and chemical derivatization techniques [3] are being adapted for space flight use to transform reactive or fragile molecules that would not have been detected by the Viking GCMS instruments, into species that are sufficiently volatile to be detected by GCMS. Recent experiments carried out at NASA Goddard have shown that using this derivatization technique all of the targeted compounds

  2. Crystal Structures of Non-Natural Nucleobase Pairs in A- and B-DNA†

    PubMed Central

    Georgiadis, Millie M.; Singh, Isha; Kellett, Whitney F.; Hoshika, Shuichi; Benner, Steven A.; Richards, Nigel G. J.

    2015-01-01

    The extent to which synthetic biology can be used to expand genetic information systems compatible with natural enzymes and cells will depend on the extent to which multiple and contiguous non-natural nucleobase pairs fit within the standard double helical conformations of DNA. Toward this goal, two non-standard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) were designed to form a Z:P pair with a standard “edge on” Watson-Crick geometry, but with rearranged hydrogen bond donor and acceptor groups. Here, we present the crystal structures of two self-complementary 16-mer oligonucleotides containing Z:P pairs. The first contained two consecutive Z:P nucleobase pairs and was found to crystallize within a host-guest complex in B-form. The second contained six consecutive Z:P pairs; it was found to crystallize as an A-form DNA duplex, although it can adopt B-form in solution as inferred from circular dichroism spectra. Although Z:P pairs have some structural properties that are similar to those of G:C pairs, unique features include stacking of the nitro group on Z with the adjacent heterocyclic nucleobase ring in A-DNA. In both B-and A-DNA, major groove widths associated with the Z:P pairs are approximately 1 Å wider than those of comparable G:C pairs potentially due to the presence of the nitro group in Z. Thus, our structural studies suggest that multiple and consecutive Z:P pairs are readily accommodated in DNA duplex structures recognized by natural polymerases, and therefore the GACTZP synthetic genetic system has the requisite properties to expand sequence space. PMID:25961938

  3. The photochemistry of pyrimidine in realistic astrophysical ices and the production of nucleobases

    SciTech Connect

    Nuevo, Michel; Materese, Christopher K.; Sandford, Scott A.

    2014-10-01

    Nucleobases, together with deoxyribose/ribose and phosphoric acid, are the building blocks of DNA and RNA for all known life. The presence of nucleobase-like compounds in carbonaceous chondrites delivered to the Earth raises the question of an extraterrestrial origin for the molecules that triggered life on our planet. Whether these molecules are formed in interstellar/protostellar environments, in small parent bodies in the solar system, or both, is currently unclear. Recent experiments show that the UV irradiation of pyrimidine (C{sub 4}H{sub 4}N{sub 2}) in H{sub 2}O-rich ice mixtures that contain NH{sub 3}, CH{sub 3}OH, or CH{sub 4} leads to the formation of the pyrimidine-based nucleobases uracil, cytosine, and thymine. In this work, we discuss the low-temperature UV irradiation of pyrimidine in realistic astrophysical ice mixtures containing H{sub 2}O, CH{sub 3}OH, and NH{sub 3}, with or without CH{sub 4}, to search for the production of nucleobases and other prebiotic compounds. These experiments show the presence of uracil, urea, glycerol, hexamethylenetetramine, small amino acids, and small carboxylic acids in all samples. Cytosine was only found in one sample produced from ices irradiated with a higher UV dose, while thymine was not found in any sample, even after irradiation with a higher UV dose. Results are discussed to evaluate the role of the photochemistry of pyrimidine in the inventory of organic molecules detected in meteorites and their astrophysical/astrobiological implications.

  4. Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging

    PubMed Central

    Afsari, Hamid Samareh; Cardoso Dos Santos, Marcelina; Lindén, Stina; Chen, Ting; Qiu, Xue; van Bergen en Henegouwen, Paul M. P.; Jennings, Travis L.; Susumu, Kimihiro; Medintz, Igor L.; Hildebrandt, Niko; Miller, Lawrence W.

    2016-01-01

    Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide–mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions. PMID:27386579

  5. Nucleobase-templated polymerization: copying the chain length and polydispersity of living polymers into conjugated polymers.

    PubMed

    Lo, Pik Kwan; Sleiman, Hanadi F

    2009-04-01

    Conjugated polymers synthesized by step polymerization mechanisms typically suffer from poor molecular weight control and broad molecular weight distributions. We report a new method which uses nucleobase recognition to read out and efficiently copy the controlled chain length and narrow molecular weight distribution of a polymer template generated by living polymerization, into a daughter conjugated polymer. Aligning nucleobase-containing monomers on their complementary parent template using hydrogen-bonding interactions, and subsequently carrying out a Sonogashira polymerization, leads to the templated synthesis of a conjugated polymer. Remarkably, this daughter strand is found to possess a narrow molecular weight distribution and a chain length nearly equivalent to that of the parent template. On the other hand, nontemplated polymerization or polymerization with the incorrect template generates a short conjugated oligomer with a significantly broader molecular weight distribution. Hence, nucleobase-templated polymerization is a useful tool in polymer synthesis, in this case allowing the use of a large number of polymers generated by living methods, such as anionic polymerization, controlled radical polymerizations (NMP, ATRP, and RAFT) and other mechanisms to program the structure, length, and molecular weight distribution of polymers normally generated by step polymerization methods and significantly enhance their properties.

  6. The origin of efficient triplet state population in sulfur-substituted nucleobases

    PubMed Central

    Mai, Sebastian; Pollum, Marvin; Martínez-Fernández, Lara; Dunn, Nicholas; Marquetand, Philipp; Corral, Inés; Crespo-Hernández, Carlos E.; González, Leticia

    2016-01-01

    Elucidating the photophysical mechanisms in sulfur-substituted nucleobases (thiobases) is essential for designing prospective drugs for photo- and chemotherapeutic applications. Although it has long been established that the phototherapeutic activity of thiobases is intimately linked to efficient intersystem crossing into reactive triplet states, the molecular factors underlying this efficiency are poorly understood. Herein we combine femtosecond transient absorption experiments with quantum chemistry and nonadiabatic dynamics simulations to investigate 2-thiocytosine as a necessary step to unravel the electronic and structural elements that lead to ultrafast and near-unity triplet-state population in thiobases in general. We show that different parts of the potential energy surfaces are stabilized to different extents via thionation, quenching the intrinsic photostability of canonical DNA and RNA nucleobases. These findings satisfactorily explain why thiobases exhibit the fastest intersystem crossing lifetimes measured to date among bio-organic molecules and have near-unity triplet yields, whereas the triplet yields of canonical nucleobases are nearly zero. PMID:27703148

  7. Nucleobase and amino acid formation through impacts of meteorites on the early ocean

    NASA Astrophysics Data System (ADS)

    Furukawa, Yoshihiro; Nakazawa, Hiromoto; Sekine, Toshimori; Kobayashi, Takamichi; Kakegawa, Takeshi

    2015-11-01

    The emergence of life's building blocks on the prebiotic Earth was the first crucial step for the origins of life. Extraterrestrial delivery of intact amino acids and nucleobases is the prevailing hypothesis for their availability on prebiotic Earth because of the difficulties associated with the production of these organics from terrestrial carbon and nitrogen sources under plausible prebiotic conditions. However, the variety and amounts of these intact organics delivered by meteorites would have been limited. Previous shock-recovery experiments have demonstrated that meteorite impact reactions could have generated organics on the prebiotic Earth. Here, we report on the simultaneous formation of nucleobases (cytosine and uracil) found in DNA and/or RNA, various proteinogenic amino acids (glycine, alanine, serine, aspartic acid, glutamic acid, valine, leucine, isoleucine, and proline), non-proteinogenic amino acids, and aliphatic amines in experiments simulating reactions induced by extraterrestrial objects impacting on the early oceans. To the best of our knowledge, this is the first report of the formation of nucleobases from inorganic materials by shock conditions. In these experiments, bicarbonate was used as the carbon source. Bicarbonate, which is a common dissolved carbon species in CO2-rich atmospheric conditions, was presumably the most abundant carbon species in the early oceans and in post-impact plumes. Thus, the present results expand the possibility that impact-induced reactions generated various building blocks for life on prebiotic Earth in large quantities through the use of terrestrial carbon reservoirs.

  8. Understanding the interaction of DNA-RNA nucleobases with different ZnO nanomaterials.

    PubMed

    Saha, Supriya; Sarkar, Pranab

    2014-08-01

    Due to the potential application of different nanostructure materials in biomedical nanotechnologies, understanding the interaction between the inorganic nanoparticles and biological molecules at the atomic level is of paramount importance. We present here the results of our theoretical investigation of the interaction of different nucleotide bases--adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)--with different ZnO nanoparticles, such as ZnO nanowires (NWs), nanotubes (NTs), surfaces and quantum dots (QDs). As the size of the systems we studied is relatively large, we have used the self-consistent-charge density-functional tight-binding (SCC-DFTB) method to optimize the complex systems. We have studied in detail the site-specific binding nature and the adsorption strength of these nucleobases with different ZnO nanoparticles. The calculated binding energy order and the interaction strength of nucleobases are very much dependent on the nature of the nanoparticle surfaces and are different for different nanostructures. In most of the cases ZnO prefers to bind either through the top site of the nucleobases or with the ring nitrogen atom having a lone pair relative to other binding sites of the bases. PMID:24942064

  9. Determination of HDV ribozyme N(-1) nucleobase and functional group specificity using internal competition kinetics

    PubMed Central

    Kellerman, Daniel L; Simmons, Kandice S; Pedraza, Mayra; Piccirilli, Joseph A; York, Darrin M; Harris, Michael E

    2015-01-01

    Biological catalysis involves interactions distant from the site of chemistry that can position the substrate for reaction. Catalysis of RNA 2′-O-transphosphorylation by the HDV ribozyme is sensitive to the identity of the N(-1) nucleotide flanking the reactive phosphoryl group. However, the interactions that affect the conformation of this position, and in turn the 2′O nucleophile, are unclear. Here, we describe the application of multiple substrate internal competition kinetic analyses to understand how the N(-1) nucleobase contributes to HDV catalysis, and to test the utility of this approach for RNA structure-function studies. Internal competition reactions containing all four substrate sequence variants at the N(-1) position in reactions using ribozyme active site mutations at A77 and A78 were used to test a proposed basepairing interaction. Mutants A78U, A78G and A79G retain significant catalytic activity, but do not alter the specificity for the N(-1) nucleobase. Effects of nucleobase analog substitutions at N(-1) indicate that U is preferred due to the ability to donate an H-bond in the Watson-Crick face and avoid minor groove steric clash. The results provide information essential for evaluating models of the HDV active site, and illustrate multiple-substrate kinetic analyses as a practical approach for characterizing structure-function relationships in RNA reactions. PMID:25937290

  10. Studies on effective atomic numbers and electron densities of nucleobases in DNA

    NASA Astrophysics Data System (ADS)

    Kumar, Ashok

    2016-10-01

    Various parameters of dosimetric importance such as effective atomic numbers (Zeff) and electron densities (Nel) of nucleobases in DNA have been calculated for the total and partial photon interaction processes in the wide energy range of 1 keV-100 GeV. The variations of Zeff and Nel with energy are shown graphically for all partial and total interaction processes and are found to be similar. Up to 10 keV, Zeff and Nel show a sharp increase for cytosine-guanine and thymine-adenine whereas for all the other nucleobases, it is almost constant. Then there is sharp decrease in Zeff and Nel with energy up to 100 keV for all the nucleobases. From 100 keV to 6 MeV, Zeff and Nel are almost independent of energy. From 6 MeV to 100 MeV, there is regular increase in Zeff and Nel with photon energy. Above 400 MeV, Zeff and Nel remain almost constant. The obtained results are due to the dominance of photoelectric absorption, Compton scattering and pair production in different energy regions as respectively stated above and their dependences on the chemical compositions of the interacting media.

  11. Formation of Nucleobases from the UV Irradiation of Pyrimidine in Astrophysical Ice Analogs

    NASA Technical Reports Server (NTRS)

    Sandford, Scott A.; Nuevo, Michel; Materese, Christopher K.

    2014-01-01

    Nucleobases are the informational subunits of DNA and RNA. They consist of Nheterocycles that belong to either the pyrimidine-base group (uracil, cytosine, and thymine) or the purinebase group (adenine and guanine). Several nucleobases, mostly purine bases, have been detected in meteorites [1-3], with isotopic signatures consistent with an extraterrestrial origin [4]. Uracil is the only pyrimidine-base compound formally reported in meteorites [2], though the presence of cytosine cannot be ruled out [5,6]. However, the actual process by which the uracil was made and the reasons for the non-detection of thymine in meteorites have yet to be fully explained. Although no N-heterocycles have ever been observed in the ISM [7,8], the positions of the 6.2-µm interstellar emission features suggest a population of such molecules is likely to be present [9]. In this work we study the formation of pyrimidine-based molecules, including the three nucleobases uracil, cytosine, and thymine from the ultraviolet (UV) irradiation of pyrimidine in ices consisting of several combinations of H(sub2)O, NH(sub3), CH(sub3)OH, and CH(sub4) at low temperature, in order to simulate the astrophysical conditions under which prebiotic species may be formed in the interstellar medium, in the protosolar nebula, and on icy bodies of the Solar System.

  12. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes

    PubMed Central

    Mishra, Sourav; Lahiri, Hiya; Banerjee, Siddhartha; Mukhopadhyay, Rupa

    2016-01-01

    So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored. PMID:27025649

  13. Biodegradable analogues of DDT*

    PubMed Central

    Metcalf, Robert L.; Kapoor, Inder P.; Hirwe, Asha S.

    1971-01-01

    Despite the immense utility of DDT for vector control its usefulness is prejudiced by its stability in the environment and by the low rate at which it can be degraded biologically. Metabolic studies in insects, in mice, and in a model ecosystem with several food chains have shown that DDT analogues with substituent groups readily attacked by multifunction oxidases undergo a substantial degree of biological degradation and do not appear to be stored readily in animal tissues or concentrated in food chains. Detailed metabolic pathways have been worked out and it is clear that comparative biochemistry can be used to develop DDT analogues that are adequately persistent yet biodegradable. A number of new DDT analogues have been evaluated for insecticidal activity against flies and mosquitos and for their potential usefulness as safe, persistent, and biodegradable insecticides. PMID:5315354

  14. Cyclic wear behavior (fretting) of a tempered martensite steel

    NASA Astrophysics Data System (ADS)

    Gaul, D. J.; Duquette, D. J.

    1980-09-01

    Cyclic wear experiments were conducted on a 4130 steel with a tempered martensitic structure in laboratory air and in flowing argon. Temperatures of the sliding interfaces were measured and were of the order of + 10 K above the ambient. The interface temperatures were observed to be cyclic and correlated with maxima in sliding velocities between the surfaces. Microhardness measurements and optical and electron metallographic observations of fretted surfaces indicated selective surface hardening and the formation of large numbers of thin metallic flakes. The thickness of the flakes corresponded to the thickness of the hardened layer and were formed from subsurface crack initiation processes. The results of this investigation do not support many previously proposed models for fretting damage. Rather a micromechanical cracking model is proposed which appears to explain observed results in terms of a delamination model.

  15. Nucleobases and other Prebiotic Species from the Ultraviolet Irradiation of Pyrimidine in Astrophysical Ices

    NASA Technical Reports Server (NTRS)

    Sandford, S. A.; Nuevo, M.; Materese, C. K.; Milam, S. N.

    2012-01-01

    Nucleobases are N-heterocycles that are the informational subunits of DNA and RNA, and are divided into two families: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites and their extraterrestrial origin confirmed by isotope measurement. Although no Nheterocycles have ever been observed in the ISM, the positions of the 6.2-m interstellar emission features suggest a population of such molecules is likely to be present. In this work we study the formation of pyrimidine-based molecules, including nucleobases, as well as other species of prebiotic interest, from the ultraviolet (UV) irradiation of pyrimidine in combinations of H2O, NH3, CH3OH, and CH4 ices at low temperature, in order to simulate the astrophysical conditions under which prebiotic species may be formed in the interstellar medium and icy bodies of the Solar System. Experimental: Gas mixtures are prepared in a glass mixing line (background pressure approx. 10(exp -6)-10(exp -5) mbar). Relative proportions between mixture components are determined by their partial pressures. Gas mixtures are then deposited on an aluminum foil attached to a cold finger (15-20 K) and simultaneously irradiated with an H2 lamp emitting UV photons (Lyman and a continuum at approx.160 nm). After irradiation samples are warmed to room temperature, at which time the remaining residues are recovered to be analyzed with liquid and gas chromatographies. Results: These experiments showed that the UV irradiation of pyrimidine mixed in these ices at low temperature leads to the formation of several photoproducts derived from pyrimidine, including the nucleobases uracil and cytosine, as well as their precursors 4(3H)-pyrimidone and 4-aminopyrimidine (Fig. 1). Theoretical quantum calculations on the formation of 4(3H)-pyrimidone and uracil from the irradiation of pyrimidine in pure H2O ices are in agreement with their experimental formation pathways. In

  16. Dual Readout BRET/FRET Sensors for Measuring Intracellular Zinc

    PubMed Central

    2016-01-01

    Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn2+. However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative. In this work, we used the bright and stable luciferase NanoLuc to create the first genetically encoded BRET sensors for measuring intracellular Zn2+. Using a new sensor approach, the NanoLuc domain was fused to the Cerulean donor domain of two previously developed FRET sensors, eCALWY and eZinCh-2. In addition to preserving the excellent Zn2+ affinity and specificity of their predecessors, these newly developed sensors enable both BRET- and FRET-based detection. While the dynamic range of the BRET signal for the eCALWY-based BLCALWY-1 sensor was limited by the presence of two competing BRET pathways, BRET/FRET sensors based on the eZinCh-2 scaffold (BLZinCh-1 and -2) yielded robust 25–30% changes in BRET ratio. In addition, introduction of a chromophore-silencing mutation resulted in a BRET-only sensor (BLZinCh-3) with increased BRET response (50%) and an unexpected 10-fold increase in Zn2+ affinity. The combination of robust ratiometric response, physiologically relevant Zn2+ affinities, and stable and bright luminescence signal offered by the BLZinCh sensors allowed monitoring of intracellular Zn2+ in plate-based assays as well as intracellular BRET-based imaging in single living cells in real time. PMID:27547982

  17. Multiphoton FLIM: a reliable FRET detection tool in cell biological applications

    NASA Astrophysics Data System (ADS)

    Krishnan, Ramanujan V.; Biener, Eva; Centonze, Victoria E.; Gertler, Arieh; Herman, Brian A.

    2004-06-01

    Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation is emerging as a reliable quantitative tool for measuring fluorescence resonance energy transfer (FRET) in living cells. By virtue of being free from spectroscopic artifacts encountered in conventional FRET detection methods, multiphoton FLIM methods offer the advantages of high spatial and temporal resolution, faster data acquisition and data analysis. We compare the FRET results obtained by two different methods namely (i) multiphoton excitation lifetime-based FRET and (ii) single photon excitation intensity-based acceptor photobleaching FRET. Using the same biological samples, we apply these two different methods in understanding the growth hormone receptor dimerization kinetics at the cell surface of human embryonic kidney cells. We conclude that the multiphoton FLIM using the streak-camera approach provides the best ability to monitor FRET in dynamic situations where high temporal and spatial resolution are required with minimal photodamage/phototoxicity.

  18. Fretting wear of iron, nickel, and titanium under varied environmental conditions

    NASA Technical Reports Server (NTRS)

    Bill, R. C.

    1978-01-01

    Fretting wear experiments were conducted on high purity iron, nickel and titanium in air under conditions of varied humidity and temperature, and in nitrogen. For iron and titanium, maximum fretting occurred at 10 and 30 percent relative humidity respectively. Nickel showed a minimum in fretting wear at about 10 percent relative humidity. With increasing temperature, all three metals initially showed reduced fretting wear, with increasing wear observed as temperatures increased beyond 200-300 C. For titanium, dramatically reduced fretting wear was observed at temperatures above 500 C, relatable to a change in oxidation kinetics. All three metals showed much less fretting wear in N2 with the presence of moisture in N2 having a proportionally stronger effect than in air.

  19. Fretting Wear of Ti-48Al-2Cr-2Nb

    NASA Technical Reports Server (NTRS)

    Miyoshi, Kazuhisa; Lerch, Bradley A.; Draper, Susan L.

    2001-01-01

    An investigation was conducted to examine the wear behavior of gamma titanium aluminide (Ti-48Al-2Cr-2Nb in atomic percent) in contact with a typical nickel-base superalloy under repeated microscopic vibratory motion in air at temperatures from 296-823 K. The surface damage observed on the interacting surfaces of both Ti-48Al-2Cr-2Nb and superalloy consisted of fracture pits, oxides, metallic debris, scratches, craters, plastic deformation, and cracks. The Ti-48Al-2Cr-2Nb transferred to the superalloy at all fretting conditions and caused scuffing or galling. The increasing rate of oxidation at elevated temperatures led to a drop in Ti-48Al-2Cr-2Nb wear at 473 K. Mild oxidative wear was observed at 473 K. However, fretting wear increased as the temperature was increased from 473-823 K. At 723 and 823 K, oxide disruption generated cracks, loose wear debris, and pits on the Ti-48Al-2Cr-2Nb wear surface. Ti-48Al-2Cr-2Nb wear generally decreased with increasing fretting frequency. Both increasing slip amplitude and increasing load tended to produce more metallic wear debris, causing severe abrasive wear in the contacting metals. Keywords

  20. Quantitative structural information from single-molecule FRET.

    PubMed

    Beckers, M; Drechsler, F; Eilert, T; Nagy, J; Michaelis, J

    2015-01-01

    Single-molecule studies can be used to study biological processes directly and in real-time. In particular, the fluorescence energy transfer between reporter dye molecules attached to specific sites on macromolecular complexes can be used to infer distance information. When several measurements are combined, the information can be used to determine the position and conformation of certain domains with respect to the complex. However, data analysis schemes that include all experimental uncertainties are highly complex, and the outcome depends on assumptions about the state of the dye molecules. Here, we present a new analysis algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering termed Fast-NPS that can analyse large smFRET networks in a relatively short time and yields the position of the dye molecules together with their respective uncertainties. Moreover, we show what effects different assumptions about the dye molecules have on the outcome. We discuss the possibilities and pitfalls in structure determination based on smFRET using experimental data for an archaeal transcription pre-initiation complex, whose architecture has recently been unravelled by smFRET measurements. PMID:26407323

  1. General FRET-based coding for application in multiplexing methods.

    PubMed

    Giestas, Letícia; Petrov, Vesselin; Baptista, Pedro V; Lima, João Carlos

    2009-08-01

    FRET can be used as a strategy to assign different simultaneous events in the same sample but "cross-talk" problems are a limitation. Here we present a contribution for the better understanding of the "cross-talk" in FRET experiments that include several pairs in the same sample. Using oligonucleotide probes labeled with fluorescent dyes which can be selectively excited at a specific wavelength, and using target oligonucleotides tagged with a fluorescent dye with specific characteristics that allow only it to emit light upon selective excitation of a specific probe by energy transfer (FRET), we aim to identify the exact probe-target hybridized pair. When using three donors to probe the presence of complementary targets, only 20% of possible donor/acceptor combinations give straightforward signals readily identifiable with the sample composition, while in the remaining cases severe cross-excitation prevents the direct identification of the sample composition. To correctly resolve the samples identity, we developed a theoretical model that enables the unequivocal attribution of a sample composition to a given set of fluorescence signals, in the presence of three donors.

  2. Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions

    PubMed Central

    Sung, Uhna; Sepehri-Rad, Masoud; Piao, Hong Hua; Jin, Lei; Hughes, Thomas; Cohen, Lawrence B.; Baker, Bradley J.

    2015-01-01

    FRET (Förster Resonance Energy Transfer)-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP) voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different “Nabi1” constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms) and signal decay (~3 ms). We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP) and mRuby2 (acceptor FP) to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz. PMID:26587834

  3. Conformational Dynamics of Titin PEVK Explored with FRET Spectroscopy

    PubMed Central

    Huber, Tamás; Grama, László; Hetényi, Csaba; Schay, Gusztáv; Fülöp, Lívia; Penke, Botond; Kellermayer, Miklós S.Z.

    2012-01-01

    The proline-, glutamate-, valine-, and lysine-rich (PEVK) domain of the giant muscle protein titin is thought to be an intrinsically unstructured random-coil segment. Various observations suggest, however, that the domain may not be completely devoid of internal interactions and structural features. To test the validity of random polymer models for PEVK, we determined the mean end-to-end distances of an 11- and a 21-residue synthetic PEVK peptide, calculated from the efficiency of the fluorescence resonance energy transfer (FRET) between an N-terminal intrinsic tryptophan donor and a synthetically added C-terminal IAEDANS acceptor obtained in steady-state and time-resolved experiments. We find that the contour-length scaling of mean end-to-end distance deviates from predictions of a purely statistical polymer chain. Furthermore, the addition of guanidine hydrochloride decreased, whereas the addition of salt increased the FRET efficiency, pointing at the disruption of structure-stabilizing interactions. Increasing temperature between 10 and 50°C increased the normalized FRET efficiency in both peptides but with different trajectories, indicating that their elasticity and conformational stability are different. Simulations suggest that whereas the short PEVK peptide displays an overall random structure, the long PEVK peptide retains residual, loose helical configurations. Transitions in the local structure and dynamics of the PEVK domain may play a role in the modulation of passive muscle mechanics. PMID:23062340

  4. An improved cyan fluorescent protein variant useful for FRET.

    PubMed

    Rizzo, Mark A; Springer, Gerald H; Granada, Butch; Piston, David W

    2004-04-01

    Many genetically encoded biosensors use Förster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation. PMID:14990965

  5. Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET

    PubMed Central

    Subach, Fedor V.; Zhang, Lijuan; Gadella, Theodorus W.J.; Gurskaya, Nadya G.; Lukyanov, Konstantin A.; Verkhusha, Vladislav V.

    2010-01-01

    SUMMARY We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or non-fluorescent state (OFF state), respectively. The ON and OFF states exhibit absorbance maxima at 567 and 440 nm, respectively. Due to the photoswitchable absorbance, rsTagRFP can be used as an acceptor for a photochromic Förster resonance energy transfer (pcFRET). The photochromic acceptor facilitates determination of a protein-protein interaction by providing an internal control for FRET. Using pcFRET with EYFP as a donor we observed an interaction between epidermal growth factor receptor and growth factor receptor-binding protein 2 in live cells by detecting the modulation of both the fluorescence intensity and lifetime of the EYFP donor upon the ON-OFF photoswitching of the rsTagRFP acceptor. PMID:20659687

  6. Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

    PubMed

    Sung, Uhna; Sepehri-Rad, Masoud; Piao, Hong Hua; Jin, Lei; Hughes, Thomas; Cohen, Lawrence B; Baker, Bradley J

    2015-01-01

    FRET (Förster Resonance Energy Transfer)-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP) voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different "Nabi1" constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms) and signal decay (~3 ms). We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP) and mRuby2 (acceptor FP) to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz. PMID:26587834

  7. Conformational dynamics of titin PEVK explored with FRET spectroscopy.

    PubMed

    Huber, Tamás; Grama, László; Hetényi, Csaba; Schay, Gusztáv; Fülöp, Lívia; Penke, Botond; Kellermayer, Miklós S Z

    2012-10-01

    The proline-, glutamate-, valine-, and lysine-rich (PEVK) domain of the giant muscle protein titin is thought to be an intrinsically unstructured random-coil segment. Various observations suggest, however, that the domain may not be completely devoid of internal interactions and structural features. To test the validity of random polymer models for PEVK, we determined the mean end-to-end distances of an 11- and a 21-residue synthetic PEVK peptide, calculated from the efficiency of the fluorescence resonance energy transfer (FRET) between an N-terminal intrinsic tryptophan donor and a synthetically added C-terminal IAEDANS acceptor obtained in steady-state and time-resolved experiments. We find that the contour-length scaling of mean end-to-end distance deviates from predictions of a purely statistical polymer chain. Furthermore, the addition of guanidine hydrochloride decreased, whereas the addition of salt increased the FRET efficiency, pointing at the disruption of structure-stabilizing interactions. Increasing temperature between 10 and 50°C increased the normalized FRET efficiency in both peptides but with different trajectories, indicating that their elasticity and conformational stability are different. Simulations suggest that whereas the short PEVK peptide displays an overall random structure, the long PEVK peptide retains residual, loose helical configurations. Transitions in the local structure and dynamics of the PEVK domain may play a role in the modulation of passive muscle mechanics.

  8. Endo-S-c-di-GMP analogues-polymorphism and binding studies with class I riboswitch.

    PubMed

    Zhou, Jie; Sayre, David A; Wang, Jingxin; Pahadi, Nirmal; Sintim, Herman O

    2012-01-01

    C-di-GMP, a cyclic guanine dinucleotide, has been shown to regulate biofilm formation as well as virulence gene expression in a variety of bacteria. Analogues of c-di-GMP have the potential to be used as chemical probes to study c-di-GMP signaling and could even become drug leads for the development of anti-biofilm compounds. Herein we report the synthesis and biophysical studies of a series of c-di-GMP analogues, which have both phosphate and sugar moieties simultaneously modified (called endo-S-c-di-GMP analogues). We used computational methods to predict the relative orientation of the guanine nucleobases in c-di-GMP and analogues. DOSY NMR of the endo-S-c-di-GMP series showed that the polymorphism of c-di-GMP can be tuned with conservative modifications to the phosphate and sugar moieties (conformational steering). Binding studies with Vc2 RNA (a class I c-di-GMP riboswitch) revealed that conservative modifications to the phosphate and 2'-positions of c-di-GMP dramatically affected binding to class I riboswitch. PMID:23143150

  9. Considerations for sensitivity analysis, uncertainty quantification, and data assimilation for grid-to-rod fretting

    SciTech Connect

    Michael Pernice

    2012-10-01

    Grid-to-rod fretting is the leading cause of fuel failures in pressurized water reactors, and is one of the challenge problems being addressed by the Consortium for Advanced Simulation of Light Water Reactors to guide its efforts to develop a virtual reactor environment. Prior and current efforts in modeling and simulation of grid-to-rod fretting are discussed. Sources of uncertainty in grid-to-rod fretting are also described.

  10. Natural Analogue Synthesis Report

    SciTech Connect

    A. M. Simmons

    2002-05-01

    The purpose of this report is to present analogue studies and literature reviews designed to provide qualitative and quantitative information to test and provide added confidence in process models abstracted for performance assessment (PA) and model predictions pertinent to PA. This report provides updates to studies presented in the ''Yucca Mountain Site Description'' (CRWMS M and O 2000 [151945], Section 13) and new examples gleaned from the literature, along with results of quantitative studies conducted specifically for the Yucca Mountain Site Characterization Project (YMP). The intent of the natural analogue studies was to collect corroborative evidence from analogues to demonstrate additional understanding of processes expected to occur during postclosure at a potential Yucca Mountain repository. The report focuses on key processes by providing observations and analyses of natural and anthropogenic (human-induced) systems to improve understanding and confidence in the operation of these processes under conditions similar to those that could occur in a nuclear waste repository. The process models include those that represent both engineered and natural barrier processes. A second purpose of this report is to document the various applications of natural analogues to geologic repository programs, focusing primarily on the way analogues have been used by the YMP. This report is limited to providing support for PA in a confirmatory manner and to providing corroborative inputs for process modeling activities. Section 1.7 discusses additional limitations of this report. Key topics for this report are analogues to emplacement drift degradation, waste form degradation, waste package degradation, degradation of other materials proposed for the engineered barrier, seepage into drifts, radionuclide flow and transport in the unsaturated zone (UZ), analogues to coupled thermal-hydrologic-mechanical-chemical processes, saturated zone (SZ) transport, impact of radionuclide

  11. Fretting Wear Mechanisms in A216 Plain Carbon Steel

    NASA Astrophysics Data System (ADS)

    Maich, Alyssa Anne

    The subsurface and surface microstructures during pin-on-disk fretting wear of A216 steel disks under various loading conditions and times are investigated. The corresponding pins are fabricated from 410 stainless steel to simulate in-service conditions found in such engineering components as the Siemens W501FD engine row-2 diaphragm of a Siemens turbine engine, which is known to be prone to failure by fretting wear. Loading conditions range from 2N to 15N and times from 1 hour to a maximum of 69 hours, when steady state is confirmed. Wear track depth is quantitatively determined by optical profilometry, and found to range from 3 to 11 microns dependent upon load. Wear depth increases from 2N to 10N load, but decreases when increased to 15N load, due to heavier transfer of pin material to disk, as can be seen by EDS images of chromium transfer on A216 disk. Microstructures are evaluated by transmission electron microscopy of samples prepared by focused ion beam machining to pinpoint wear tracks and expose them in cross-section. EDS is used, in conjunction with TEM, to elucidate primary wear mechanisms at each stage of fretting wear. Microstructures in the subsurface of wear tracks are found to be heavily dislocated and layered, features that vary with both applied load and time. The microstructure eventually evolves into stable dislocation cells with cell walls aligned parallel to the surface. Penetration depth of the damaged layers increases with applied load, associated with a non-uniform maximum shear stress distribution that varies with depth. Primary oxide appears to evolve from Fe2O3 to Fe3O4, with increasing fretting time, leading to a uniform oxide on the surface of the A216 disk. Oxidation rate may be increased with the evolution of this subsurface dislocation cell structure. It is concluded that fretting wear failure is likely associated with a synergy between oxidative wear and crack initiation and propagation along dislocation cell walls under high

  12. A communication theoretical analysis of FRET-based mobile ad hoc molecular nanonetworks.

    PubMed

    Kuscu, Murat; Akan, Ozgur B

    2014-09-01

    Nanonetworks refer to a group of nanosized machines with very basic operational capabilities communicating to each other in order to accomplish more complex tasks such as in-body drug delivery, or chemical defense. Realizing reliable and high-rate communication between these nanomachines is a fundamental problem for the practicality of these nanonetworks. Recently, we have proposed a molecular communication method based on Förster Resonance Energy Transfer (FRET) which is a nonradiative excited state energy transfer phenomenon observed among fluorescent molecules, i.e., fluorophores. We have modeled the FRET-based communication channel considering the fluorophores as single-molecular immobile nanomachines, and shown its reliability at high rates, and practicality at the current stage of nanotechnology. In this study, for the first time in the literature, we investigate the network of mobile nanomachines communicating through FRET. We introduce two novel mobile molecular nanonetworks: FRET-based mobile molecular sensor/actor nanonetwork (FRET-MSAN) which is a distributed system of mobile fluorophores acting as sensor or actor node; and FRET-based mobile ad hoc molecular nanonetwork (FRET-MAMNET) which consists of fluorophore-based nanotransmitter, nanoreceivers and nanorelays. We model the single message propagation based on birth-death processes with continuous time Markov chains. We evaluate the performance of FRET-MSAN and FRET-MAMNET in terms of successful transmission probability and mean extinction time of the messages, system throughput, channel capacity and achievable communication rates.

  13. Probing Nucleobase Interactions and Predicting Mechanisms of Synthetic Interest Using Computational Chemistry, and Furthering the Development of BVI Education in Chemistry

    ERIC Educational Resources Information Center

    Harrison, Jason Gordon

    2013-01-01

    Quantum mechanical (QM) and molecular docking methods are used to probe systems of biological and synthetic interest. Probing interactions of nucleobases within proteins, and properly modeling said interactions toward novel nucleobase development, is extremely difficult, and of great utility in RNA interference (RNAi) therapeutics. The issues in…

  14. H-bonding-directed self-assembly of synthetic copolymers containing nucleobases: organization and colloidal fusion in a noncompetitive solvent.

    PubMed

    Lutz, Jean-François; Pfeifer, Sebastian; Chanana, Munish; Thünemann, Andreas F; Bienert, Ralf

    2006-08-15

    The self-organization of random copolymers composed of a nucleobase monomer (either 1-(4-vinylbenzyl)thymine or 9-(4-vinylbenzyl)adenine) and dodecyl methacrylate (DMA) was studied in dilute chloroform solutions. The balance between the molar fractions of the nucleobase monomer (leading to intermolecular H-bonding) and DMA (soluble moiety in chloroform) in the polymer chains was found to be the parameter that principally influences the self-organization. DMA-rich copolymers are molecularly soluble in chloroform, whereas nucleobase-rich copolymers are insoluble in this solvent. Copolymers possessing an equimolar comonomer composition self-assemble into micrometer-sized particles physically cross-linked by intermolecular H-bonds (either thymine-thymine or adenine-adenine interactions, depending on the studied copolymer). Nevertheless, when mixed together, thymine- and adenine-based colloids fuse into thermodynamically stable microspheres cross linked by adenine-thymine interactions.

  15. Nonempirically Tuned Range-Separated DFT Accurately Predicts Both Fundamental and Excitation Gaps in DNA and RNA Nucleobases

    PubMed Central

    2012-01-01

    Using a nonempirically tuned range-separated DFT approach, we study both the quasiparticle properties (HOMO–LUMO fundamental gaps) and excitation energies of DNA and RNA nucleobases (adenine, thymine, cytosine, guanine, and uracil). Our calculations demonstrate that a physically motivated, first-principles tuned DFT approach accurately reproduces results from both experimental benchmarks and more computationally intensive techniques such as many-body GW theory. Furthermore, in the same set of nucleobases, we show that the nonempirical range-separated procedure also leads to significantly improved results for excitation energies compared to conventional DFT methods. The present results emphasize the importance of a nonempirically tuned range-separation approach for accurately predicting both fundamental and excitation gaps in DNA and RNA nucleobases. PMID:22904693

  16. Ab Initio Inverstagation of the Excited States of Nucleobases and Nucleosides

    NASA Astrophysics Data System (ADS)

    Szalay, Péter G.; Fogarasi, Géza; Watson, Thomas; Perera, Ajith; Lotrich, Victor; Bartlett, Rod J.

    2011-06-01

    Most living bodies are exposed to sunlight, essential life sustaining processes are using this natural radiation. Sunlight has, however, several components (has a broad "spectrum") and in particular the invisible component (UV, ultraviolet) is harmful for living organisms. Scientists around the word are busy to understand what happens in the cell when it is exposed to light: it seems that the building blocks of cells and in particular those carrying the genetic information (DNA and RNA) are highly protected against this exposition. Our research focuses on the spectral properties of the building blocks of DNA and RNA, the so called nucleobases and nucleosides, in order to understand this mechanism. Due to improvement in computer technology both at hardware and software side we are now able to use the most accurate methods of ab initio quantum chemistry to investigate the spectroscopic properties of these building blocks. These calculations provide direct information on the properties of these molecules but also provide important benchmarks for cheaper methods which can be used for even larger systems. We have calculated the excited state properties for the nucleobases (cytosine, guanine and adenine), their complexes with water and with each other (Watson-Crick base pairs and stacks) as well as corresponding nucleosides at the EOM-CCSD(T)/aug-cc-pVDZ level of theory and try to answer the following questions: (1) how the order of excited states varies in different nucleobases; (2) how hydration influences the excitation energy and order of excited states; (3) is there any effect of the sugar substituent; (4) how do close lying other bases change the spectrum. The calculations involve over hundred correlated electrons and up to thousand basis functions. Such calculations are now routinely available with the recently developed ACESIII code and can make use of hundreds or even several thousand of processors. V. Lotrich, N. Flocke, M. Ponton, A. Yau, A. Perera, E. Deumens

  17. Aspartame and Its Analogues

    NASA Astrophysics Data System (ADS)

    Pavlova, L. A.; Komarova, T. V.; Davidovich, Yurii A.; Rogozhin, S. V.

    1981-04-01

    The results of studies on the biochemistry of the sweet taste are briefly reviewed. The methods of synthesis of "aspartame" — a sweet dipeptide — are considered, its structural analogues are described, and quantitative estimates are made of the degree of sweetness relative to sucrose. Attention is concentrated mainly on problems of the relation between the structure of the substance and its taste in the series of aspartyl derivatives. The bibliography includes 118 references.

  18. Ice world: the origin of nucleobases in ice-liquid water coexistence conditions.

    NASA Astrophysics Data System (ADS)

    Menor Salvan, C.

    2013-09-01

    We could define the ice world as the chemical evolution in the range between freezing point of water and the limit of stability of liquid brines, ≈273 to 210 K. In this environment, the synthesis of nitrogen heterocycles using urea as nitrogen source and methane as precursor of active intermediates is favorable from a prebiotic chemistry standpoint, leading to a mixture dominated by pyrimidines and hydantoins. Hence, the synthesis in ice matrix constitutes an experimental model for the study of origin of nucleobases in Solar System bodies.

  19. Quantum analogue computing.

    PubMed

    Kendon, Vivien M; Nemoto, Kae; Munro, William J

    2010-08-13

    We briefly review what a quantum computer is, what it promises to do for us and why it is so hard to build one. Among the first applications anticipated to bear fruit is the quantum simulation of quantum systems. While most quantum computation is an extension of classical digital computation, quantum simulation differs fundamentally in how the data are encoded in the quantum computer. To perform a quantum simulation, the Hilbert space of the system to be simulated is mapped directly onto the Hilbert space of the (logical) qubits in the quantum computer. This type of direct correspondence is how data are encoded in a classical analogue computer. There is no binary encoding, and increasing precision becomes exponentially costly: an extra bit of precision doubles the size of the computer. This has important consequences for both the precision and error-correction requirements of quantum simulation, and significant open questions remain about its practicality. It also means that the quantum version of analogue computers, continuous-variable quantum computers, becomes an equally efficient architecture for quantum simulation. Lessons from past use of classical analogue computers can help us to build better quantum simulators in future.

  20. Mass Spectrometric Investigation of Anions Formed upon Free Electron Attachment to Nucleobase Molecules and Clusters Embedded in Superfluid Helium Droplets

    SciTech Connect

    Denifl, Stephan; Zappa, Fabio; Maehr, Ingo; Lecointre, Julien; Probst, Michael; Maerk, Tilmann D.; Scheier, Paul

    2006-07-28

    Here we report the first mass spectrometric study of negative ions formed via free electron attachment (EA) to nucleobases (NBs) embedded in helium clusters. Pure and mixed clusters of adenine and thymine have been formed by pickup of isolated NB molecules by cold helium droplets. In contrast to EA of isolated molecules in the gas phase we observe a long-lived parent anion NB{sup -} and, in addition, parent cluster ions NB{sub n}{sup -} up to size n=6. Moreover, we show that a low energy electron penetrating into a doped helium droplet causes efficient damage of the embedded nucleobases via resonant, site selective, dissociative electron attachment.

  1. Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins

    SciTech Connect

    Lan, Tien-Hung; Wu, Guangyu; Lambert, Nevin A.

    2015-08-14

    Diffusion can enhance Förster resonance energy transfer (FRET) when donors or acceptors diffuse distances that are similar to the distances separating them during the donor's excited state lifetime. Lanthanide donors remain in the excited state for milliseconds, which makes them useful for time-resolved FRET applications but also allows time for diffusion to enhance energy transfer. Here we show that diffusion dramatically enhances FRET between membrane proteins labeled with lanthanide donors. This phenomenon complicates interpretation of experiments that use long-lived donors to infer association or proximity of mobile membrane proteins, but also offers a method of monitoring diffusion in membrane domains in real time in living cells. - Highlights: • Diffusion enhances TR-FRET from membrane proteins labeled with lanthanide donors. • Diffusion-dependent FRET can overshadow FRET due to oligomerization or clustering. • FRET studies using lanthanide-tagged membrane proteins should consider diffusion. • FRET from lanthanide donors can be used to monitor membrane protein diffusion.

  2. Prediction of Fretting Crack Location and Orientation in a Single Crystal Nickel Alloy

    NASA Technical Reports Server (NTRS)

    Matlik, J. F.; Farris, T. N.; Haynes, J.; Swanson, G. R.; Ham-Battista, G.

    2005-01-01

    Fretting is a structural damage mechanism arising between two nominally clamped surfaces subjected to an oscillatory loading. A critical location for fretting induced damage has been identified at the blade/disk and blade/damper interfaces of gas turbine engine turbomachinery and space propulsion components. The high- temperature, high-frequency loading environment seen by these components lead to severe stress gradients at the edge-of-contact that could potentially foster crack growth leading to component failure. These contact stresses drive crack nucleation in fretting and are very sensitive to the geometry of the contacting bodies, the contact loads, materials, temperature, and contact surface tribology (friction). Recently, a high-frequency, high-temperature load frame has been designed for experimentally investigating fretting damage of single crystal nickel materials employed in aircraft and spacecraft turbomachinery. A modeling method for characterizing the fretting stresses of the spherical fretting contact stress behavior in this experiment is developed and described. The calculated fretting stresses for a series of experiments are then correlated to the observed fretting damage. Results show that knowledge of the normal stresses and resolved shear stresses on each crystal plane can aid in predicting crack locations and orientations.

  3. Evaluating the Relationship between FRET Changes and Distance Changes Using DNA Length and Restriction Enzyme Specificity

    ERIC Educational Resources Information Center

    Pazhani, Yogitha; Horn, Abigail E.; Grado, Lizbeth; Kugel, Jennifer F.

    2016-01-01

    FRET (Fo¨rster resonance energy transfer) involves the transfer of energy from an excited donor fluorophore to an acceptor molecule in a manner that is dependent on the distance between the two. A biochemistry laboratory experiment is described that teaches students how to use FRET to evaluate distance changes in biological molecules. Students…

  4. DsRed as a Potential FRET Partner with CFP and GFP

    PubMed Central

    Erickson, Michael G.; Moon, Daniel L.; Yue, David T.

    2003-01-01

    Fluorescence resonance energy transfer (FRET) between mutant green fluorescent proteins (GFP) provides powerful means to monitor in vivo protein-protein proximity and intracellular messengers. However, the leading FRET pair of this class (CFP/YFP) entails suboptimal donor excitation by Argon lasers, thereby hindering FRET imaging on many confocal microscopes. Further challenges arise from the large spectral overlap of CFP/YFP emission. By contrast, DsRed, along with other members of a growing family of red-shifted sea coral fluorophores, features spectra that could obviate such limitations, using DsRed as FRET acceptor, and GFP or CFP as donor. Nonetheless, DsRed suffers from slow chromophore maturation, which confounds quantitative FRET. Here, we develop strategies minimizing the resulting complexity: 1), Pulsed activation of inducible promoters, driving expression of DsRed-tagged molecules, yields a uniform bolus of mature fluorophore; 2), The 33-FRET detection algorithm, adapted for CFP/DsRed and GFP/DsRed, proves insensitive to distortion by slow maturation. We thus show that DsRed supports strong FRET in CFP-DsRed or GFP-DsRed concatemers. These results reveal the promise of sea coral fluorophores like DsRed as FRET partners with GFP or CFP. PMID:12829514

  5. The effect of surface roughness on the fretting corrosion of 316L stainless steel biomaterial surfaces

    NASA Astrophysics Data System (ADS)

    Shenoy, Aarti

    The medical device industry is still seeking answers to the mechanically-assisted corrosion (MAC) problem, which becomes increasingly important due to modularity in design. MAC manifests in various forms, some of which are fretting corrosion, crevice corrosion and stress corrosion. Several studies have been conducted to understand the causes and the factors that affect fretting corrosion. Some of the factors are the applied load, surface potential, oxide film characteristics and solution chemistry near the interface. Surface properties such as surface roughness determine the topography of the surface and the nature of asperity-asperity contact, which is a factor that would determine the mechanically assisted corrosion behavior of the interface, like the stem-neck and head-neck taper junctions in modular hip replacement devices. This study aims to understand the correlation between surface roughness of 316L stainless steel samples and fretting corrosion behavior using a variable load pin-on-disc test. It was found that the smoother surfaces are associated with lower fretting currents. However, smoother surfaces also created the conditions for fretting initiated crevice corrosion to occur more readily. Fretting corrosion regimes and the severity are thus dependent upon the surface roughness. A possible explanation could be due to the inverse relationship between the interasperity distance parameter, Delta, and fretting currents. The coefficient of friction between the two surfaces in contact however remained unaffected by surface roughness, but decreased with increasing load. Smoother surfaces, while lowering fretting corrosion reactions can enhance crevice corrosion reactions in 316L stainless steel interfaces.

  6. FRET-based small-molecule fluorescent probes: rational design and bioimaging applications.

    PubMed

    Yuan, Lin; Lin, Weiying; Zheng, Kaibo; Zhu, Sasa

    2013-07-16

    Fluorescence imaging has emerged as a powerful tool for monitoring biomolecules within the context of living systems with high spatial and temporal resolution. Researchers have constructed a large number of synthetic intensity-based fluorescent probes for bio-imaging. However, intensity-based fluorescent probes have some limitations: variations in probe concentration, probe environment, and excitation intensity may influence the fluorescence intensity measurements. In principle, the use of ratiometric fluorescent probes can alleviate this shortcoming. Förster resonance energy transfer (FRET) is one of the most widely used sensing mechanisms for ratiometric fluorescent probes. However, the development of synthetic FRET probes with favorable photophysical properties that are also suitable for biological imaging applications remains challenging. In this Account, we review the rational design and biological applications of synthetic FRET probes, focusing primarily on studies from our laboratory. To construct useful FRET probes, it is a pre-requisite to develop a FRET platform with favorable photophysical properties. The design criteria of a FRET platform include (1) well-resolved absorption spectra of the donor and acceptor, (2) well-separated emission spectra of the donor and acceptor, (3) donors and acceptors with comparable brightness, (4) rigid linkers, and (5) near-perfect efficiency in energy transfer. With an efficient FRET platform in hand, it is then necessary to modulate the donor-acceptor distance or spectral overlap integral in an analyte-dependent fashion for development of FRET probes. Herein, we emphasize our most recent progress on the development of FRET probes by spectral overlap integral, in particular by changing the molar absorption coefficient of the donor dyes such as rhodamine dyes, which undergo unique changes in the absorption profiles during the ring-opening and -closing processes. Although partial success has been obtained in design of

  7. Application of FRET Technology to the In Vivo Evaluation of Therapeutic Nucleic Acids (ANTs)

    NASA Astrophysics Data System (ADS)

    Benítez-Hess, María Luisa; Alvarez-Salas, Luis Marat

    2007-02-01

    Developing applications for therapeutic nucleic acids (TNAs) (i.e. ribozymes, antisense oligodeoxynucleotides (AS-ODNs), siRNA and aptamers) requires a reporter system designed to rapidly evaluate their in vivo effect. To this end we designed a reporter system based on the fluorescence resonance energy transfer (FRET) engineered to release the FRET effect produced by two green fluorescent protein (GFP) variants linked by a TNA target site. Because the FRET effect occurs instantaneously when two fluorophores are very close to each other (>100nm) stimulating emission of the acceptor fluorophore by the excitation of the donor fluorophore it has been widely use to reveal interactions between molecules. The present system (FRET2) correlates the FRET effect with the in vivo activity of distinct types of TNAs based on a model consisting of RNA from human papillomavirus type 16 (HPV-16) previously shown accessible to TNAs. HPV-16 is the most common papillomavirus associated with cervical cancer, the leading cause of death by cancer in México. The FRET2 system was first tested in vitro and then used in bacteria in which transcription is linked to translation allowing controlled expression and rapid evaluation of the FRET2 protein. To assure accessibility of the target mRNA to TNAs, the FRET2 mRNA was probed by RNaseH assays prior FRET testing. The fluorescence features of the FRET2 system was tested with different FRET-producing GFP donor-acceptor pairs leading to selection of green (donor) and yellow (acceptor) variants of GFP as the most efficient. Modifications in aminoacid composition and linker length of the target sequence did not affect FRET efficiency. In vivo AS-ODN-mediated destruction of the chimerical FRET2 reporter mRNA resulted in the recovery of GFP fluorescent spectrum in a concentration and time dependent manner. Reported anti-HPV ribozymes were also tested with similar results. Therefore, we conclude that the FRET effect can be a useful tool in the

  8. The design of Förster (fluorescence) resonance energy transfer (FRET)-based molecular sensors for Ran GTPase

    PubMed Central

    Kalab, Petr; Soderholm, Jon

    2010-01-01

    The application of FRET-based molecular bio-sensors provided confirmation of the central model of Ran GTPase function and led to important new insights into its physiological role. In many fields of cell biology, methods employing FRET are a standard approach that is becoming increasingly accessible due to advances in instrumentation and available fluorophores. However, the optimal design of a FRET sensor remains to be the cornerstone of any successful FRET application. Utilizing the recent literature on FRET applications and our studies on Ran, we outline the basic considerations involved in designing molecular FRET sensors. We point to several broadly applicable principles that were used in many different FRET sensors that can detect a wide range of molecular events. Using the FRET sensors for Ran that we created as examples, we then focus on the practical aspects of FRET assays. We describe the preparation of a bipartite FRET sensor consisting of ECFP-Ran and EYFP-importin β and its validation as a reporter for FRET-based high throughput screening in small molecule libraries. Finally, we review the design and optimization of monomolecular FRET sensors that monitor the RanGTP-RanBP1 interaction, and of sensors detecting the RanGTP-regulated importin β cargo release. PMID:20096786

  9. A Versatile Multiple Target Detection System Based on DNA Nano-assembled Linear FRET Arrays

    PubMed Central

    Li, Yansheng; Du, Hongwu; Wang, Wenqian; Zhang, Peixun; Xu, Liping; Wen, Yongqiang; Zhang, Xueji

    2016-01-01

    DNA molecules have been utilized both as powerful synthetic building blocks to create nanoscale architectures and as inconstant programmable templates for assembly of biosensors. In this paper, a versatile, scalable and multiplex detection system is reported based on an extending fluorescent resonance energy transfer (FRET) cascades on a linear DNA assemblies. Seven combinations of three kinds of targets are successfully detected through the changes of fluorescence spectra because of the three-steps FRET or non-FRET continuity mechanisms. This nano-assembled FRET-based nanowire is extremely significant for the development of rapid, simple and sensitive detection system. The method used here could be extended to a general platform for multiplex detection through more-step FRET process. PMID:27230484

  10. A Versatile Multiple Target Detection System Based on DNA Nano-assembled Linear FRET Arrays.

    PubMed

    Li, Yansheng; Du, Hongwu; Wang, Wenqian; Zhang, Peixun; Xu, Liping; Wen, Yongqiang; Zhang, Xueji

    2016-05-27

    DNA molecules have been utilized both as powerful synthetic building blocks to create nanoscale architectures and as inconstant programmable templates for assembly of biosensors. In this paper, a versatile, scalable and multiplex detection system is reported based on an extending fluorescent resonance energy transfer (FRET) cascades on a linear DNA assemblies. Seven combinations of three kinds of targets are successfully detected through the changes of fluorescence spectra because of the three-steps FRET or non-FRET continuity mechanisms. This nano-assembled FRET-based nanowire is extremely significant for the development of rapid, simple and sensitive detection system. The method used here could be extended to a general platform for multiplex detection through more-step FRET process.

  11. A Versatile Multiple Target Detection System Based on DNA Nano-assembled Linear FRET Arrays

    NASA Astrophysics Data System (ADS)

    Li, Yansheng; Du, Hongwu; Wang, Wenqian; Zhang, Peixun; Xu, Liping; Wen, Yongqiang; Zhang, Xueji

    2016-05-01

    DNA molecules have been utilized both as powerful synthetic building blocks to create nanoscale architectures and as inconstant programmable templates for assembly of biosensors. In this paper, a versatile, scalable and multiplex detection system is reported based on an extending fluorescent resonance energy transfer (FRET) cascades on a linear DNA assemblies. Seven combinations of three kinds of targets are successfully detected through the changes of fluorescence spectra because of the three-steps FRET or non-FRET continuity mechanisms. This nano-assembled FRET-based nanowire is extremely significant for the development of rapid, simple and sensitive detection system. The method used here could be extended to a general platform for multiplex detection through more-step FRET process.

  12. Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM.

    PubMed

    Chennell, George; Willows, Robin J W; Warren, Sean C; Carling, David; French, Paul M W; Dunsby, Chris; Sardini, Alessandro

    2016-01-01

    We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation. PMID:27548185

  13. Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

    PubMed Central

    Chennell, George; Willows, Robin J. W.; Warren, Sean C.; Carling, David; French, Paul M. W.; Dunsby, Chris; Sardini, Alessandro

    2016-01-01

    We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation. PMID:27548185

  14. Interaction of nucleobases with silicon doped and defective silicon doped graphene and optical properties.

    PubMed

    Mudedla, Sathish Kumar; Balamurugan, Kanagasabai; Kamaraj, Manoharan; Subramanian, Venkatesan

    2016-01-01

    The interaction of nucleobases (NBs) with the surface of silicon doped graphene (SiGr) and defective silicon doped graphene (dSiGr) has been studied using electronic structure methods. A systematic comparison of the calculated interaction energies (adsorption strength) of NBs with the surface of SiGr and dSiGr with those of pristine graphene (Gr) has also been made. The doping of graphene with silicon increases the adsorption strength of NBs. The introduction of defects in SiGr further enhances the strength of interaction with NBs. The appreciable stability of complexes (SiGr-NBs and dSiGr-NBs) arises due to the partial electrostatic and covalent (Si···O(N)) interaction in addition to π-π stacking. The interaction energy increases with the size of graphene models. The strong interaction between dSiGr-NBs and concomitant charge transfer causes significant changes in the electronic structure of dSiGr in contrast to Gr and SiGr. Further, the calculated optical properties of all the model systems using time dependent density functional theory (TD-DFT) reveal that absorption spectra of SiGr and dSiGr undergo appreciable changes after adsorption of NBs. Thus, the significant variations in the HOMO-LUMO gap and absorption spectra of dSiGr after interaction with the NBs can be exploited for possible applications in the sensing of DNA nucleobases.

  15. New approach for designing single-chain magnets: organization of chains via hydrogen bonding between nucleobases.

    PubMed

    Zhang, Wei-Xiong; Shiga, Takuya; Miyasaka, Hitoshi; Yamashita, Masahiro

    2012-04-25

    Two one-dimensional (1D) manganese complexes, [Mn(2)(naphtmen)(2)(L)](ClO(4))·2Et(2)O·2MeOH·H(2)O (1) and [Mn(2)(naphtmen)(2)(HL)](ClO(4))(2)·MeOH (2), were synthesized by using a bridging ligand with a nucleobase moiety, 6-amino-9-β-carboxyethylpurine, and a salen-type manganese(III) dinuclear complex, [Mn(2)(naphtmen)(2)(H(2)O)(2)](ClO(4))(2) (naphtmen(2-) = N,N'-(1,1,2,2-tetramethylethylene)bis(naphthylideneiminato) dianion). In 1 and 2, the carboxylate-bridged Mn(III) dinuclear units are alternately linked by two kinds of weak Mn···O interactions into 1D chains. As a result, canted antiferromagnetic and ferromagnetic interactions are alternately present along the chains, leading to a 1D chain with non-cancellation of anisotropic spins. Since the chains connected via H-bonds between nucleobase moieties are magnetically isolated, both 1 and 2 act as single-chain magnets (SCMs). More importantly, this result shows the smaller canting angles hinder long-range ordering in favor of SCM dynamics.

  16. Enthalpy-Entropy Tuning in the Adsorption of Nucleobases at the Au(111) Surface.

    PubMed

    Rosa, Marta; Corni, Stefano; Di Felice, Rosa

    2014-04-01

    The interaction of DNA molecules with hard substrates is of paramount importance both for the study of DNA itself and for the variety of possible technological applications. Interaction with inorganic surfaces strongly modifies the helical shape of DNA. Hence, an accurate understanding of DNA structure and function at interfaces is a fundamental question with enormous impact in science and society. This work sets the fundamentals for the simulation of entire DNA oligomers on gold surfaces in dry and wet conditions. Thanks to the new GolDNA-AMBER force field, which was derived from first principles and includes dispersion interactions and polarization effects, we simulated self-assembled guanine and adenine monolayers on Au(111) in vacuo and the adsorption of all nucleobases on the same substrate in aqueous conditions. The periodic monolayers obtained from classical simulations match very well those from first principle calculations and experiments, assessing the robustness of the force field and motivating the application to more complex systems for which quantum calculations are not affordable and experiments are elusive. The energetics of nucleobases on Au(111) in solution reveal fundamental physicochemical effects: we find that the adsorption paradigm shifts from purely enthalpic to dominantly entropic by changing the environment and aggregation phase.

  17. Catalytic Role of Manganese Oxides in Prebiotic Nucleobases Synthesis from Formamide.

    PubMed

    Bhushan, Brij; Nayak, Arunima; Kamaluddin

    2016-06-01

    Origin of life processes might have begun with the formation of important biomonomers, such as amino acids and nucleotides, from simple molecules present in the prebiotic environment and their subsequent condensation to biopolymers. While studying the prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide, the manganese oxides demonstrated not only good binding for formamide but demonstrated novel catalytic activity. A novel one pot manganese oxide catalyzed synthesis of pyrimidine nucleobases like thymine is reported along with the formation of other nucleobases like purine, 9-(hydroxyacetyl) purine, cytosine, 4(3 H)-pyrimidinone and adenine in acceptable amounts. The work reported is significant in the sense that the synthesis of thymine has exhibited difficulties especially under one pot conditions and also such has been reported only under the catalytic activity of TiO2. The lower oxides of manganese were reported to show higher potential as catalysts and their existence were favored by the reducing atmospheric conditions prevalent on early Earth; thereby confirming the hypothesis that mineral having metals in reduced form might have been more active during the course of chemical evolution. Our results further confirm the role of formamide as a probable precursor for the formation of purine and pyrimidine bases during the course of chemical evolution and origin of life.

  18. Understanding prebiotic chemistry through the analysis of extraterrestrial amino acids and nucleobases in meteorites.

    PubMed

    Burton, Aaron S; Stern, Jennifer C; Elsila, Jamie E; Glavin, Daniel P; Dworkin, Jason P

    2012-08-21

    The discoveries of amino acids of extraterrestrial origin in many meteorites over the last 50 years have revolutionized the Astrobiology field. A variety of non-terrestrial amino acids similar to those found in life on Earth have been detected in meteorites. A few amino acids have even been found with chiral excesses, suggesting that meteorites could have contributed to the origin of homochirality in life on Earth. In addition to amino acids, which have been productively studied for years, sugar-like molecules, activated phosphates, and nucleobases have also been determined to be indigenous to numerous meteorites. Because these molecules are essential for life as we know it, and meteorites have been delivering them to the Earth since accretion, it is plausible that the origin(s) of life on Earth were aided by extraterrestrially-synthesized molecules. Understanding the origins of life on Earth guides our search for life elsewhere, helping to answer the question of whether biology is unique to Earth. This tutorial review focuses on meteoritic amino acids and nucleobases, exploring modern analytical methods and possible formation mechanisms. We will also discuss the unique window that meteorites provide into the chemistry that preceded life on Earth, a chemical record we do not have access to on Earth due to geologic recycling of rocks and the pervasiveness of biology across the planet. Finally, we will address the future of meteorite research, including asteroid sample return missions.

  19. Ultraviolet Irradiation of Pyrimidine in Interstellar Ice Analogs: Formation and Photo-Stability of Nucleobases

    NASA Technical Reports Server (NTRS)

    Nuevo, Michel; Milam, Stefanie N.; Sandford, Scott A.; Elsila, Jamie E.; Dworkin, Jason P.

    2010-01-01

    Astrochemistry laboratory experiments recently showed that molecules of prebiotic interest can potentially form in space, as supported by the detection of amino acids in organic residues formed by the UV photolysis of ices simulating interstellar and cometary environments (H2O, CO, CO2, CH3OH, NH3, etc.). Although the presence of amino acids in the interstellar medium (ISM) is still under debate, experiments and the detection of amino acids in meteorites both support a scenario in which prebiotic molecules could be of extraterrestrial origin, before they are delivered to planets by comets, asteroids, and interplanetary dust particles. Nucleobases, the informational subunits of DNA and RNA, have also been detected in meteorites, although they have not yet been observed in the ISM. Thus, these molecules constitute another family of prebiotic compounds that can possibly form via abiotical processes in astrophysical environments. Nucleobases are nitrogen-bearing cyclic aromatic species with various functional groups attached, which are divided into two classes: pyrimidines (uracil, cytosine, and thymine) and purines (adenine and guanine). In this work, we study how UV irradiation affects pyrimidine mixed in interstellar ice analogs (H2O, NH3, CH3OH). In particular, we show that the UV irradiation of H2O:pyrimidine mixtures leads to the production of oxidized compounds including uracil, and show that both uracil and cytosine are formed upon irradiation of H2O:NH3:pyrimidine mixtures. We also study the photostability of pyrimidine and its photoproducts formed during these experiments.

  20. Physisorption of nucleobases on graphene: a comparative van der Waals study.

    PubMed

    Le, Duy; Kara, Abdelkader; Schröder, Elsebeth; Hyldgaard, Per; Rahman, Talat S

    2012-10-24

    The physisorption of the nucleobases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) on graphene is studied using several variants of the density functional theory (DFT): the generalized gradient approximation with the inclusion of van der Waals interaction (vdW) based on the TS approach (Tkatchenko and Scheffer 2009 Phys. Rev. Lett. 102 073005) and our simplified version of this approach (here called sTS), the van der Waals density functional vdW-DF (Dion et al 2004 Phys. Rev. Lett. 92 246401) and vdW-DF2 (Lee et al 2010 Phys. Rev. B 82 081101), and DFT-D2 (Grimme 2006 J. Comput. Chem. 27 1787) and DFT-D3 (Grimme et al 2010 J. Chem. Phys. 132 154104) methods. The binding energies of nucleobases on graphene are found to be in the following order: G > A > T > C > U within TS, sTS, vdW-DF, and DFT-D2, and in the following order: G > A > T ~ C > U within DFT-D3 and vdW-DF2. The binding separations are found to be different within different methods and in the following order: DFT-D2 < TS < DFT-D3 ~ vdW-DF2 < vdW-DF. We also comment on the efficiency of combining the DFT-D approach and vdW-DF to study systems with van der Waals interactions.

  1. Physisorption of nucleobases on graphene: a comparative van der Waals study

    NASA Astrophysics Data System (ADS)

    Le, Duy; Kara, Abdelkader; Schröder, Elsebeth; Hyldgaard, Per; Rahman, Talat S.

    2012-10-01

    The physisorption of the nucleobases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) on graphene is studied using several variants of the density functional theory (DFT): the generalized gradient approximation with the inclusion of van der Waals interaction (vdW) based on the TS approach (Tkatchenko and Scheffer 2009 Phys. Rev. Lett. 102 073005) and our simplified version of this approach (here called sTS), the van der Waals density functional vdW-DF (Dion et al 2004 Phys. Rev. Lett. 92 246401) and vdW-DF2 (Lee et al 2010 Phys. Rev. B 82 081101), and DFT-D2 (Grimme 2006 J. Comput. Chem. 27 1787) and DFT-D3 (Grimme et al 2010 J. Chem. Phys. 132 154104) methods. The binding energies of nucleobases on graphene are found to be in the following order: G > A > T > C > U within TS, sTS, vdW-DF, and DFT-D2, and in the following order: G > A > T ˜ C > U within DFT-D3 and vdW-DF2. The binding separations are found to be different within different methods and in the following order: DFT-D2 < TS < DFT-D3 ˜ vdW-DF2 < vdW-DF. We also comment on the efficiency of combining the DFT-D approach and vdW-DF to study systems with van der Waals interactions.

  2. The search for and identification of amino acids, nucleobases and nucleosides in samples returned from Mars

    NASA Technical Reports Server (NTRS)

    Gehrke, Charles W.; Ponnamperuma, Cyril; Kuo, Kenneth C.; Stalling, David L.; Zumwalt, Robert W.

    1989-01-01

    An investigation of the returned Mars samples for biologically important organic compounds, with emphasis on amino acid, the puring and pyrimidine bases, and nucleosides is proposed. These studies would be conducted on subsurface samples obtained by drilling past the surface oxidizing layer with emphasis on samples containing the larges quantities of organic carbon as determined by the rover gas chromatographic mass spectrometer (GCMS). Extraction of these molecules from the returned samples will be performed using the hydrothermal extraction technique described by Cheng and Ponnamperuma. More rigorous extraction methods will be developed and evaluated. For analysis of the extract for free amino acids or amino acids present in a bound or peptidic form, aliquots will be analyzed by capillary GCMS both before and after hydrolysis with 6N hydrochloric acid. Establishment of the presence of amino acids would then lead to the next logical step which would be the use of chiral stationary gas chromatography phases to determine the enatiomeic composition of the amino acids present, and thus potentially establish their biotic or abiotic origin. Confirmational analyses for amino acids would include ion-exchange and reversed-phase liquid chromatographic analysis. For analyses of the returned Mars samples for nucleobases and nucleosides, affinity and reversed-phase liquid chromatography would be utilized. This technology coupled with scanning UV detection for identification, presents a powerful tool for nucleobase and nucleoside analysis. Mass spectrometric analysis of these compounds would confirm their presence in samples returned form Mars.

  3. Exploring the Roles of Nucleobase Desolvation and Shape Complementarity during the Misreplication of O6-Methylguanine

    PubMed Central

    Chavarria, Delia; Ramos-Serrano, Andrea; Hirao, Ichiro; Berdis, Anthony J.

    2011-01-01

    O6-methylguanine is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and non-natural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a large role in controlling the extension of various mispairs containing O6-methylguanine. This is evident as the rate constants for extension correlate with proper interglycosyl distances and symmetry between the base angles of the formed mispair. Base pairs not conforming to an acceptable geometry within the polymerase’s active site are refractory to elongation and are processed via exonuclease proofreading. The collective data set encompassing nucleotide incorporation, extension, and excision is used to generate a model accounting for the mutagenic potential of O6-methylguanine observed in vivo. In addition, kinetic studies monitoring the incorporation and extension of non-natural nucleotides identified an analog that displays high selectivity for incorporation opposite O6-methylguanine compared to unmodified purines. The unusual selectivity of this analog for replicating damaged DNA provides a novel biochemical tool to study translesion DNA synthesis. PMID:21819995

  4. Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase

    PubMed Central

    Liu, Xinran; Musser, Derek M.; Lee, Cheri A.; Yang, Xiaorong; Arnold, Jamie J.; Cameron, Craig E.; Boehr, David D.

    2015-01-01

    The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes (i.e., D53N, Y73H, K250E, and T362I) in the RNA-dependent RNA polymerase (RdRp). We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes (i.e., D53N, Y73H, K250E) help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation. PMID:26516899

  5. Identification of the distribution of adenosine phosphates, nucleosides and nucleobases in royal jelly.

    PubMed

    Wu, Liming; Chen, Lanzhen; Selvaraj, Jonathan Nimal; Wei, Yue; Wang, Yong; Li, Yi; Zhao, Jing; Xue, Xiaofeng

    2015-04-15

    Nucleotides, nucleosides and nucleobases play a greater role in the physiological activity of organisms which are highly present in royal jelly (RJ). The objective of the present study is to develop a HPLC method to simultaneous determine nucleotides, nucleosides and nucleobases in RJ and access them in fresh and commercial RJ samples. The LOD and LOQ were 12.2-99.6 μg/L and 40.8-289.4 μg/L, respectively with nearly 100.9% recoveries. Except uric acid, all other compounds were found in RJ samples. Significant difference in the average content of compounds in fresh (2682.93 mg/kg) and commercial samples (3152.78 mg/kg) were observed. AMP, adenosine and adenine were found predominant in all the samples. Significant higher levels of ATP, ADP and AMP was seen in fresh RJ samples, and IMP, uridine, guanosine, and thymidine was seen in commercial RJ samples. The investigated compounds can be used as indexes for assessment RJ freshness and quality.

  6. Meteoritic Input of Amino Acids and Nucleobases: Methodology and Implications for the Origins of Life

    NASA Technical Reports Server (NTRS)

    Burton, Aaron S.; Stern, Jennifer C.; Elsila, Jamie E.; Glavin, Daniel P.; Dworkin, Jason P.

    2012-01-01

    The discoveries of amino acids of extraterrestrial origin in many meteorites over the last 40 years have revolutionized the Astrobiology field. A variety of non-terrestrial amino acids similar to those found in life on Earth have been detected in meteorites. A few amino acids have even been found with chiral excesses, suggesting that meteorites could have contributed to the origin of homochirality in life on Earth. In addition to amino acids, which have been productively studied for years, sugar-like molecules, activated phosphates, and nucleobases have also been determined to be indigenous to numerous meteorites. Because these molecules are essential for life as we know it, and meteorites have been delivering them to the Earth since accretion, it is plausible that the origin(s) of life on Earth were aided by extraterrestrially-synthesized molecules. Understanding the origins of life on Earth guides our search for life elsewhere, helping to answer the question of whether biology is unique to Earth. This tutorial review focuses on meteoritic amino acids and nucleobases, exploring modern analytical methods and possible formation mechanisms. We will also discuss the unique window that meteorites provide into the chemistry that preceded life on Earth, a chemical record we do not have access to on Earth due to geologic recycling of rocks and the pervasiveness of biology across the planet. Finally, we will address the future of meteorite research, including asteroid sample return mIssIons.

  7. Understanding Prebiotic Chemistry Through the Analysis of Extraterrestrial Amino Acids and Nucleobases in Meteorites

    NASA Technical Reports Server (NTRS)

    Burton, Aaron S.; Stern, Jennifer C.; Elsila, Jamie E.; Glavin, Daniel P.; Dworkin, Jason P.

    2012-01-01

    The discoveries of amino acids of extraterrestrial origin in many meteorites over the last 50 years have revolutionized the Astrobiology field. A variety of non-terrestrial amino acids similar to those found in life on Earth have been detected in meteorites. A few amino acids have even been found with chiral excesses, suggesting that meteorites could have contributed to the origin of homochirality in life on Earth. In addition to amino acids, which have been productively studied for years, sugar-like molecules, activated phosphates, and nucleobases have also been determined to be indigenous to numerous meteorites. Because these molecules are essential for life as we know it, and meteorites have been delivering them to the Earth since accretion, it is plausible that the origines) of life on Earth were aided by extrataterrestrially-synthesized molecules. Understanding the origins of life on Earth guides our search for life elsewhere, helping to answer the question of whether biology is unique to Earth. This tutorial focuses on meteoritic amino acids and nucleobases, exploring modern analytical methods and possible formation mechanisms. We will also discuss the unique window that meteorites provide into the chemistry that preceded life on Earth, a chemical record we do not have access to on Earth due to geologic recycling of rocks and the pervasiveness of biology across the planet. Finally. we will address the future of meteorite research, including asteroid sample return missions.

  8. Catalytic Role of Manganese Oxides in Prebiotic Nucleobases Synthesis from Formamide

    NASA Astrophysics Data System (ADS)

    Bhushan, Brij; Nayak, Arunima; Kamaluddin

    2016-06-01

    Origin of life processes might have begun with the formation of important biomonomers, such as amino acids and nucleotides, from simple molecules present in the prebiotic environment and their subsequent condensation to biopolymers. While studying the prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide, the manganese oxides demonstrated not only good binding for formamide but demonstrated novel catalytic activity. A novel one pot manganese oxide catalyzed synthesis of pyrimidine nucleobases like thymine is reported along with the formation of other nucleobases like purine, 9-(hydroxyacetyl) purine, cytosine, 4(3 H)-pyrimidinone and adenine in acceptable amounts. The work reported is significant in the sense that the synthesis of thymine has exhibited difficulties especially under one pot conditions and also such has been reported only under the catalytic activity of TiO2. The lower oxides of manganese were reported to show higher potential as catalysts and their existence were favored by the reducing atmospheric conditions prevalent on early Earth; thereby confirming the hypothesis that mineral having metals in reduced form might have been more active during the course of chemical evolution. Our results further confirm the role of formamide as a probable precursor for the formation of purine and pyrimidine bases during the course of chemical evolution and origin of life.

  9. Engineering Dark Chromoprotein Reporters for Photoacoustic Microscopy and FRET Imaging

    NASA Astrophysics Data System (ADS)

    Li, Yan; Forbrich, Alex; Wu, Jiahui; Shao, Peng; Campbell, Robert E.; Zemp, Roger

    2016-03-01

    A subset of the family of fluorescent proteins are the non-fluorescent chromoproteins which are promising probe molecules for use in photoacoustic imaging and as acceptor chromophores in Förster resonance energy transfer (FRET)-based biosensors. Typical approaches for fluorescent protein optimization by screening of large libraries of variants cannot be effectively applied to chromoproteins due to their characteristic lack of fluorescence. To address this challenge, we have developed a directed evolution method to iteratively screen large libraries of protein variants on the basis of their photoacoustic signal levels. By applying this procedure to the promising Ultramarine and cjBlue chromoprotein templates, we were able to identify improved variants with a 02-04 fold increase in photoacoustic signal-to-noise ratio after only a few evolutionary steps. These improved variants enable more accurate spectral de-mixing and localization of protein-producing bacteria in vivo and serve as effective FRET acceptors for both fluorescence- and photoacoustic-based detection of protease activity.

  10. Engineering Dark Chromoprotein Reporters for Photoacoustic Microscopy and FRET Imaging

    PubMed Central

    Li, Yan; Forbrich, Alex; Wu, Jiahui; Shao, Peng; Campbell, Robert E.; Zemp, Roger

    2016-01-01

    A subset of the family of fluorescent proteins are the non-fluorescent chromoproteins which are promising probe molecules for use in photoacoustic imaging and as acceptor chromophores in Förster resonance energy transfer (FRET)-based biosensors. Typical approaches for fluorescent protein optimization by screening of large libraries of variants cannot be effectively applied to chromoproteins due to their characteristic lack of fluorescence. To address this challenge, we have developed a directed evolution method to iteratively screen large libraries of protein variants on the basis of their photoacoustic signal levels. By applying this procedure to the promising Ultramarine and cjBlue chromoprotein templates, we were able to identify improved variants with a 02–04 fold increase in photoacoustic signal-to-noise ratio after only a few evolutionary steps. These improved variants enable more accurate spectral de-mixing and localization of protein-producing bacteria in vivo and serve as effective FRET acceptors for both fluorescence- and photoacoustic-based detection of protease activity. PMID:26926390

  11. Ligament Rupture Pressure of Fretted SG Tubes of PWRs

    SciTech Connect

    Seong Sik Hwang; Man Kyo Jung; Hong Pyo Kim; Joung Soo Kim

    2006-07-01

    A fretting/wear degradation at the tube support in the U-bend region of a steam generator (SG) of a pressurized water reactor (PWR) has been reported. Simulated fretted flaws were machined on SG tubes of 195 mm in length. A pressure test was carried out with the tubes at room temperature by using a high pressure test facility which consisted of a water pressurizing pump, a test specimen section and a control unit. Water leak rates just after a ligament rupture or a burst were measured. Tubes degraded by up to 70% of the tube wall (TW) showed a high safety margin in terms of the burst pressure during normal operating conditions. Tubes degraded by up to 50% of the TW did not show a burst. Burst pressure depended on the defect depths rather than on the wrap angles. The tube with a wrap angle of 0 deg. showed a fish mouth fracture, whereas the tube with a 45 deg. wrap angle showed a three way fracture. (authors)

  12. Paths to Förster's resonance energy transfer (FRET) theory

    NASA Astrophysics Data System (ADS)

    Masters, B. R.

    2014-02-01

    Theodor Förster (1910-1974) developed a phenomenological theory of nonradiative resonance energy transfer which proved to be transformative in the fields of chemistry, biochemistry, and biology. This paper explores the experimental and the theoretical antecedents of Förster's theory of resonance energy transfer (FRET). Early studies of sensitized fluorescence, fluorescence depolarization, and photosynthesis demonstrated the phenomena of long-range energy transfer. At the same time physicists developed theoretical models which contained common physical mechanisms and parameters: oscillating dipoles as models for the atoms or molecules, dipole-dipole coupling for the interaction, and a distance R0 that is optimal for resonance energy transfer. Early theories predicted R0 that was too large as compared to experiments. Finally, in 1946 Förster developed a classical theory and in 1948 he developed a quantum mechanical theory; both theories predicted an inverse sixth power dependence of the rate of energy transfer and a R0 that agreed with experiments. This paper attempts to determine why Förster succeeded when the other theoreticians failed to develop the correct theory. The putative roles of interdisciplinary education and collaborative research are discussed. Furthermore, I explore the role of science journals and their specific audiences in the popularization of FRET to a broad interdisciplinary community.

  13. Using and interpreting analogue designs.

    PubMed

    Cook, Bryan G; Rumrill, Phillip D

    2005-01-01

    Researchers in rehabilitation counseling and disability studies sometimes use analogue research, which involves materials that approximate or describe reality (e.g., written vignettes, videotaped exemplars) rather than investigating phenomena in real-world settings. Analogue research often utilizes experimental designs, and it thereby frequently possesses a high degree of internal validity. Analogue research allows investigators to exercise tight control over the implementation of the independent or treatment variable and over potentially confounding variables, which enables them to isolate the effects of those treatment variables on selected outcome measures. However, the simulated nature of analogue research presents an important threat to external validity. As such, the generalizability of analogue research to real-life settings and situations may be problematic. These and other issues germane to analogue research in vocational rehabilitation are discussed in this article, illustrated with examples from the contemporary literature.

  14. Simultaneous determination of 16 nucleosides and nucleobases by hydrophilic interaction chromatography and its application to the quality evaluation of Ganoderma.

    PubMed

    Chen, Yi; Bicker, Wolfgang; Wu, Junyan; Xie, Mingyong; Lindner, Wolfgang

    2012-05-01

    In order to develop a simple, efficient, and sensitive method for comprehensive analysis of the nucleosides and nucleobases in natural products, a zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) method for the simultaneous determination of 16 nucleosides and nucleobases has been studied. A mechanistic study confirmed that ZIC-HILIC separation showed a mixed-mode effect of both hydrophilic and electrostatic interactions. This method was validated to be precise, accurate, and sensitive with overall precision (intra- and interday) less than 1.8% (RSD), and LOD and LOQ was in the range of 0.005-0.029 μg/mL and 0.018-0.096 μg/mL, respectively. With this method, the nucleosides and nucleobases in Ganoderma of different species (G. atrum, G. lucidum, and G. sinense) and origins were quantified. The results showed that the contents varied with the species and origins. With the aid of hierarchical cluster analysis (HCA), cultivated Ganoderma from different origins and species were successfully discriminated. It is for the first time that the content of nucleosides and nucleobases in G. atrum is reported and compared. Our data showed that HILIC had advantages as a useful and potential tool for the study of the bioactive components in Ganoderma as well as their quality control, and could therefore be used for the determination of the analytes in other natural products.

  15. Analogue-to-Digital and Digital-to-Analogue Conversion.

    ERIC Educational Resources Information Center

    Gregory, Martin

    1997-01-01

    Discusses circuits for three-bit and four-bit analogue digital converters and digital analogue converters. These circuits feature slow operating speeds that enable the circuitry to be used to demonstrate the mode of operation using oscilloscopes and signal generators. (DDR)

  16. MIL-L-87177 and CLT:X-10 Lubricants Improve Electrical Connector Fretting Corrosion Behavior

    SciTech Connect

    AUKLAND,NEIL R.; HANLON,JAMES T.

    1999-10-12

    We have conducted a fretting research project using MIL-L-87177 and CLT: X-10 lubricants on Nano-miniature connectors. When they were fretted without lubricant, individual connectors first exceeded our 0.5 ohm failure criteria from 2,341 to 45,238 fretting cycles. With additional fretting, their contact resistance increased to more than 100,000 ohms. Unmodified MIL-L-87177 lubricant delayed the onset of first failure to between 430,000 and over 20,000,000 fretting cycles. MIL-L-87177 modified by addition of Teflon powder delayed first failure to beyond 5 million fretting cycles. Best results were obtained when Teflon was used and also when both the straight and modified lubricants were poured into and then out of the connector. CLT: X-10 lubricant delayed the onset of first failure to beyond 55 million cycles in one test where a failure was actually observed and to beyond 20 million cycles in another that was terminated without failure. CLT: X-10 recovered an unlubricated connector driven deeply into failure, with six failed pins recovering immediately and four more recovering during an additional 420 thousand fretting cycles. MIL-L-87177 was not able to recover a connector under similar conditions.

  17. N-way FRET microscopy of multiple protein-protein interactions in live cells.

    PubMed

    Hoppe, Adam D; Scott, Brandon L; Welliver, Timothy P; Straight, Samuel W; Swanson, Joel A

    2013-01-01

    Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells. PMID:23762252

  18. Partially reduced graphene oxide based FRET on fiber-optic interferometer for biochemical detection.

    PubMed

    Yao, B C; Wu, Y; Yu, C B; He, J R; Rao, Y J; Gong, Y; Fu, F; Chen, Y F; Li, Y R

    2016-03-24

    Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerful technique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel 'FRET on Fiber' concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiber-optic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 μM and 1 pM, respectively. Such a prGO based 'FRET on fiber' configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated 'FRET on Fiber' sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response.

  19. Partially reduced graphene oxide based FRET on fiber-optic interferometer for biochemical detection

    PubMed Central

    Yao, B. C.; Wu, Y.; Yu, C. B.; He, J. R.; Rao, Y. J.; Gong, Y.; Fu, F.; Chen, Y. F.; Li, Y. R.

    2016-01-01

    Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerful technique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel ‘FRET on Fiber’ concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiber-optic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 μM and 1 pM, respectively. Such a prGO based ‘FRET on fiber’ configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated ‘FRET on Fiber’ sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response PMID:27010752

  20. Partially reduced graphene oxide based FRET on fiber-optic interferometer for biochemical detection

    NASA Astrophysics Data System (ADS)

    Yao, B. C.; Wu, Y.; Yu, C. B.; He, J. R.; Rao, Y. J.; Gong, Y.; Fu, F.; Chen, Y. F.; Li, Y. R.

    2016-03-01

    Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerful technique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel ‘FRET on Fiber’ concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiber-optic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 μM and 1 pM, respectively. Such a prGO based ‘FRET on fiber’ configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated ‘FRET on Fiber’ sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response

  1. Simultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light

    PubMed Central

    Niino, Yusuke; Hotta, Kohji; Oka, Kotaro

    2009-01-01

    Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility. PMID:19551140

  2. MagFRET: The First Genetically Encoded Fluorescent Mg2+ Sensor

    PubMed Central

    Oortwijn, Jorn; Aper, Stijn J. A.; Merkx, Maarten

    2013-01-01

    Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg2+, MagFRET-1. This sensor is based on the high-affinity Mg2+ binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg2+-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg2+ affinity (Kd = 148 µM) with a 50% increase in emission ratio upon Mg2+ binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg2+ affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg2+ concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg2+ concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca2+, this affinity is sufficiently attenuated (Kd of 10 µM) to make the sensor insensitive to known Ca2+ stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg2+ imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg2+ sensors could prove very useful in understanding intracellular Mg2+ homeostasis and signaling. PMID:24312622

  3. Detection and Quantification of Intracellular Signaling Using FRET-Based Biosensors and High Content Imaging.

    PubMed

    Halls, Michelle L; Poole, Daniel P; Ellisdon, Andrew M; Nowell, Cameron J; Canals, Meritxell

    2015-01-01

    Förster resonance energy transfer (FRET) biosensors represent invaluable tools to detect the spatiotemporal context of second messenger production and intracellular signaling that cannot be attained using traditional methods. Here, we describe a detailed protocol for the use of high content imaging in combination with FRET biosensors to assess second messenger production and intracellular signaling in a time-effective manner. We use four different FRET biosensors to measure cAMP levels, kinase (ERK and PKC), and GTPase activity. Importantly, we provide the protocols to express and measure these sensors in a variety of model cell lines and primary dorsal root ganglia neurons.

  4. Laser-induced shockwave paired with FRET: a method to study cell signaling.

    PubMed

    Gomez-Godinez, Veronica; Preece, Daryl; Shi, Linda; Khatibzadeh, Nima; Rosales, Derrick; Pan, Yijia; Lei, Lie; Wang, Yingxiao; Berns, Michael W

    2015-03-01

    Cells within the body are subject to various forces; however, the details concerning the way in which cells respond to mechanical stimuli are not well understood. We demonstrate that laser-induced shockwaves (LIS) combined with biosensors based on fluorescence resonance energy transfer (FRET) is a promising new approach to study biological processes in single live cells. As "proof-of-concept," using a FRET biosensor, we show that in response to LIS, cells release intracellular calcium. With the parameters used, cells retain their morphology and remain viable. LIS combined with FRET permits observation of the cells immediate response to a sudden shear force.

  5. Time-resolved FRET and PCT in cationic conjugated polymer/dye-labeled DNA complex

    NASA Astrophysics Data System (ADS)

    Kim, Inhong; Kim, Jihoon; Kim, Bumjin; Kang, Mijeong; Woo, Han Young; Kyhm, Kwangseuk

    2011-12-01

    The energy transfer mechanism between cationic conjugated polyelectrolytes and a single stranded DNA labeled with fluorescein was investigated in terms of Förster resonance energy transfer (FRET) and photo-induced charge transfer (PCT) by time-resolved fluorescence. Both FRET and PCT rate efficiencies were obtained by phenomenological coupled rate equations, which are in excellent agreement with experiments. We found the total energy transfer in the complex is maximized as a consequence of FRET and PCT at an optimum distance 32.7Å.

  6. High-temperature, high-frequency fretting fatigue of a single crystal nickel alloy

    NASA Astrophysics Data System (ADS)

    Matlik, John Frederick

    Fretting is a structural damage mechanism arising from a combination of wear, corrosion, and fatigue between two nominally clamped surfaces subjected to an oscillatory loading. A critical location for fretting induced damage has been identified at the blade/disk and blade/damper interfaces of gas turbine engine turbomachinery and space propulsion components. The high-temperature, high-frequency loading environment seen by these components lead to severe stress gradients at the edge-of-contact that could potentially foster crack growth leading to component failure. These contact stresses drive crack nucleation in fretting and are very sensitive to the geometry of the contacting bodies, the contact loads, materials, temperature, and contact surface tribology (friction). To diagnose the threat that small and relatively undetectable fretting fatigue cracks pose to damage tolerance and the ensuing structural integrity of aerospace components, a strong motivation exists to develop a quantitative mechanics based understanding of fretting crack nucleation in advanced aerospace alloys. In response to this need, the objective of this work is to characterize the fretting behavior exhibited by a polycrystalline/single crystal nickel contact subjected to elevated frequency and temperature. The effort to meet this objective is two fold: (1) to develop a well-characterized experimental fretting rig to investigate fretting behavior of advanced aerospace alloys at high frequency and high temperature, and (2) to develop the associated contact modeling tools for calculating contact stresses given in-situ experimentally measured remote contact loads. By coupling the experimental results and stress analysis, this effort aims to correlate the fretting crack nucleation behavior with the local contact stresses calculated from the devised three dimensional, anisotropic, dissimilar material contact model. The experimental effort is first motivated by a survey of recent fretting issues and

  7. Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins.

    PubMed

    Lan, Tien-Hung; Wu, Guangyu; Lambert, Nevin A

    2015-08-14

    Diffusion can enhance Förster resonance energy transfer (FRET) when donors or acceptors diffuse distances that are similar to the distances separating them during the donor's excited state lifetime. Lanthanide donors remain in the excited state for milliseconds, which makes them useful for time-resolved FRET applications but also allows time for diffusion to enhance energy transfer. Here we show that diffusion dramatically enhances FRET between membrane proteins labeled with lanthanide donors. This phenomenon complicates interpretation of experiments that use long-lived donors to infer association or proximity of mobile membrane proteins, but also offers a method of monitoring diffusion in membrane domains in real time in living cells.

  8. Study of fretting wear of Al-Fe-Ce-SiC composites

    SciTech Connect

    Bai, M.; Xue, Q.

    1996-07-15

    Due to adoption of rapid solidification technology (RST) in conjunction with powder metallurgy processing, aluminum matrix composites (AMC) offer significant improvements in density, strength, stiffness, fracture resistance, and/or higher use temperature which translates into improved aerospace vehicle performance. Fretting wear is a wear phenomena occurring between two surfaces having oscillatory relative motion of small amplitude. The objective of this paper was to investigate the effects of SiC reinforcement and fretting parameters on the fretting wear resistance of Al-7.8Fe-6.1Ce (AFC) alloys and to clarify the mechanism of material removal.

  9. Detection and Quantification of Intracellular Signaling Using FRET-Based Biosensors and High Content Imaging.

    PubMed

    Halls, Michelle L; Poole, Daniel P; Ellisdon, Andrew M; Nowell, Cameron J; Canals, Meritxell

    2015-01-01

    Förster resonance energy transfer (FRET) biosensors represent invaluable tools to detect the spatiotemporal context of second messenger production and intracellular signaling that cannot be attained using traditional methods. Here, we describe a detailed protocol for the use of high content imaging in combination with FRET biosensors to assess second messenger production and intracellular signaling in a time-effective manner. We use four different FRET biosensors to measure cAMP levels, kinase (ERK and PKC), and GTPase activity. Importantly, we provide the protocols to express and measure these sensors in a variety of model cell lines and primary dorsal root ganglia neurons. PMID:26260599

  10. Laser-induced shockwave paired with FRET: a method to study cell signaling.

    PubMed

    Gomez-Godinez, Veronica; Preece, Daryl; Shi, Linda; Khatibzadeh, Nima; Rosales, Derrick; Pan, Yijia; Lei, Lie; Wang, Yingxiao; Berns, Michael W

    2015-03-01

    Cells within the body are subject to various forces; however, the details concerning the way in which cells respond to mechanical stimuli are not well understood. We demonstrate that laser-induced shockwaves (LIS) combined with biosensors based on fluorescence resonance energy transfer (FRET) is a promising new approach to study biological processes in single live cells. As "proof-of-concept," using a FRET biosensor, we show that in response to LIS, cells release intracellular calcium. With the parameters used, cells retain their morphology and remain viable. LIS combined with FRET permits observation of the cells immediate response to a sudden shear force. PMID:25639252

  11. Correlating Calmodulin Landscapes with Chemical Catalysis in Neuronal Nitric Oxide Synthase using Time-Resolved FRET and a 5-Deazaflavin Thermodynamic Trap

    PubMed Central

    2016-01-01

    A major challenge in enzymology is the need to correlate the dynamic properties of enzymes with, and understand the impact on, their catalytic cycles. This is especially the case with large, multicenter enzymes such as the nitric oxide synthases (NOSs), where the importance of dynamics has been inferred from a variety of structural, single-molecule, and ensemble spectroscopic approaches but where motions have not been correlated experimentally with mechanistic steps in the reaction cycle. Here we take such an approach. Using time-resolved spectroscopy employing absorbance and Förster resonance energy transfer (FRET) and exploiting the properties of a flavin analogue (5-deazaflavin mononucleotide (5-dFMN)) and isotopically labeled nicotinamide coenzymes, we correlate the timing of CaM structural changes when bound to neuronal nitric oxide synthase (nNOS) with the nNOS catalytic cycle. We show that remodeling of CaM occurs early in the electron transfer sequence (FAD reduction), not at later points in the reaction cycle (e.g., FMN reduction). Conformational changes are tightly correlated with FAD reduction kinetics and reflect a transient “opening” and then “closure” of the bound CaM molecule. We infer that displacement of the C-terminal tail on binding NADPH and subsequent FAD reduction are the likely triggers of conformational change. By combining the use of cofactor/coenzyme analogues and time-resolved FRET/absorbance spectrophotometry, we show how the reaction cycles of complex enzymes can be simplified, enabling a detailed study of the relationship between protein dynamics and reaction cycle chemistry—an approach that can also be used with other complex multicenter enzymes. PMID:27563493

  12. Mammary Analogue Secretory Carcinoma.

    PubMed

    Stevens, Todd M; Parekh, Vishwas

    2016-09-01

    Mammary analogue secretory carcinoma (MASC) is a recently described salivary gland tumor that shares the same histologic appearance and ETV6 gene (12p13) rearrangement as secretory carcinoma of the breast. Prior to its recognition, MASC cases were commonly labeled acinic cell carcinoma and adenocarcinoma, not otherwise specified. Despite distinctive histologic features, MASC may be difficult to distinguish from other salivary gland tumors, in particular zymogen-poor acinic cell carcinoma and low-grade salivary duct carcinoma. Although characteristic morphologic and immunohistochemical features form the basis of a diagnosis of MASC, the presence of an ETV6-NTRK3 gene fusion is confirmatory. Given its recent recognition the true prognostic import of MASC is not yet clearly defined. PMID:27575269

  13. FRET-based real-time DNA microarrays.

    PubMed

    Hassibi, Arjang; Vikalo, Haris; Riechmann, José Luis; Hassibi, Babak

    2012-01-01

    We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e., real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation, washing artifacts, microarray spot-to-spot variations, and other intensity-affecting impediments. We demonstrate in both theory and practice that the time-constant of target capturing is inversely proportional to the concentration of the target analyte, which we take advantage of as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to experimentally validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays. PMID:22130990

  14. Mars Global Surveyor observations of Martian fretted terrain

    USGS Publications Warehouse

    Carr, M.H.

    2001-01-01

    The Martian fretted terrain between latitudes 30?? and 50?? N and between 315?? and 360?? W has been reexamined in light of new Mars Orbiter Camera (MOC) and Mars Orbiter Laser Altimeter (MOLA) data from Mars Global Surveyor. Much of the terrain in the 30??-50?? latitude belt in both hemispheres has a characteristic stippled or pitted texture at MOC (1.5 m) scale. The texture appears to result from partial removal of a formerly smooth, thin deposit as a result of sublimation and deflation. A complex history of deposition and exhumation is indicated by remnants of a former, thicker cover of layered deposits. In some hollows and on some slopes, particularly those facing the pole, are smooth textured deposits outlined by an outward facing escarpment. Throughout the study area are numerous escarpments with debris flows at their base. The escarpments typically have slopes in the 20??-30?? range. At the base of the escarpment is commonly a deposit with striae oriented at right angles to the escarpment. Outside this deposit is the main debris apron with a surface that typically slopes 2??-3?? and complex surface textures suggestive of compression, sublimation, and deflation. The presence of undeformed impact craters indicates that the debris flows are no longer forming. Fretted valleys contain lineated fill and are poorly graded. They likely form from fluvial valleys that were initially like those elsewhere on the planet but were subsequently widened and filled by the same mass-wasting processes that formed the debris aprons. Slope reversals indicate that downvalley flow of the lineated fill is minor. The ubiquitous presence of breaks in slope formed by mass wasting and the complex surface textures that result from mass wasting, deflation, and sublimation decreases the recognizability of the shorelines formerly proposed for this area.

  15. Synthesis Structure and Imaging of Oligodeoxyribonucleotides with Tellurium-nucleobase Derivatization

    SciTech Connect

    J Sheng; A Hassan; W Zhang; J Zhou; B Xu; A Soares; Z Huang

    2011-12-31

    We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.

  16. Synthesis, structure and imaging of oligodeoxyribonucleotides with tellurium-nucleobase derivatization

    SciTech Connect

    Sheng, J.; Soares, A.; Hassan, A. E. A.; Zhang, W.; Zhou, J.; Xu, B.; Huang, Z.

    2011-05-01

    We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.

  17. New size-expanded RNA nucleobase analogs: A detailed theoretical study

    NASA Astrophysics Data System (ADS)

    Zhang, Laibin; Zhang, Zhenwei; Ren, Tingqi; Tian, Jianxiang; Wang, Mei

    2015-04-01

    Fluorescent nucleobase analogs have attracted much attention in recent years due to their potential applications in nucleic acids research. In this work, four new size-expanded RNA base analogs were computationally designed and their structural, electronic, and optical properties are investigated by means of DFT calculations. The results indicate that these analogs can form stable Watson-Crick base pairs with natural counterparts and they have smaller ionization potentials and HOMO-LUMO gaps than natural ones. Particularly, the electronic absorption spectra and fluorescent emission spectra are calculated. The calculated excitation maxima are greatly red-shifted compared with their parental and natural bases, allowing them to be selectively excited. In gas phase, the fluorescence from them would be expected to occur around 526, 489, 510, and 462 nm, respectively. The influences of water solution and base pairing on the relevant absorption spectra of these base analogs are also examined.

  18. DNA-nucleobases: gate dielectric/passivation layer for flexible GFET-based sensor applications

    NASA Astrophysics Data System (ADS)

    Williams, Adrienne D.; Ouchen, Fahima; Kim, Steve S.; Elhamri, Said; Naik, Rajesh R.; Grote, James

    2015-09-01

    The main goal of this research was to maintain the bulk charge carrier mobility of graphene, after deposition of the gate dielectric layer used for making transistor devices. The approach was introducing a thin film of deoxyribonucleic acid (DNA) nucleobase purine guanine, deposited by physical vapor deposition (PVD), onto layers of graphene that were transferred onto various flexible substrates. Several test platforms were fabricated with guanine as a standalone gate dielectric, as the control, and guanine as a passivation layer between the graphene and PMMA. It was found that the bulk charge carrier mobility of graphene was best maintained and most stable using guanine as a passivation layer between the graphene and PMMA. Other transport properties, such as charge carrier concentration, conductivity type and electrical resistivity were investigated as well. This is an important first step to realizing high performance graphene-based transistors that have potential use in bio and environmental sensors, computer-processing and electronics.

  19. Synthesis of nucleobase-functionalized carbon nanotubes and their hybridization with single-stranded DNA.

    PubMed

    Hwu, Jih Ru; Kapoor, Mohit; Li, Rou-Ying; Lin, Yung-Chieh; Horng, Jia-Cherng; Tsay, Shwu-Chen

    2014-12-01

    For the first time ssDNA (25-aptamer of mixed dA, dT, dG, and dC) was wrapped around functionalized single-walled carbon nanotubes (SWCNTs), whose external surfaces were attached to multiple triazole-(ethylene glycol)-dA ligands. This method of hybridization involved the formation of hydrogen bonds between dT of ssDNA and dA of functionalized SWCNTs. It deviates from the reported π-π stacking between the nucleobases of DNA and the external sidewalls of nanotubes. The structural properties of the functionalized SWCNTs and its ssDNA complex were characterized by spectroscopic (including CD and Raman), thermogravimetric, and microscopic (TEM) methods. The results thus obtained establish a new platform of DNA delivery by use of nanotubes as a new vehicle with great potential in biomedical applications and drug development.

  20. Nanostructured gel scaffolds for osteogenesis through biological assembly of biopolymers via specific nucleobase pairing.

    PubMed

    Fan, Ming; Yan, Jingxuan; Tan, Huaping; Ben, Dandan; He, Qiuling; Huang, Zhongwei; Hu, Xiaohong

    2014-11-01

    Biopolymer-based gel scaffolds have great potential in the field of tissue regenerative medicine. In this work, a nanostructured biopolymer gel scaffold via specific pairing of functionalized nucleobases was developed for specifically targeted drug delivery and in vitro osteogenesis. The biopolymer gel system was established by the Watson-Crick base pairing between thymine and adenine via the hydrogen bonding. As gel scaffold precursors, opposite charged polysaccharide derivatives, e.g. quaternized cellulose and heparin, could be additionally crosslinked by extra electrostatic interactions. The potential application of this gel scaffold in bone tissue engineering was confirmed by encapsulation behavior of osteoblasts. In combination with cell growth factor, e.g. bone morphogenetic protein, the nanostructured gel scaffold exhibited beneficial effects on osteoblast activity and differentiation, which suggested a promising future for local treatment of pathologies involving bone loss.

  1. Carbon nanotube-nucleobase hybrids: nanorings from uracil-modified single-walled carbon nanotubes.

    PubMed

    Singh, Prabhpreet; Toma, Francesca Maria; Kumar, Jitendra; Venkatesh, V; Raya, Jesus; Prato, Maurizio; Verma, Sandeep; Bianco, Alberto

    2011-06-01

    Single-walled carbon nanotubes (SWCNTs) have been covalently functionalized with uracil nucleobase. The hybrids have been characterized by using complementary spectroscopic and microscopic techniques including solid-state NMR spectroscopy. The uracil-functionalized SWCNTs are able to self-assemble into regular nanorings with a diameter of 50-70 nm, as observed by AFM and TEM. AFM shows that the rings do not have a consistent height and thickness, which indicates that they may be formed by separate bundles of CNTs. The simplest model for the nanoring formation likely involves two bundles of CNTs interacting with each other via uracil-uracil base-pairing at both CNT ends. These nanorings can be envisaged for the development of advanced electronic circuits.

  2. Electron Detachment as a Probe of Intrinsic Nucleobase Dynamics in Dianion-Nucleobase Clusters: Photoelectron Spectroscopy of the Platinum II Cyanide Dianion Bound to Uracil, Thymine, Cytosine and Adenine

    SciTech Connect

    Sen, Ananya; Hou, Gao-Lei; Wang, Xue B.; Dessent, Caroline

    2015-08-05

    We report the first low-temperature photodetachment photoelectron spectra of isolated gas-phase complexes of the platinum II cyanide dianion bound to nucleobases. These systems are model systems for understanding platinum-complex photodynamic therapies, and knowledge of the intrinsic photodetachment properties is crucial for understanding their broader photophysical properties. Well-resolved, distinct peaks are observed in the spectra consistent with the complexes where the Pt(CN)42- moiety is largely intact. The adiabatic electron detachment energies for the dianion-nucleobase complexes are measured to be between 2.39-2.46 eV. The magnitudes of the repulsive Coulomb barriers of the complexes are estimated to be between 1.9 and 2.1 eV, values that are lower than for the bare Pt(CN)42- dianion as a result of charge solvation by the nucleobases. In addition to the resolved spectral features, broad featureless bands indicative of delayed electron detachment are observed in the 193 nm photodetachment spectra of the four nucleobase-dianion complexes, and also in the 266 nm spectra of the Pt(CN)42-∙thymine and Pt(CN)42-∙adenine complexes. The selective excitation of these features in the 266 nm spectra is attributed to one-photon excitation of [Pt(CN)42-∙T]* and [Pt(CN)42-∙A]* long-lived excited states that can effectively couple to the electron detachment continuum, producing strong electron detachment signals. We attribute the resonant electron detachment bands observed here for Pt(CN)42-∙T and Pt(CN)42-∙A but not for Pt(CN)42-∙U and Pt(CN)42-∙C to fundamental differences in the individual nucleobase photophysics following 266 nm excitation. This indicates that the Pt(CN)42- dianion in the Pt(CN)42-∙M clusters can be viewed as a “dynamic tag” which has the propensity to emit electrons when the attached nucleobase disaplys a long-lived excited state.

  3. NASA/ESMD Analogue Mission Plans

    NASA Technical Reports Server (NTRS)

    Hoffman, Stephen J.

    2007-01-01

    A viewgraph presentation exploring Earth and its analogues is shown. The topics include: 1) ESMD Goals for the Use of Earth Analogues; 2) Stakeholders Summary; 3) Issues with Current Analogue Situation; 4) Current state of Analogues; 5) External Implementation Plan (Second Step); 6) Recent Progress in Utilizing Analogues; 7) Website Layout Example-Home Page; 8) Website Layout Example-Analogue Site; 9) Website Layout Example-Analogue Mission; 10) Objectives of ARDIG Analog Initiatives; 11) Future Plans; 12) Example: Cold-Trap Sample Return; 13) Example: Site Characterization Matrix; 14) Integrated Analogue Studies-Prerequisites for Human Exploration; and 15) Rating Scale Definitions.

  4. Asymmetric Synthesis and Binding Study of New Long-Chain HPA-12 Analogues as Potent Ligands of the Ceramide Transfer Protein CERT.

    PubMed

    Ďuriš, Andrej; Daïch, Adam; Santos, Cécile; Fleury, Laurence; Ausseil, Frédéric; Rodriguez, Frédéric; Ballereau, Stéphanie; Génisson, Yves; Berkeš, Dušan

    2016-05-01

    A series of 12 analogues of the Cer transfer protein (CERT) antagonist HPA-12 with long aliphatic chains were prepared as their (1R,3S)-syn and (1R,3R)-anti stereoisomers from pivotal chiral oxoamino acids. The enantioselective access to these intermediates as well as their ensuing transformation relied on a practical crystallization-induced asymmetric transformation (CIAT) process. Sonogashira coupling followed by triple bond reduction and thiophene ring hydrodesulfurization (HDS) into the corresponding alkane moieties was then implemented to complete the synthetic routes delivering the targeted HPA-12 analogues in concise 4- to 6-step reaction sequences. Ten compounds were evaluated regarding their ability to bind to the CERT START domain by using the recently developed time-resolved FRET-based homogeneous (HTR-FRET) binding assay. The introduction of a lipophilic appendage on the phenyl moiety led to an overall 10- to 1000-fold enhancement of the protein binding, with the highest effect being observed for a n-hexyl residue in the meta position. The importance of the phenyl ring for the activity was indicated by the reduced potency of the 3-deoxyphytoceramide aliphatic analogues. The 1,3-syn stereoisomers were systematically more potent than their 1,3-anti analogues. In silico studies were used to rationalized these trends, leading to a model of protein recognition coherent with the stronger binding of (1R,3S)-syn HPAs.

  5. Quantum dot/methylene blue FRET mediated NIR fluorescent nanomicelles with large Stokes shift for bioimaging.

    PubMed

    Li, Li; Liu, Jianbo; Yang, Xiaohai; Peng, Zhihong; Liu, Wei; Xu, Jianguo; Tang, Jinlu; He, Xiaoxiao; Wang, Kemin

    2015-10-01

    Here we present a novel large Stokes shifting NIR fluorescent nanomicelle through the encapsulation of a quantum dot/methylene blue FRET pair, which is employed as an excellent contrast reagent for NIR fluorescence bioimaging. PMID:26267557

  6. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

    PubMed Central

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N. P.; Riedmayr, Lisa M.; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S.; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  7. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments.

    PubMed

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N P; Riedmayr, Lisa M; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  8. A new pair for inter- and intra-molecular FRET measurement

    SciTech Connect

    Yang Xiaofei; Xu Pingyong; Xu Tao . E-mail: xutao@sun5.ibp.ac.cn

    2005-05-13

    Fluorescence resonance energy transfer between mutant green fluorescent proteins provides powerful means to monitor in vivo protein-protein proximity and intracellular signaling. However, the current widely applied FRET pair of this class (CFP/YFP) requires excitation by expensive UV lasers, thereby hindering FRET imaging on many confocal microscopes. Further challenges arise from the large spectral overlap of CFP/YFP emission. Another FRET pair GFP/DsRed could obviate such limitations. However, the use of DsRed as a FRET acceptor is hampered by several critical problems, including a slow and incomplete maturation and obligate tetramerization. A tandem dimer mutant of DsRed (TDimer2) has similar spectral properties as those of DsRed. The rapid maturation and non-oligomerization make TDimer2 a promising substitute for DsRed in FRET experiments. Here, we have explored the possibility of using TDimer2 as a FRET acceptor for the donor EGFP. FRET was demonstrated between the EGFP-TDimer2 chimeric fusion protein. By substituting CFP/YFP in the Ca{sup 2+}-sensor cameleon with EGFP/TDimer2, dynamic changes in cytosolic free Ca{sup 2+} concentrations were observed with 488 nm excitation under conventional wide-field microscopy. The EGFP/TDimer2 pair was further successfully employed to monitor inter-molecular interaction between Syntaxin and SNAP25. These results reveal EGFP/TDimer2 as a promising FRET pair in monitoring intra-molecular conformation change as well as inter-molecular interaction.

  9. Ultrafast FRET in a room temperature ionic liquid microemulsion: a femtosecond excitation wavelength dependence study.

    PubMed

    Adhikari, Aniruddha; Das, Dibyendu Kumar; Sasmal, Dibyendu Kumar; Bhattacharyya, Kankan

    2009-04-23

    Fluorescence resonance energy transfer (FRET) from coumarin 480 (C480) to rhodamine 6G (R6G) is studied in a room temperature ionic liquid (RTIL) microemulsion by picosecond and femtosecond emission spectroscopy. The microemulsion is comprised of the RTIL 1-pentyl-3-methylimidazolium tetraflouroborate, [pmim][BF4], in TX-100/ benzene. We have studied the microemulsion with and without water. The time constants of FRET were obtained from the risetime of the acceptor (R6G) emission. In the RTIL microemulsion, FRET occurs on multiple time scales: 1, 250, and 3900 ps. In water containing RTIL microemulsion, the rise components are 1.5, 250, and 3900 ps. The 1 and 1.5 ps components are assigned to FRET at a close contact of donor and acceptor (RDA approximately 12 A). This occurs within the highly polar (RTIL/water) pool of the microemulsion. With increase in the excitation wavelength (lambdaex) from 375 to 435 nm, the relative contribution of the ultrafast component of FRET (1 ps) increases from 4% to 100% in the RTIL microemulsion and 12% to 100% in the water containing RTIL microemulsion. It is suggested that at lambdaex = 435 nm, mainly the highly polar RTIL pool is probed where FRET is very fast due to the close proximity of the donor and the acceptor. The very long 3900 ps (RDA approximately 45 A) component may arise from FRET from a donor in the outer periphery of the microemulsion to an acceptor in the polar RTIL pool. The 250 ps component (RDA approximately 29 A) is assigned to FRET from a donor inside the surfactant chains. PMID:19127996

  10. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

    PubMed

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections.

  11. A Simple, Rapid and Sensitive FRET Assay for Botulinum Neurotoxin Serotype B Detection

    PubMed Central

    Li, Xuechen; Chen, Sheng

    2014-01-01

    Botulinum neurotoxins (BoNTs), the most potent naturally-occurring neurotoxins known to humans, comprise seven distinct serotypes (BoNT/A-G), each of which exhibits unique substrate specificity. Many methods have been developed for BoNT detection, in particular for BoNT/A, with various complexity and sensitivity, while substrate based FRET assay is considered as the most widely used approach due to its simplicity and sensitivity. In this study, we designed a vesicle-associated membrane protein 2 (VAMP2) based FRET assay based on the understanding of the VAMP2 and light chain/B (LC/B) interactions in our previous studies. The current design constituted the shortest peptide, VAMP2 (63–85), with FRET dyes (EDAN and Dabcyl) labelled at position 76 and 85, respectively, which showed minimal effect on VAMP2 substrate catalysis by LC/B and therefore enhanced the sensitivity of the assay. The FRET peptide, designated as FVP-B, was specific to LC/B, with a detection sensitivity as low as ∼20 pM in 2 h. Importantly, FVP-B showed the potential to be scaled up and used in high throughput screening of LC/B inhibitor. The currently developed FRET assay is one of the most economic and rapid FRET assays for LC/B detection. PMID:25437190

  12. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

    PubMed

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections. PMID:26908592

  13. Formation of nucleobases from formamide in the presence of iron oxides: implication in chemical evolution and origin of life.

    PubMed

    Shanker, Uma; Bhushan, Brij; Bhattacharjee, G; Kamaluddin

    2011-04-01

    Simple compounds like HCN, which have one C and one N, are proposed as the probable precursors for biomonomers. Formamide, a hydrolysis product of HCN, is known as the precursor of various biologically important compounds, for example, nucleobases (purines and pyrimidines). In this paper, we report our results on the synthesis of nucleobases, adenine, cytosine, purine, 9-(hydroxyacetyl) purine, and 4(3H)-pyrimidinone from formamide, using iron oxide (hematite) and oxide hydroxides (goethite and akaganeite) as a catalyst. Goethite and hematite produced purine in higher yield. The products formed were characterized by high-performance liquid chromatography and electrospray ionization mass spectrometry techniques. Results of our study reveal that iron oxides might have worked as efficient prebiotic catalysts.

  14. A Search for Amino Acids and Nucleobases in the Martian Meteorite Roberts Massif 04262 Using Liquid Chromatography-Mass Spectrometry

    NASA Technical Reports Server (NTRS)

    Callahan, Michael P.; Burton, Aaron S.; Elsila, Jamie E.; Baker, Eleni M.; Smith, Karen E.; Glavin, Daniel P.; Dworkin, Jason P.

    2013-01-01

    The investigation into whether Mars contains signatures of past or present life is of great interest to science and society. Amino acids and nucleobases are compounds that are essential for all known life on Earth and are excellent target molecules in the search for potential Martian biomarkers or prebiotic chemistry. Martian meteorites represent the only samples from Mars that can be studied directly in the laboratory on Earth. Here, we analyzed the amino acid and nucleobase content of the shergottite Roberts Massif (RBT) 04262 using liquid chromatography-mass spectrometry. We did not detect any nucleobases above our detection limit in formic acid extracts; however, we did measure a suite of protein and nonprotein amino acids in hot-water extracts with high relative abundances of beta-alanine and gamma-amino-eta-butyric acid. The presence of only low (to absent) levels of several proteinogenic amino acids and a lack of nucleobases suggest that this meteorite fragment is fairly uncontaminated with respect to these common biological compounds. The distribution of straight-chained amine-terminal eta-omega-amino acids in RBT 04262 resembled those previously measured in thermally altered carbonaceous meteorites. A carbon isotope ratio of -24(0/00) +/- 6(0/00) for beta-alanine in RBT 04262 is in the range of reduced organic carbon previously measured in Martian meteorites (Steele et al. 2012). The presence of eta-omega-amino acids may be due to a high temperature Fischer-Tropschtype synthesis during igneous processing on Mars or impact ejection of the meteorites from Mars, but more experimental data are needed to support these hypotheses.

  15. A search for amino acids and nucleobases in the Martian meteorite Roberts Massif 04262 using liquid chromatography-mass spectrometry

    NASA Astrophysics Data System (ADS)

    Callahan, Michael P.; Burton, Aaron S.; Elsila, Jamie E.; Baker, Eleni M.; Smith, Karen E.; Glavin, Daniel P.; Dworkin, Jason P.

    2013-05-01

    The investigation into whether Mars contains signatures of past or present life is of great interest to science and society. Amino acids and nucleobases are compounds that are essential for all known life on Earth and are excellent target molecules in the search for potential Martian biomarkers or prebiotic chemistry. Martian meteorites represent the only samples from Mars that can be studied directly in the laboratory on Earth. Here, we analyzed the amino acid and nucleobase content of the shergottite Roberts Massif (RBT) 04262 using liquid chromatography-mass spectrometry. We did not detect any nucleobases above our detection limit in formic acid extracts; however, we did measure a suite of protein and nonprotein amino acids in hot-water extracts with high relative abundances of β-alanine and γ-amino-n-butyric acid. The presence of only low (to absent) levels of several proteinogenic amino acids and a lack of nucleobases suggest that this meteorite fragment is fairly uncontaminated with respect to these common biological compounds. The distribution of straight-chained amine-terminal n-ω-amino acids in RBT 04262 resembled those previously measured in thermally altered carbonaceous meteorites (Burton et al. 2012; Chan et al. 2012). A carbon isotope ratio of -24‰ ± 6‰ for β-alanine in RBT 04262 is in the range of reduced organic carbon previously measured in Martian meteorites (Steele et al. 2012). The presence of n-ω-amino acids may be due to a high temperature Fischer-Tropsch-type synthesis during igneous processing on Mars or impact ejection of the meteorites from Mars, but more experimental data are needed to support these hypotheses.

  16. Three-Dimensional Reconstruction of Three-Way FRET Microscopy Improves Imaging of Multiple Protein-Protein Interactions

    PubMed Central

    Scott, Brandon L.; Hoppe, Adam D.

    2016-01-01

    Fluorescence resonance energy transfer (FRET) microscopy is a powerful tool for imaging the interactions between fluorescently tagged proteins in two-dimensions. For FRET microscopy to reach its full potential, it must be able to image more than one pair of interacting molecules and image degradation from out-of-focus light must be reduced. Here we extend our previous work on the application of maximum likelihood methods to the 3-dimensional reconstruction of 3-way FRET interactions within cells. We validated the new method (3D-3Way FRET) by simulation and fluorescent protein test constructs expressed in cells. In addition, we improved the computational methods to create a 2-log reduction in computation time over our previous method (3DFSR). We applied 3D-3Way FRET to image the 3D subcellular distributions of HIV Gag assembly. Gag fused to three different FPs (CFP, YFP, and RFP), assembled into viral-like particles and created punctate FRET signals that become visible on the cell surface when 3D-3Way FRET was applied to the data. Control experiments in which YFP-Gag, RFP-Gag and free CFP were expressed, demonstrated localized FRET between YFP and RFP at sites of viral assembly that were not associated with CFP. 3D-3Way FRET provides the first approach for quantifying multiple FRET interactions while improving the 3D resolution of FRET microscopy data without introducing bias into the reconstructed estimates. This method should allow improvement of widefield, confocal and superresolution FRET microscopy data. PMID:27023704

  17. Formamide-based synthesis of nucleobases by metal(II) octacyanomolybdate(IV): implication in prebiotic chemistry.

    PubMed

    Kumar, Anand; Sharma, Rachana; Kamaluddin

    2014-09-01

    We propose that double metal cyanides that formed in primeval seas might have played a vital role in chemical evolution and the origin of life. An array of metal octacyanomolybdates (MOCMos) has been synthesized, and their role as catalyst in the formation of nucleobases from formamide has been studied. Formamide, a hydrolysis product of HCN, was taken as starting material for the formation of nucleobases. Recent studies support the presence of formamide on some celestial bodies. Metal octacyanomolybdates, MOCMos (M = Mn, Fe, Co, Ni, Cu, Zn, Cd), are found to be highly efficient catalysts in the conversion of formamide into different nucleobases. Neat formamide is converted to purine, 4(3H)-pyrimidinone, cytosine, adenine, 9-(hydroxyacetyl)-purine, and thymine in good yield when using MOCMos. The products formed were characterized by high-performance liquid chromatography and electrospray ionization mass spectrometry techniques. The results of our study show that insoluble double metal cyanides might have acted as efficient catalysts in the synthesis of various biologically important compounds (e.g., purines, pyrimidines) under primeval seas on Earth or elsewhere in our solar system.

  18. Emergent functionality of nucleobase radical cations in duplex DNA: prediction of reactivity using qualitative potential energy landscapes.

    PubMed

    Joseph, Joshy; Schuster, Gary B

    2006-05-10

    The one-electron oxidation of a series of DNA oligonucleotides was examined. Each oligomer contains a covalently linked anthraquinone (AQ) group. Irradiation of the AQ group with near-UV light results in a one-electron oxidation of the DNA that generates a radical cation (electron "hole"). The radical cation migrates through the DNA by a hopping mechanism and is trapped by reaction with water or molecular oxygen, which results in chemical reaction at particular nucleobases. This reaction is revealed as strand cleavage when the irradiated oligonucleotide is treated with piperidine. The specific oligomers examined reveal the existence of three categories of nucleobase sequences: charge shuttles, charge traps, and barriers to charge migration. The characterization of a sequence is not independent of the identity of other sequences in the oligonucleotide, and for this reason, the function of a particular sequence emerges from an analysis of the entire structure. Qualitative potential energy landscapes are introduced as a tool to assist in the rationalization and prediction of the reactions of nucleobases in oxidized DNA. PMID:16669676

  19. Determination of DNA adducts by combining acid-catalyzed hydrolysis and chromatographic analysis of the carcinogen-modified nucleobases.

    PubMed

    Leung, Elvis M K; Deng, Kailin; Wong, Tin-Yan; Chan, Wan

    2016-01-01

    The commonly used method of analyzing carcinogen-induced DNA adducts involves the hydrolysis of carcinogen-modified DNA samples by using a mixture of enzymes, followed by (32)P-postlabeling or liquid chromatography (LC)-based analyses of carcinogen-modified mononucleotides/nucleosides. In the present study, we report the development and application of a new approach to DNA adduct analysis by combining the H(+)/heat-catalyzed release of carcinogen-modified nucleobases and the use of LC-based methods to analyze DNA adducts. Results showed that heating the carcinogen-modified DNA samples at 70 °C for an extended period of 4 to 6 h in the presence of 0.05% HCl can efficiently induce DNA depurination, releasing the intact carcinogen-modified nucleobases for LC analyses. After optimizing the hydrolysis conditions, DNA samples with C8- and N (2) -modified 2'-deoxyguanosine, as well as N (6) -modified 2'-deoxyadenosine, were synthesized by reacting DNA with 1-nitropyrene, acetaldehyde, and aristolochic acids, respectively. These samples were then hydrolyzed, and the released nucleobase adducts were analyzed using LC-based analytical methods. Analysis results demonstrated a dose-dependent release of target DNA adducts from carcinogen-modified DNA samples, indicating that the developed H(+)/heat-catalyzed hydrolysis method was quantitative. Comparative studies with enzymatic digestion method on carcinogen-modified DNA samples revealed that the two hydrolysis methods did not yield systematically different results.

  20. Formamide-based synthesis of nucleobases by metal(II) octacyanomolybdate(IV): implication in prebiotic chemistry.

    PubMed

    Kumar, Anand; Sharma, Rachana; Kamaluddin

    2014-09-01

    We propose that double metal cyanides that formed in primeval seas might have played a vital role in chemical evolution and the origin of life. An array of metal octacyanomolybdates (MOCMos) has been synthesized, and their role as catalyst in the formation of nucleobases from formamide has been studied. Formamide, a hydrolysis product of HCN, was taken as starting material for the formation of nucleobases. Recent studies support the presence of formamide on some celestial bodies. Metal octacyanomolybdates, MOCMos (M = Mn, Fe, Co, Ni, Cu, Zn, Cd), are found to be highly efficient catalysts in the conversion of formamide into different nucleobases. Neat formamide is converted to purine, 4(3H)-pyrimidinone, cytosine, adenine, 9-(hydroxyacetyl)-purine, and thymine in good yield when using MOCMos. The products formed were characterized by high-performance liquid chromatography and electrospray ionization mass spectrometry techniques. The results of our study show that insoluble double metal cyanides might have acted as efficient catalysts in the synthesis of various biologically important compounds (e.g., purines, pyrimidines) under primeval seas on Earth or elsewhere in our solar system. PMID:25192494

  1. Fretting and Corrosion in Modular Shoulder Arthroplasty: A Retrieval Analysis.

    PubMed

    Eckert, Johannes A; Mueller, Ulrike; Jaeger, Sebastian; Panzram, Benjamin; Kretzer, J Philippe

    2016-01-01

    Tribocorrosion in taper junctions of retrieved anatomic shoulder arthroplasty implants was evaluated. A comparison of the tribocorrosion between cobalt-chromium and titanium alloy stems was conducted and the observations were correlated with the individual's clinical data. Adverse effects caused by metal debris and subsequent elevated serum metal ion levels are frequently reported in total hip arthroplasty. In total shoulder arthroplasty, to date only a small number of retrieval analyses are available and even fewer address the issue of tribocorrosion at the taper junctions. A total of 36 retrieved hemiarthroplasties and total shoulder arthroplasties were assessed using the modified Goldberg score. The prevalence of fretting and corrosion was confirmed in this cohort. Titanium stems seem to be more susceptible to damage caused by tribocorrosion than cobalt-chromium stems. Furthermore, stemless designs offered less tribocorrosion at the taper junction than stemmed designs. A weak correlation between time to revision and increased levels of tribocorrosion was seen. Whether or not tribocorrosion can lead to adverse clinical reactions and causes failure of shoulder arthroplasties remains to be examined. PMID:27433471

  2. Interference Assembly and Fretting Wear Analysis of Hollow Shaft

    PubMed Central

    2014-01-01

    Fretting damage phenomenon often appears in the interference fit assembly. The finite element model of hollow shaft and shaft sleeve was established, and the equivalent stress and contact stress were computed after interference assembly. The assembly body of hollow shaft and shaft sleeve was in whirling bending load, and the contact status (sticking, sliding, and opening) and the distribution of stress along one typical contact line were computed under different loads, interferences, hollow degrees, friction coefficient, and wear quantity. Judgment formula of contact state was fixed by introducing the corrected coefficient k. The computation results showed that the “edge effect” appears in the contact surface after interference fit. The size of slip zone is unchanged along with the increase of bending load. The greater the interference value, the bigger the wear range. The hollow degree does not influence the size of stick zone but controls the position of the junction point of slip-open. Tangential contact stress increases with the friction coefficient, which has a little effect on normal contact stress. The relationship between open size and wear capacity is approximately linear. PMID:24955422

  3. Fretting and Corrosion in Modular Shoulder Arthroplasty: A Retrieval Analysis

    PubMed Central

    Panzram, Benjamin

    2016-01-01

    Tribocorrosion in taper junctions of retrieved anatomic shoulder arthroplasty implants was evaluated. A comparison of the tribocorrosion between cobalt-chromium and titanium alloy stems was conducted and the observations were correlated with the individual's clinical data. Adverse effects caused by metal debris and subsequent elevated serum metal ion levels are frequently reported in total hip arthroplasty. In total shoulder arthroplasty, to date only a small number of retrieval analyses are available and even fewer address the issue of tribocorrosion at the taper junctions. A total of 36 retrieved hemiarthroplasties and total shoulder arthroplasties were assessed using the modified Goldberg score. The prevalence of fretting and corrosion was confirmed in this cohort. Titanium stems seem to be more susceptible to damage caused by tribocorrosion than cobalt-chromium stems. Furthermore, stemless designs offered less tribocorrosion at the taper junction than stemmed designs. A weak correlation between time to revision and increased levels of tribocorrosion was seen. Whether or not tribocorrosion can lead to adverse clinical reactions and causes failure of shoulder arthroplasties remains to be examined. PMID:27433471

  4. FRET-Assisted Determination of CLN3 Membrane Topology

    PubMed Central

    Ramos-Moreno, Juliana; Ruonala, Mika O.

    2014-01-01

    Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene, which encodes for a putative lysosomal transmembrane protein with thus far undescribed structure and function. Here we investigate the membrane topology of human CLN3 protein with a combination of advanced molecular cloning, spectroscopy, and in silico computation. Using the transposomics cloning method we first created a library of human CLN3 cDNA clones either with a randomly inserted eGFP, a myc-tag, or both. The functionality of the clones was evaluated by assessing their ability to revert a previously reported lysosomal phenotype in immortalized cerebellar granular cells derived from Cln3Δex7/8 mice (CbCln3Δex7/8). The double-tagged clones were expressed in HeLa cells, and FRET was measured between the donor eGFP and an acceptor DyLight547 coupled to a monoclonal α-myc antibody to assess their relative membrane orientation. The data were used together with previously reported experimental data to compile a constrained membrane topology model for hCLN3 using TOPCONS consensus membrane prediction algorithm. Our model with six transmembrane domains and cytosolic N- and C-termini largely agrees with those previously suggested but differs in terms of the transmembrane domain positions as well as in the size of the luminal loops. This finding improves understanding the function of the native hCLN3 protein. PMID:25051496

  5. Time-resolved FRET strategy to screen GPCR ligand library.

    PubMed

    Oueslati, Nadia; Hounsou, Candide; Belhocine, Abderazak; Rodriguez, Thieric; Dupuis, Elodie; Zwier, Jurriaan M; Trinquet, Eric; Pin, Jean-Philippe; Durroux, Thierry

    2015-01-01

    Screening chemical libraries to find specific drugs for G protein-coupled receptors is still of major interest. Indeed, because of their major roles in all physiological functions, G protein-coupled receptors remain major targets for drug development programs. Currently, interest in GPCRs as drug targets has been boosted by the discovery of biased ligands, thus allowing the development of drugs not only specific for one target but also for the specific signaling cascade expected to have the therapeutic effect. Such molecules are then expected to display fewer side effects. To reach such a goal, there is much interest in novel, efficient, simple, and direct screening assays that may help identify any drugs interacting with the target, these being then analyzed for their biased activity. Here, we present an efficient strategy to screen ligands on their binding properties. The method described is based on time-resolved FRET between a receptor and a ligand. This method has already been used to develop new assays called Tag-lite(®) binding assays for numerous G protein-coupled receptors, proving its broad application and its power.

  6. FRETsg: Biomolecular structure model building from multiple FRET experiments

    NASA Astrophysics Data System (ADS)

    Schröder, G. F.; Grubmüller, H.

    2004-04-01

    Fluorescence energy transfer (FRET) experiments of site-specifically labelled proteins allow one to determine distances between residues at the single molecule level, which provide information on the three-dimensional structural dynamics of the biomolecule. To systematically extract this information from the experimental data, we describe a program that generates an ensemble of configurations of residues in space that agree with the experimental distances between these positions. Furthermore, a fluctuation analysis allows to determine the structural accuracy from the experimental error. Program summaryTitle of program: FRETsg Catalogue identifier: ADTU Program obtainable from: CPC Program Library, Queen's University of Belfast, N. Ireland Program summary URL:http://cpc.cs.qub.ac.uk/summaries/ADTU Computer: SGI Octane, Pentium II/III, Athlon MP, DEC Alpha Operating system: Unix, Linux, Windows98/NT/XP Programming language used: ANSI C No. of bits in a word: 32 or 64 No. of processors used: 1 No. of bytes in distributed program, including test data, etc.: 11407 No. of lines in distributed program, including test data, etc.: 1647 Distribution format: gzipped tar file Nature of the physical problem: Given an arbitrary number of distance distributions between an arbitrary number of points in three-dimensional space, find all configurations (set of coordinates) that obey the given distances. Method of solution: Each distance is described by a harmonic potential. Starting from random initial configurations, their total energy is minimized by steepest descent. Fluctuations of positions are chosen to generate distance distribution widths that best fit the given values.

  7. Nucleosome immobilization strategies for single-pair FRET microscopy.

    PubMed

    Koopmans, Wiepke J A; Schmidt, Thomas; van Noort, John

    2008-10-01

    All genomic transactions in eukaryotes take place in the context of the nucleosome, the basic unit of chromatin, which is responsible for DNA compaction. Overcoming the steric hindrance that nucleosomes present for DNA-processing enzymes requires significant conformational changes. The dynamics of these have been hard to resolve. Single-pair Fluorescence Resonance Energy Transfer (spFRET) microscopy is a powerful technique for observing conformational dynamics of the nucleosome. Nucleosome immobilization allows the extension of observation times to a limit set only by photobleaching, and thus opens the possibility of studying processes occurring on timescales ranging from milliseconds to minutes. It is crucial however, that immobilization itself does not introduce artifacts in the dynamics. Here we report on various nucleosome immobilization strategies, such as single-point attachment to polyethylene glycol (PEG) or surfaces coated with bovine serum albumin (BSA), and confinement in porous agarose or polyacrylamide gels. We compare the immobilization specificity and structural integrity of immobilized nucleosomes. A crosslinked star polyethylene glycol coating performs best with respect to tethering specificity and nucleosome integrity, and enables us to reproduce for the first time bulk nucleosome unwrapping kinetics in single nucleosomes without immobilization artifacts.

  8. Single color FRET based measurements of conformational changes of proteins resulting from translocation inside cells.

    PubMed

    Gahl, Robert F; Tekle, Ephrem; Tjandra, Nico

    2014-03-15

    Translocation of proteins to different parts of the cell is necessary for many cellular mechanisms as a means for regulation and a variety of other functions. Identifying how these proteins undergo conformational changes or interact with various partners during these events is critical to understanding how these mechanisms are executed. A protocol is presented that identifies conformational changes in a protein that occur during translocation while overcoming challenges in extracting distance information in very different environments of a living cell. Only two samples are required to be prepared and are observed with one optical setup. Live-cell FRET imaging has been applied to identify conformational changes between two native cysteines in Bax, a member of the Bcl-2 family of proteins that regulates apoptosis. Bax exists in the cytosol and translocates to the mitochondria outer membrane upon apoptosis induction. The distance, r, between the two native cysteines in the cytosolic structure of Bax necessitates the use of a FRET donor-accepter pair with R0~r as the most sensitive probe for identifying structural changes at these positions. Alexa Fluor 546 and Dabcyl, a dark acceptor, were used as FRET pairs - resulting in single color intensity variations of Alexa-546 as a measure of FRET efficiency. An internal reference, conjugated to Bax, was employed to normalize changes in fluorescence intensity of Alexa Fluor 546 due to inherent inhomogeneities in the living cell. This correction allowed the true FRET effects to be measured with increased precision during translocation. Normalization of intensities to the internal reference identified a FRET efficiency of 0.45±0.14 in the cytosol and 0.11±0.20 in the mitochondria. The procedure for the conjugation of the internal reference and FRET probes as well as the data analysis is presented.

  9. Hybridization accompanying FRET event in labeled natural nucleoside-unnatural nucleoside containing chimeric DNA duplexes.

    PubMed

    Bag, Subhendu Sekhar; Das, Suman K; Pradhan, Manoj Kumar; Jana, Subhashis

    2016-09-01

    Förster resonance energy transfer (FRET) is a highly efficient strategy in illuminating the structures, structural changes and dynamics of DNA, proteins and other biomolecules and thus is being widely utilized in studying such phenomena, in designing molecular/biomolecular probes for monitoring the hybridization event of two single stranded DNA to form duplex, in gene detection and in many other sensory applications in chemistry, biology and material sciences. Moreover, FRET can give information about the positional status of chromophores within the associated biomolecules with much more accuracy than other methods can yield. Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene ((TPhen)BDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, (Per)U or (OxoPy)U or (Per)U, forming two stable chimeric DNA duplexes. The pairing selectivity and the thermal duplex stability of the chimeric duplexes are higher than any of the duplexes with natural nucleoside formed. The hybridization results in a Förster resonance energy transfer (FRET) from donor triazolylphenanthrene of (TPhen)BDo to acceptor oxopyrene of (OxoPy)U and/or to perylene chromophore of (Per)U, respectively, in two chimeric DNA duplexes. Therefore, we have established the FRET process in two chimeric DNA duplexes wherein a fluorescently labeled natural nucleoside ((OxoPy)U or (Per)U) paired against an unnatural nucleoside ((TPhen)BDo) without sacrificing the duplex stability and B-DNA conformation. The hybridization accompanying FRET event in these classes of interacting fluorophores is new. Moreover, there is no report of such designed system of chimeric DNA duplex. Our observed phenomenon and the design can potentially be exploited in designing more of such efficient FRET pairs for useful application in the detection and analysis of biomolecular interactions and in material science application. PMID:27498231

  10. Steam generator fretting-wear damage: A summary of recent findings

    SciTech Connect

    Guerout, F.M.; Fisher, N.J.

    1999-08-01

    Flow-induced vibration of steam generator (SG) tubes may sometimes result in fretting-wear damage at the tube-to-support locations. Fretting-wear damage predictions are largely based on experimental data obtained at representative test conditions. Fretting-wear of SG materials has been studied at the Chalk River Laboratories for two decades. Tests are conducted in fretting-wear test machines that simulate SG environmental conditions and tube-to-support dynamic interactions. A new high-temperature force and displacement measuring system was developed to monitor tube-to-support interaction (i.e., work-rate) at operating conditions. This improvement in experimental fretting-wear technology was used to perform a comprehensive study of the effect of various environment and design parameters on SG tube wear damage. This paper summarizes the results of tests performed over the past 4 yr to study the effect of temperature, water chemistry, support geometry, and tube material on fretting-wear. The results show a significant effect of temperature on tube wear damage. Therefore, fretting-wear tests must be performed at operating temperatures in order to be relevant. No significant effect of the type of water treatment on tube wear damage was observed. For predominantly impacting motion, the wear of SG tubes in contact with 410 stainless steel is similar regardless of whether Alloy 690 or Alloy 800 is used as tubing material or whether lattice bars or broached hole supports are used. Based on results presented in this paper, an average wear coefficient value is recommended that is used for the prediction of SG tube wear depth versus time.

  11. Hybridization accompanying FRET event in labeled natural nucleoside-unnatural nucleoside containing chimeric DNA duplexes.

    PubMed

    Bag, Subhendu Sekhar; Das, Suman K; Pradhan, Manoj Kumar; Jana, Subhashis

    2016-09-01

    Förster resonance energy transfer (FRET) is a highly efficient strategy in illuminating the structures, structural changes and dynamics of DNA, proteins and other biomolecules and thus is being widely utilized in studying such phenomena, in designing molecular/biomolecular probes for monitoring the hybridization event of two single stranded DNA to form duplex, in gene detection and in many other sensory applications in chemistry, biology and material sciences. Moreover, FRET can give information about the positional status of chromophores within the associated biomolecules with much more accuracy than other methods can yield. Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene ((TPhen)BDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, (Per)U or (OxoPy)U or (Per)U, forming two stable chimeric DNA duplexes. The pairing selectivity and the thermal duplex stability of the chimeric duplexes are higher than any of the duplexes with natural nucleoside formed. The hybridization results in a Förster resonance energy transfer (FRET) from donor triazolylphenanthrene of (TPhen)BDo to acceptor oxopyrene of (OxoPy)U and/or to perylene chromophore of (Per)U, respectively, in two chimeric DNA duplexes. Therefore, we have established the FRET process in two chimeric DNA duplexes wherein a fluorescently labeled natural nucleoside ((OxoPy)U or (Per)U) paired against an unnatural nucleoside ((TPhen)BDo) without sacrificing the duplex stability and B-DNA conformation. The hybridization accompanying FRET event in these classes of interacting fluorophores is new. Moreover, there is no report of such designed system of chimeric DNA duplex. Our observed phenomenon and the design can potentially be exploited in designing more of such efficient FRET pairs for useful application in the detection and analysis of biomolecular interactions and in material science application.

  12. Ranking of Molecular Biomarker Interaction with Targeted DNA Nucleobases via Full Atomistic Molecular Dynamics

    PubMed Central

    Zhang, Wenjun; Wang, Ming L.; Cranford, Steven W.

    2016-01-01

    DNA-based sensors can detect disease biomarkers, including acetone and ethanol for diabetes and H2S for cardiovascular diseases. Before experimenting on thousands of potential DNA segments, we conduct full atomistic steered molecular dynamics (SMD) simulations to screen the interactions between different DNA sequences with targeted molecules to rank the nucleobase sensing performance. We study and rank the strength of interaction between four single DNA nucleotides (Adenine (A), Guanine (G), Cytosine (C), and Thymine (T)) on single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with acetone, ethanol, H2S and HCl. By sampling forward and reverse interaction paths, we compute the free-energy profiles of eight systems for the four targeted molecules. We find that dsDNA react differently than ssDNA to the targeted molecules, requiring more energy to move the molecule close to DNA as indicated by the potential of mean force (PMF). Comparing the PMF values of different systems, we obtain a relative ranking of DNA base for the detection of each molecule. Via the same procedure, we could generate a library of DNA sequences for the detection of a wide range of chemicals. A DNA sensor array built with selected sequences differentiating many disease biomarkers can be used in disease diagnosis and monitoring. PMID:26750747

  13. Preliminary studies on unusual polymorphs of thymine: Structural comparison with other nucleobases

    NASA Astrophysics Data System (ADS)

    Chennuru, Ramanaiah; Muthudoss, Prakash; Ramakrishnan, Srividya; Mohammad, Amjad Basha; Ravi Chandra Babu, R.; Mahapatra, Sudarshan; Nayak, Susanta K.

    2016-09-01

    Two polymorphs Form-R2 and Form-R4 of anhydrous thymine, one of the four nucleobases in the nucleic acid of DNA were obtained via sublimation crystallization and desolvation technique respectively. Form-R2 crystallizes in monoclinic C 2/c with a = 25.107(7) Å, b = 6.846(2) Å, c = 6.715(2) Å, β = 90.529(6)⁰ and V = 1154.1(5) Å3. The supramolecular assembly in Form-R2 is a sheet of hydrogen bonded network similar to that found in the crystal structures of other reported anhydrous form of thymine (Form-R1). Interestingly the thermal behavior is similar for these two forms with a minor difference in powder X-ray diffraction pattern. Further thymine Form-R2 closely matches with one of the predicted form of thymine using Polymorph module of Accelrys. Form-R4 is obtained by the dehydration of the mono hydrated form (Form-R3) and characterized by powder X-ray diffraction, FTIR spectroscopic techniques and thermal analysis.

  14. Low-energy positron scattering from DNA nucleobases: the effects from permanent dipoles

    NASA Astrophysics Data System (ADS)

    Franz, Jan; Gianturco, Francesco Antonio

    2014-10-01

    Ab initio quantum calculations for low-energy positron scattering from gas-phase isolated molecular nucleobases which are part of the DNA structure are presented and discussed over the range of 1 eV to 25 eV. The calculations report the integral cross sections (ICSs) and the momentum-transfer cross sections (MTCSs) for Adenine, Guanine, Thymine and Cytosine. The calculations show very clearly the important role of the dominant long-range interaction between the positron projectile and the permanent dipole-moments of the target molecules in deciding the relative sizes of the ICSs and MTCSs for the present series of molecules. Such results confirm the largely repulsive interaction between positron and DNA bases, which is nevertheless producing very large cross sections and marked deflection functions from the latter molecules. Contribution to the Topical Issue "Nano-scale Insights into Ion-beam Cancer Therapy", edited by Andrey V. Solov'yov, Nigel Mason, Paulo Limão-Vieira and Malgorzata Smialek-Telega.

  15. High-resolution photoelectron spectra of the pyrimidine-type nucleobases

    SciTech Connect

    Fulfer, K. D.; Hardy, D.; Poliakoff, E. D.; Aguilar, A. A.

    2015-06-14

    High-resolution photoelectron spectra of the gas phase pyrimidine-type nucleobases, thymine, uracil, and cytosine, were collected using synchrotron radiation over the photon energy range 17 ≤ hν ≤ 150 eV. These data provide the highest resolution photoelectron spectra of thymine, uracil, and cytosine published to date. By comparing integrated regions of the energy dependent photoelectron spectra of thymine, the ionization potentials of the first four ionic states of thymine were estimated to be 8.8, 9.8, 10.3, and 10.8 eV. The thymine data also show evidence for low energy shape resonances in three of the outermost valence electronic states. Comparing the uracil spectrum with the thymine spectrum, the four outermost valence electronic states of uracil likely begin at binding energies 9.3, 9.9, 10.5, and 11.0 eV. High-resolution spectra indicate only one tautomeric form of cytosine contributes significantly to the spectrum with the four outermost valence electronic states beginning at binding energies 8.9, 9.9, 10.4, and 10.85 eV.

  16. Ranking of Molecular Biomarker Interaction with Targeted DNA Nucleobases via Full Atomistic Molecular Dynamics

    NASA Astrophysics Data System (ADS)

    Zhang, Wenjun; Wang, Ming L.; Cranford, Steven W.

    2016-01-01

    DNA-based sensors can detect disease biomarkers, including acetone and ethanol for diabetes and H2S for cardiovascular diseases. Before experimenting on thousands of potential DNA segments, we conduct full atomistic steered molecular dynamics (SMD) simulations to screen the interactions between different DNA sequences with targeted molecules to rank the nucleobase sensing performance. We study and rank the strength of interaction between four single DNA nucleotides (Adenine (A), Guanine (G), Cytosine (C), and Thymine (T)) on single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with acetone, ethanol, H2S and HCl. By sampling forward and reverse interaction paths, we compute the free-energy profiles of eight systems for the four targeted molecules. We find that dsDNA react differently than ssDNA to the targeted molecules, requiring more energy to move the molecule close to DNA as indicated by the potential of mean force (PMF). Comparing the PMF values of different systems, we obtain a relative ranking of DNA base for the detection of each molecule. Via the same procedure, we could generate a library of DNA sequences for the detection of a wide range of chemicals. A DNA sensor array built with selected sequences differentiating many disease biomarkers can be used in disease diagnosis and monitoring.

  17. High-energy chemistry of formamide: A unified mechanism of nucleobase formation

    PubMed Central

    Ferus, Martin; Nesvorný, David; Šponer, Jiří; Kubelík, Petr; Michalčíková, Regina; Shestivská, Violetta; Šponer, Judit E.; Civiš, Svatopluk

    2015-01-01

    The coincidence of the Late Heavy Bombardment (LHB) period and the emergence of terrestrial life about 4 billion years ago suggest that extraterrestrial impacts could contribute to the synthesis of the building blocks of the first life-giving molecules. We simulated the high-energy synthesis of nucleobases from formamide during the impact of an extraterrestrial body. A high-power laser has been used to induce the dielectric breakdown of the plasma produced by the impact. The results demonstrate that the initial dissociation of the formamide molecule could produce a large amount of highly reactive CN and NH radicals, which could further react with formamide to produce adenine, guanine, cytosine, and uracil. Based on GC-MS, high-resolution FTIR spectroscopic results, as well as theoretical calculations, we present a comprehensive mechanistic model, which accounts for all steps taking place in the studied impact chemistry. Our findings thus demonstrate that extraterrestrial impacts, which were one order of magnitude more abundant during the LHB period than before and after, could not only destroy the existing ancient life forms, but could also contribute to the creation of biogenic molecules. PMID:25489115

  18. Quantum Mechanical Studies on the Photophysics and the Photochemistry of Nucleic Acids and Nucleobases.

    PubMed

    Improta, Roberto; Santoro, Fabrizio; Blancafort, Lluís

    2016-03-23

    The photophysics and photochemistry of DNA is of great importance due to the potential damage of the genetic code by UV light. Quantum mechanical studies have played a key role in interpretating the results of modern time-resolved pump-probe spectroscopy, and in elucidating the main photoactivated reactive paths. This review provides a concise, complete picture of the computational studies carried out, approximately, in the past decade. We start with an overview of the photophysics of the nucleobases in the gas phase and in solution. We discuss the proposed mechanisms for ultrafast decay to the ground state, that involve conical intersections, consider the role of triplet states, and analyze how the solvent modulates the photophysics. Then we move to larger systems, from dinucleotides to single- and double-stranded oligonucleotides. We focus on the possible role of charge transfer and delocalized or excitonic states in the photophysics of these systems and discuss the main photochemical paths. We finish with an outlook on the current challenges in the field and future directions of research.

  19. Association of poly(N-isopropylacrylamide) containing nucleobase multiple hydrogen bonding of adenine for DNA recognition

    NASA Astrophysics Data System (ADS)

    Yang, Hsiu-Wen; Chen, Jem-Kun; Cheng, Chih-Chia; Kuo, Shiao-Wei

    2013-04-01

    In this study we used the poly(N-isopropylacrylamide) (PNIPAAm) as a medium to generate PNIPAAm-adenine supramolecular complexes. A nucleobase-like hydrogen bonding (NLHB) between PNIPAAm and adenine was found that changed the morphology, crystalline structure, and temperature responsiveness of PNIPAAm microgels relatively to the adenine concentrations. With increasing the adenine concentration, the PNIPAAm-adenine supramolecular complexes gradually altered their morphologies from microgel particles to thin film structures and suppressed the thermodynamical coil-to-globule transition of PNIPAAm because of the NLHB existed between the PNIPAAm amide and ester groups and the adenine amide groups (Cdbnd O⋯Hsbnd N and Nsbnd H⋯Nsbnd R), verified by FTIR spectral analysis. NLHB was also diverse and extensive upon increasing the temperature; therefore, the thermoresponsive behavior of the complexes was altered with the NLBH intensity, evaluated by the inter-association equilibrium constant (Ka) above and below their LCST. Therefore, PNIPAAm can be as a medium to recognize adenine in various concentrations, which could potentially be applied in DNA recognition.

  20. High-energy chemistry of formamide: a unified mechanism of nucleobase formation.

    PubMed

    Ferus, Martin; Nesvorný, David; Šponer, Jiří; Kubelík, Petr; Michalčíková, Regina; Shestivská, Violetta; Šponer, Judit E; Civiš, Svatopluk

    2015-01-20

    The coincidence of the Late Heavy Bombardment (LHB) period and the emergence of terrestrial life about 4 billion years ago suggest that extraterrestrial impacts could contribute to the synthesis of the building blocks of the first life-giving molecules. We simulated the high-energy synthesis of nucleobases from formamide during the impact of an extraterrestrial body. A high-power laser has been used to induce the dielectric breakdown of the plasma produced by the impact. The results demonstrate that the initial dissociation of the formamide molecule could produce a large amount of highly reactive CN and NH radicals, which could further react with formamide to produce adenine, guanine, cytosine, and uracil. Based on GC-MS, high-resolution FTIR spectroscopic results, as well as theoretical calculations, we present a comprehensive mechanistic model, which accounts for all steps taking place in the studied impact chemistry. Our findings thus demonstrate that extraterrestrial impacts, which were one order of magnitude more abundant during the LHB period than before and after, could not only destroy the existing ancient life forms, but could also contribute to the creation of biogenic molecules. PMID:25489115

  1. An atlas of RNA base pairs involving modified nucleobases with optimal geometries and accurate energies

    PubMed Central

    Chawla, Mohit; Oliva, Romina; Bujnicki, Janusz M.; Cavallo, Luigi

    2015-01-01

    Posttranscriptional modifications greatly enhance the chemical information of RNA molecules, contributing to explain the diversity of their structures and functions. A significant fraction of RNA experimental structures available to date present modified nucleobases, with half of them being involved in H-bonding interactions with other bases, i.e. ‘modified base pairs’. Herein we present a systematic investigation of modified base pairs, in the context of experimental RNA structures. To this end, we first compiled an atlas of experimentally observed modified base pairs, for which we recorded occurrences and structural context. Then, for each base pair, we selected a representative for subsequent quantum mechanics calculations, to find out its optimal geometry and interaction energy. Our structural analyses show that most of the modified base pairs are non Watson–Crick like and are involved in RNA tertiary structure motifs. In addition, quantum mechanics calculations quantify and provide a rationale for the impact of the different modifications on the geometry and stability of the base pairs they participate in. PMID:26117545

  2. An atlas of RNA base pairs involving modified nucleobases with optimal geometries and accurate energies.

    PubMed

    Chawla, Mohit; Oliva, Romina; Bujnicki, Janusz M; Cavallo, Luigi

    2015-08-18

    Posttranscriptional modifications greatly enhance the chemical information of RNA molecules, contributing to explain the diversity of their structures and functions. A significant fraction of RNA experimental structures available to date present modified nucleobases, with half of them being involved in H-bonding interactions with other bases, i.e. 'modified base pairs'. Herein we present a systematic investigation of modified base pairs, in the context of experimental RNA structures. To this end, we first compiled an atlas of experimentally observed modified base pairs, for which we recorded occurrences and structural context. Then, for each base pair, we selected a representative for subsequent quantum mechanics calculations, to find out its optimal geometry and interaction energy. Our structural analyses show that most of the modified base pairs are non Watson-Crick like and are involved in RNA tertiary structure motifs. In addition, quantum mechanics calculations quantify and provide a rationale for the impact of the different modifications on the geometry and stability of the base pairs they participate in. PMID:26117545

  3. Similarity in drugs: reflections on analogue design.

    PubMed

    Wermuth, Camille G

    2006-04-01

    A survey of novel small-molecule therapeutics reveals that the majority of them result from analogue design and that their market value represents two-thirds of all small-molecule sales. In natural science, the term analogue, derived from the Latin and Greek analogia, has always been used to describe structural and functional similarity. Extended to drugs, this definition implies that the analogue of an existing drug molecule shares structural and pharmacological similarities with the original compound. Formally, this definition allows the establishment of three categories of drug analogues: analogues possessing chemical and pharmacological similarities (direct analogues); analogues possessing structural similarities only (structural analogues); and chemically different compounds displaying similar pharmacological properties (functional analogues). PMID:16580977

  4. Assembling programmable FRET-based photonic networks using designer DNA scaffolds

    PubMed Central

    Buckhout-White, Susan; Spillmann, Christopher M; Algar, W. Russ; Khachatrian, Ani; Melinger, Joseph S.; Goldman, Ellen R.; Ancona, Mario G.; Medintz, Igor L.

    2014-01-01

    DNA demonstrates a remarkable capacity for creating designer nanostructures and devices. A growing number of these structures utilize Förster resonance energy transfer (FRET) as part of the device's functionality, readout or characterization, and, as device sophistication increases so do the concomitant FRET requirements. Here we create multi-dye FRET cascades and assess how well DNA can marshal organic dyes into nanoantennae that focus excitonic energy. We evaluate 36 increasingly complex designs including linear, bifurcated, Holliday junction, 8-arm star and dendrimers involving up to five different dyes engaging in four-consecutive FRET steps, while systematically varying fluorophore spacing by Förster distance (R0). Decreasing R0 while augmenting cross-sectional collection area with multiple donors significantly increases terminal exciton delivery efficiency within dendrimers compared with the first linear constructs. Förster modelling confirms that best results are obtained when there are multiple interacting FRET pathways rather than independent channels by which excitons travel from initial donor(s) to final acceptor. PMID:25504073

  5. Gate-width impact on NIR FRET lifetime fitting using gated ICCD

    NASA Astrophysics Data System (ADS)

    Chen, Sez-Jade; Intes, Xavier

    2016-03-01

    Förster Resonance Energy Transfer (FRET) is widely used to sense molecular interactions occurring at the nanoscale. In vitro and ex vivo protocols for visualizing FRET are already well-established, but in vivo studies have proven to be more challenging. One issue that hinders in vivo visualization of FRET is the higher absorption and scattering of visible light within tissues. In this case, light in the near-infrared (NIR) spectral window is required for increased depth sensing. Moreover, due to spectral variation in optical properties as well as heterogeneous spatial distribution, lifetime-based FRET imaging is preferred. Herein, we investigate the effect of temporal acquisition settings on the lifetime-based estimation of the fraction of quenched donor molecules (A1) as well as the quenched donor lifetime (τ1). We performed in silico, in vitro, and in vivo experiments under gate widths of 300ps to 1000ps in 100ps intervals to determine the effect on quantification of A1 and τ1. Even though the NIR fluorescent dyes have shorter lifetimes then visible fluorophores, we were still able to accurately quantify FRET under all tested system gate widths and experimental conditions.

  6. Evaluating Quantum Dot Performance in Homogeneous FRET Immunoassays for Prostate Specific Antigen

    PubMed Central

    Bhuckory, Shashi; Lefebvre, Olivier; Qiu, Xue; Wegner, Karl David; Hildebrandt, Niko

    2016-01-01

    The integration of semiconductor quantum dots (QDs) into homogeneous Förster resonance energy transfer (FRET) immunoassay kits for clinical diagnostics can provide significant advantages concerning multiplexing and sensitivity. Here we present a facile and functional QD-antibody conjugation method using three commercially available QDs with different photoluminescence (PL) maxima (605 nm, 655 nm, and 705 nm). The QD-antibody conjugates were successfully applied for FRET immunoassays against prostate specific antigen (PSA) in 50 µL serum samples using Lumi4-Tb (Tb) antibody conjugates as FRET donors and time-gated PL detection on a KRYPTOR clinical plate reader. Förster distance and Tb donor background PL were directly related to the analytical sensitivity for PSA, which resulted in the lowest limits of detection for Tb-QD705 (2 ng/mL), followed by Tb-QD655 (4 ng/mL), and Tb-QD605 (23 ng/mL). Duplexed PSA detection using the Tb-QD655 and Tb-QD705 FRET-pairs demonstrated the multiplexing ability of our immunoassays. Our results show that FRET based on QD acceptors is suitable for multiplexed and sensitive biomarker detection in clinical diagnostics. PMID:26861327

  7. Construction, imaging, and analysis of FRET-based tension sensors in living cells.

    PubMed

    LaCroix, Andrew S; Rothenberg, Katheryn E; Berginski, Matthew E; Urs, Aarti N; Hoffman, Brenton D

    2015-01-01

    Due to an increased appreciation for the importance of mechanical stimuli in many biological contexts, an interest in measuring the forces experienced by specific proteins in living cells has recently emerged. The development and use of Förster resonance energy transfer (FRET)-based molecular tension sensors has enabled these types of studies and led to important insights into the mechanisms those cells utilize to probe and respond to the mechanical nature of their surrounding environment. The process for creating and utilizing FRET-based tension sensors can be divided into three main parts: construction, imaging, and analysis. First we review several methods for the construction of genetically encoded FRET-based tension sensors, including restriction enzyme-based methods as well as the more recently developed overlap extension or Gibson Assembly protocols. Next, we discuss the intricacies associated with imaging tension sensors, including optimizing imaging parameters as well as common techniques for estimating artifacts within standard imaging systems. Then, we detail the analysis of such data and describe how to extract useful information from a FRET experiment. Finally, we provide a discussion on identifying and correcting common artifacts in the imaging of FRET-based tension sensors.

  8. FRET-based protein-DNA binding assay for detection of active NF-kappa B

    SciTech Connect

    Giannetti, Ambra; Baldini, Francesco; Wabuyele, Musundi B; Vo Dinh, Tuan

    2006-01-01

    A novel method to detect the active form of NF-{kappa}B, a transcription factor regulating a battery of inflammatory genes and playing a fundamental role in the development of numerous pathological states, has been developed. In the present work, we used fluorescence resonance energy transfer (FRET) to study DNA-protein binding interaction taking place between double-strand (ds) DNA immobilized in a glass capillary wall and p50 proteins. For this purpose, we developed a regenerable FRET-based system comprising of a single-strand (ss) DNA with auto-complementary sequence that is end-labeled with Cy5 dye and is highly specific for p50 proteins. The proteins were labeled with a Black Hole Quencher (BHQ-3) to be used as FRET pair. The interaction of p50/p50 homodimer active form with its DNA binding site was demonstrated by both electrophoretic mobility shift assays and FRET studies. These preliminary results demonstrated the feasibility of the FRET-based DNA technique to detect the active form of NF-{kappa}B protein with 90% detection efficiency. In addition, we show that the system is stable and highly regenerable.

  9. Anchored FRET sensors detect local caspase activation prior to neuronal degeneration

    PubMed Central

    2011-01-01

    Background Recent studies indicate local caspase activation in dendrites or axons during development and in neurodegenerative disorders such as Alzheimer's disease (AD). Emerging evidences point to soluble oligomeric amyloid-β peptide as a causative agent in AD. Results Here we describe the design of fluorescence resonance energy transfer (FRET)-based caspase sensors, fused to the microtubule associated protein tau. Specific caspase sensors preferentially cleaved by caspase-3, -6 or -9 were expressed in differentiated human neuroblastoma SH-SY5Y cells. The anchoring of the sensors resulted in high FRET signals both in extended neurites and soma and made analysis of spatiotemporal signal propagation possible. Caspase activation was detected as loss of FRET after exposure to different stimuli. Interestingly, after staurosporine treatment caspase-6 activation was significantly delayed in neurites compared to cell bodies. In addition, we show that exposure to oligomer-enriched amyloid-β peptide resulted in loss of FRET in cells expressing sensors for caspase-3 and -6, but not -9, in both soma and neurites before neurite degeneration was observed. Conclusions Taken together, the results show that by using anchored FRET sensors it is possible to detect stimuli-dependent differential activation of caspases and to distinguish local from global caspase activation in live neuronal cells. Furthermore, in these cells oligomer-enriched amyloid-β peptide induces a global, rather than local activation of caspase-3 and -6, which subsequently leads to neuronal cell death. PMID:21605370

  10. Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

    PubMed Central

    Watkins, Herschel M.; Simon, Anna J.; Sosnick, Tobin R.; Lipman, Everett A.; Hjelm, Rex P.; Plaxco, Kevin W.

    2015-01-01

    Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant. PMID:25964362

  11. Construction, imaging and analysis of FRET-based tension sensors in living cells

    PubMed Central

    LaCroix, Andrew S.; Rothenberg, Katheryn E.; Berginski, Matthew E.; Urs, Aarti N.; Hoffman, Brenton D.

    2015-01-01

    Due to an increased appreciation for the importance of mechanical stimuli in many biological contexts, an interest in measuring the forces experienced by specific proteins in living cells has recently emerged. The development and use of Forster resonance energy transfer (FRET)-based molecular tension sensors has enabled these types of studies and led to important insights into the mechanisms those cells utilize to probe and respond to the mechanical nature of their surrounding environment. The process for creating and utilizing FRET-based tension sensors can be divided into three main parts: construction, imaging, and analysis. First we review several methods for the construction of genetically encoded FRET-based tension sensors, including restriction enzyme-based methods as well as the more recently developed overlap extension or Gibson Assembly protocols. Next, we discuss the intricacies associated with imaging tension sensors, including optimizing imaging parameters as well as common techniques for estimating artifacts within standard imaging systems. Then, we detail the analysis of such data and describe how to extract useful information from a FRET experiment. Finally, we provide a discussion on identifying and correcting common artifacts in the imaging of FRET-based tension sensors. PMID:25640429

  12. De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

    PubMed

    Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

    2012-04-01

    Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

  13. De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

    PubMed

    Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

    2012-04-01

    Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified. PMID:22474184

  14. Photochemistry of nucleic acid bases and their thio- and aza-analogues in solution.

    PubMed

    Pollum, Marvin; Martínez-Fernández, Lara; Crespo-Hernández, Carlos E

    2015-01-01

    The steady-state and time-resolved photochemistry of the natural nucleic acid bases and their sulfur- and nitrogen-substituted analogues in solution is reviewed. Emphasis is given to the experimental studies performed over the last 3-5 years that showcase topical areas of scientific inquiry and those that require further scrutiny. Significant progress has been made toward mapping the radiative and nonradiative decay pathways of nucleic acid bases. There is a consensus that ultrafast internal conversion to the ground state is the primary relaxation pathway in the nucleic acid bases, whereas the mechanism of this relaxation and the level of participation of the (1)πσ*, (1) nπ*, and (3)ππ* states are still matters of debate. Although impressive research has been performed in recent years, the microscopic mechanism(s) by which the nucleic acid bases dissipate excess vibrational energy to their environment, and the role of the N-glycosidic group in this and in other nonradiative decay pathways, are still poorly understood. The simple replacement of a single atom in a nucleobase with a sulfur or nitrogen atom severely restricts access to the conical intersections responsible for the intrinsic internal conversion pathways to the ground state in the nucleic acid bases. It also enhances access to ultrafast and efficient inter-system crossing pathways that populate the triplet manifold in yields close to unity. Determining the coupled nuclear and electronic pathways responsible for the significantly different photochemistry in these nucleic acid base analogues serves as a convenient platform to examine the current state of knowledge regarding the photodynamic properties of the DNA and RNA bases from both experimental and computational perspectives. Further investigations should also aid in forecasting the prospective use of sulfur- and nitrogen-substituted base analogues in photochemotherapeutic applications. PMID:25238718

  15. Mapping the Structure and Conformational Movements of Proteins with Transition Metal Ion FRET

    SciTech Connect

    Taraska, J.; Puljung, M; Olivier, N; Olivier, G; Zagotta, W

    2009-01-01

    Visualizing conformational dynamics in proteins has been difficult, and the atomic-scale motions responsible for the behavior of most allosteric proteins are unknown. Here we report that fluorescence resonance energy transfer (FRET) between a small fluorescent dye and a nickel ion bound to a dihistidine motif can be used to monitor small structural rearrangements in proteins. This method provides several key advantages over classical FRET, including the ability to measure the dynamics of close-range interactions, the use of small probes with short linkers, a low orientation dependence, and the ability to add and remove unique tunable acceptors. We used this 'transition metal ion FRET' approach along with X-ray crystallography to determine the structural changes of the gating ring of the mouse hyperpolarization-activated cyclic nucleotide-regulated ion channel HCN2. Our results suggest a general model for the conformational switch in the cyclic nucleotide-binding site of cyclic nucleotide-regulated ion channels.

  16. Visualization of Signaling Molecules During Neutrophil Recruitment in Transgenic Mice Expressing FRET Biosensors.

    PubMed

    Mizuno, Rei; Kamioka, Yuji; Sakai, Yoshiharu; Matsuda, Michiyuki

    2016-01-01

    A number of chemical mediators regulate neutrophil recruitment to inflammatory sites either positively or negatively. Although the actions of each chemical mediator on the intracellular signaling networks controlling cell migration have been studied with neutrophils cultured in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. To understand the mechanisms regulating neutrophil recruitment to the inflamed intestine in vivo, we recently generated transgenic mice expressing biosensors based on FRET (Förster resonance energy transfer) and set up two-photon excitation microscopy to observe the gastrointestinal tract in living mice. By measuring FRET in neutrophils, we showed activity changes of protein kinases in the neutrophils recruited to inflamed intestines. In this chapter, we describe the protocol used to visualize the protein kinase activities in neutrophils of the inflamed intestine of transgenic mice expressing the FRET biosensors. PMID:27246030

  17. Fretting of Secondary-Seal-Ring Candidate Materials in Air at Temperatures to 816 C

    NASA Technical Reports Server (NTRS)

    Bill, R. C.

    1972-01-01

    Superalloys containing chromium showed decreasing fretting damage with increasing temperature of 816 C. This trend was related to the ability of the alloys to generate self-protecting oxide films. The damage at 816 C was one-third to one-tenth of that at 23 C. Osmium, chromium, and chromium carbide platings were fretted at 23 and 450 C. Osmium was extremely protective at 23 C but oxidized excessively at 450 C. Chromium and chromium carbide gave about the same protection at 450 C as the oxide films that formed on the superalloys. High graphite and low graphite carbons were fretted at 23 and 327 C. High graphite carbon was superior at 327 C, but low graphite carbon was the best material examined at 23 C.

  18. Phanta: A Non-Fluorescent Photochromic Acceptor for pcFRET

    PubMed Central

    Don Paul, Craig; Kiss, Csaba; Traore, Daouda A. K.; Gong, Lan; Wilce, Matthew C. J.; Devenish, Rodney J.; Bradbury, Andrew; Prescott, Mark

    2013-01-01

    We have developed an orange non-fluorescent photochromic protein (quantum yield, 0.003) we call Phanta that is useful as an acceptor in pcFRET applications. Phanta can be repeatedly inter-converted between the two absorbing states by alternate exposure to cyan and violet light. The absorption spectra of Phanta in one absorbing state shows excellent overlap with the emission spectra of a number of donor green fluorescent proteins including the commonly used EGFP. We show that the Phanta-EGFP FRET pair is suitable for monitoring the activation of caspase 3 in live cells using readily available instrumentation and a simple protocol that requires the acquisition of two donor emission images corresponding to Phanta in each of its photoswitched states. This the first report of a genetically encoded non-fluorescent acceptor for pcFRET. PMID:24098733

  19. Multiplexed 3D FRET imaging in deep tissue of live embryos

    PubMed Central

    Zhao, Ming; Wan, Xiaoyang; Li, Yu; Zhou, Weibin; Peng, Leilei

    2015-01-01

    Current deep tissue microscopy techniques are mostly restricted to intensity mapping of fluorophores, which significantly limit their applications in investigating biochemical processes in vivo. We present a deep tissue multiplexed functional imaging method that probes multiple Förster resonant energy transfer (FRET) sensors in live embryos with high spatial resolution. The method simultaneously images fluorescence lifetimes in 3D with multiple excitation lasers. Through quantitative analysis of triple-channel intensity and lifetime images, we demonstrated that Ca2+ and cAMP levels of live embryos expressing dual FRET sensors can be monitored simultaneously at microscopic resolution. The method is compatible with a broad range of FRET sensors currently available for probing various cellular biochemical functions. It opens the door to imaging complex cellular circuitries in whole live organisms. PMID:26387920

  20. Fluorophores, environments and quantification techniques in the analysis of transmembrane helix interaction using FRET

    PubMed Central

    Khadria, Ambalika S.; Senes, Alessandro

    2015-01-01

    FRET has been widely used as a spectroscopic tool in vitro to study the interactions between transmembrane helices in detergent and lipid environments. This technique has been instrumental to many studies that have greatly contributed to quantitative understanding of the physical principles that govern helix-helix interaction in the membrane. These studies have also improved our understanding of the biological role of oligomerization in membrane proteins. In this review, we focus on the combinations of fluorophores used, the membrane mimetic environments and measurement techniques that have been applied to study model systems as well as biological oligomeric complexes in vitro. We highlight the different formalisms used to calculate FRET efficiency, and the challenges associated with accurate quantification. The goal is to provide the reader with a comparative summary of the relevant literature for planning and designing FRET experiments aimed at measuring transmembrane helix-helix association. PMID:25968159

  1. Visualization of Signaling Molecules During Neutrophil Recruitment in Transgenic Mice Expressing FRET Biosensors.

    PubMed

    Mizuno, Rei; Kamioka, Yuji; Sakai, Yoshiharu; Matsuda, Michiyuki

    2016-01-01

    A number of chemical mediators regulate neutrophil recruitment to inflammatory sites either positively or negatively. Although the actions of each chemical mediator on the intracellular signaling networks controlling cell migration have been studied with neutrophils cultured in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. To understand the mechanisms regulating neutrophil recruitment to the inflamed intestine in vivo, we recently generated transgenic mice expressing biosensors based on FRET (Förster resonance energy transfer) and set up two-photon excitation microscopy to observe the gastrointestinal tract in living mice. By measuring FRET in neutrophils, we showed activity changes of protein kinases in the neutrophils recruited to inflamed intestines. In this chapter, we describe the protocol used to visualize the protein kinase activities in neutrophils of the inflamed intestine of transgenic mice expressing the FRET biosensors.

  2. Novel multistep BRET-FRET energy transfer using nanoconjugates of firefly proteins, quantum dots, and red fluorescent proteins.

    PubMed

    Alam, Rabeka; Zylstra, Joshua; Fontaine, Danielle M; Branchini, Bruce R; Maye, Mathew M

    2013-06-21

    Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated.

  3. Experimental verification of the kinetic theory of FRET using optical microspectroscopy and obligate oligomers.

    PubMed

    Patowary, Suparna; Pisterzi, Luca F; Biener, Gabriel; Holz, Jessica D; Oliver, Julie A; Wells, James W; Raicu, Valerică

    2015-04-01

    Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.

  4. The effect of fretting and environment on fatigue crack initiation and early propagation in a quenched and tempered 4130 Steel

    NASA Astrophysics Data System (ADS)

    Gaul, D. J.; Duquette, D. J.

    1980-09-01

    Fretting fatigue studies were performed on quenched and tempered 4130 steel in laboratory air and in argon as functions of relative slip displacement, normal pressure and applied cyclic stress. Significant reductions in fatigue resistance were observed at all stress levels and increased with increasing normal pressures. However, a minimum in resistance was observed for relative slip magnitudes of 20 to 30 μm. Inert environments improve fatigue resistance under fretting conditions. Metallographic observations indicated that subsurface cracking was generally observed and that stress concentrations associated with this cracking resulted in deviations to and away from the faying surfaces. Fretting cracks which deviated into the alloy become initiated fatigue cracks. A mechanical model is proposed for fretting induced fatigue crack initiation which suggests that this phenomenon is a simple extension of the basic fretting process.

  5. FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET.

    PubMed

    Ingargiola, Antonino; Lerner, Eitan; Chung, SangYoon; Weiss, Shimon; Michalet, Xavier

    2016-01-01

    Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to

  6. Time resolved FRET measurement in various heterogeneous media using merocyanine dye as a donor

    NASA Astrophysics Data System (ADS)

    Kedia, Niraja; Bagchi, Sanjib

    2015-06-01

    Ultrafast fluorescence resonance energy transfer (FRET) from a merocyanine dye to a Rhodamine 6G (R6G) molecule in micelles formed by the surfactants SDS and DTAB and also in a catanionic vesicle formed by SDS and DTAB has been studied by picosecond time resolved emission spectroscopy. Here the dye acts as a donor molecule and R6G acts as the acceptor molecule. Multiple timescales of FRET have been detected, namely, an ultrafast component of 100-500 ps and relatively long component (1800-3300 ps). The different time scales are attributed to different donor-acceptor distances.

  7. Time resolved FRET measurement in various heterogeneous media using merocyanine dye as a donor.

    PubMed

    Kedia, Niraja; Bagchi, Sanjib

    2015-06-15

    Ultrafast fluorescence resonance energy transfer (FRET) from a merocyanine dye to a Rhodamine 6G (R6G) molecule in micelles formed by the surfactants SDS and DTAB and also in a catanionic vesicle formed by SDS and DTAB has been studied by picosecond time resolved emission spectroscopy. Here the dye acts as a donor molecule and R6G acts as the acceptor molecule. Multiple timescales of FRET have been detected, namely, an ultrafast component of 100-500 ps and relatively long component (1800-3300 ps). The different time scales are attributed to different donor-acceptor distances.

  8. FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET

    PubMed Central

    Lerner, Eitan; Chung, SangYoon; Weiss, Shimon; Michalet, Xavier

    2016-01-01

    Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to

  9. CASL Virtual Reactor Predictive Simulation: Grid-to-Rod Fretting Wear

    SciTech Connect

    Roger, Lu Y.; Karoutas, Zeses; Sham, Sam

    2011-01-01

    Grid-to-Rod Fretting (GTRF) wear is currently one of the main causes of fuel rod leaking in pressurized water reactors. The Consortium for Advanced Simulation of Light Water Reactors (CASL) has identified GTRF as one of the Challenge Problems that drive the requirement for the development and application of a modeling and simulation computational environment for predictive simulation of light water reactors. This paper presents fretting wear simulation methodology currently employed by Westinghouse, a CASL industrial partner, to address GTRF. The required advancements in the computational and materials science modeling areas to develop a predictive simulation environment by CASL to address GTRF are outlined.

  10. Structural, Dynamical and Electronic Transport Properties of Modified DNA Duplexes Containing Size-Expanded Nucleobases

    SciTech Connect

    Sumpter, Bobby G; Fuentes-Cabrera, Miguel A

    2011-01-01

    Among the distinct strategies proposed to expand the genetic alphabet, size-expanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. The most relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMO-LUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.

  11. Structural, Dynamical, and Electronic Transport Properties of Modified DNA Duplexes Containing Size-Expanded Nucleobases

    SciTech Connect

    Fuentes-Cabrera, Miguel A; Orozco, Modesto; Luque, Javier; Sumpter, Bobby G; Blas, Jose; Ordejon, Pablo J; Huertas, Oscar; Tabares, Carolina

    2011-01-01

    Among the distinct strategies proposed to expand the genetic alphabet, sizeexpanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. Themost relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMOLUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.

  12. Synthesis and structure of duplex DNA containing the genotoxic nucleobase lesion N7-methylguanine

    SciTech Connect

    Lee, S.; Bowman, B.R.; Ueno, Y.; Wang, S.; Verdine, G.L.

    2008-11-03

    The predominant product of aberrant DNA methylation is the genotoxic lesion N7-methyl-2{prime}-deoxyguanosine (m{sup 7}dG). M{sup 7}dG is recognized and excised by lesion-specific DNA glycosylases, namely AlkA in E. coli and Aag in humans. Structural studies of m{sup 7}dG recognition and catalysis by these enzymes have been hampered due to a lack of efficient means by which to incorporate the chemically labile m{sup 7}dG moiety site-specifically into DNA on a preparative scale. Here we report a solution to this problem. We stabilized the lesion toward acid-catalyzed and glycosylase-catalyzed depurination by 2{prime}-fluorination and toward base-catalyzed degradation using mild, nonaqueous conditions in the DNA deprotection reaction. Duplex DNA containing 2{prime}-fluoro-m{sup 7}dG (Fm{sup 7}dG) cocrystallized with AlkA as a host-guest complex in which the lesion-containing segment of DNA was nearly devoid of protein contacts, thus enabling the first direct visualization of the N7-methylguanine lesion nucleobase in DNA. The structure reveals that the base-pairing mode of Fm{sup 7}dG:C is nearly identical to that of G:C, and Fm{sup 7}dG does not induce any apparent structural disturbance of the duplex structure. These observations suggest that AlkA and Aag must perform a structurally invasive interrogation of DNA in order to detect the presence of intrahelical m{sup 7}dG lesions.

  13. Hydrated Electron Transfer to Nucleobases in Aqueous Solutions Revealed by Ab Initio Molecular Dynamics Simulations.

    PubMed

    Zhao, Jing; Wang, Mei; Fu, Aiyun; Yang, Hongfang; Bu, Yuxiang

    2015-08-01

    We present an ab initio molecular dynamics (AIMD) simulation study into the transfer dynamics of an excess electron from its cavity-shaped hydrated electron state to a hydrated nucleobase (NB)-bound state. In contrast to the traditional view that electron localization at NBs (G/A/C/T), which is the first step for electron-induced DNA damage, is related only to dry or prehydrated electrons, and a fully hydrated electron no longer transfers to NBs, our AIMD simulations indicate that a fully hydrated electron can still transfer to NBs. We monitored the transfer dynamics of fully hydrated electrons towards hydrated NBs in aqueous solutions by using AIMD simulations and found that due to solution-structure fluctuation and attraction of NBs, a fully hydrated electron can transfer to a NB gradually over time. Concurrently, the hydrated electron cavity gradually reorganizes, distorts, and even breaks. The transfer could be completed in about 120-200 fs in four aqueous NB solutions, depending on the electron-binding ability of hydrated NBs and the structural fluctuation of the solution. The transferring electron resides in the π*-type lowest unoccupied molecular orbital of the NB, which leads to a hydrated NB anion. Clearly, the observed transfer of hydrated electrons can be attributed to the strong electron-binding ability of hydrated NBs over the hydrated electron cavity, which is the driving force, and the transfer dynamics is structure-fluctuation controlled. This work provides new insights into the evolution dynamics of hydrated electrons and provides some helpful information for understanding the DNA-damage mechanism in solution.

  14. Enhanced Molecular Recognition between Nucleobases and Guanine-5'-monophosphate-disodium (GMP) by Surfactant Aggregates in Aqueous Solution.

    PubMed

    Liu, Zhang; Wang, Dong; Cao, Meiwen; Han, Yuchun; Xu, Hai; Wang, Yilin

    2015-07-15

    Only specific base pairs on DNA can bind with each other through hydrogen bonds, which is called the Watson-Crick (W/C) pairing rule. However, without the constraint of DNA chains, the nucleobases in bulk aqueous solution usually do not follow the W/C pairing rule anymore because of the strong competitive effect of water and the multi-interaction edges of nucleobases. The present work applied surfactant aggregates noncovalently functionalized by nucleotide to enhance the recognition between nucleobases without DNA chains in aqueous solution, and it revealed the effects of their self-assembling ability and morphologies on the recognition. The cationic ammonium monomeric, dimeric, and trimeric surfactants DTAB, 12-3-12, and 12-3-12-3-12 were chosen. The surfactants with guanine-5'-monophosphate-disodium (GMP) form micelles, vesicles, and fingerprint-like and plate-like aggregates bearing the hydrogen-bonding sites of GMP, respectively. The binding parameters of these aggregates with adenine (A), uracil (U), guanine (G), and cytosine(C) indicate that the surfactants can promote W/C recognitions in aqueous solution when they form vesicles (GMP/DTAB) or plate-like aggregates (GMP/12-3-12) with proper molecular packing compactness, which not only provide hydrophobic environments but also shield non-W/C recognition edges. However, the GMP/12-3-12 micelles with loose molecular packing, the GMP/12-3-12 fingerprint-like aggregates where the hydrogen bond sites of GMP are occupied by itself, and the GMP/12-3-12-3-12 vesicles with too strong self-assembling ability cannot promote W/C recognition. This work provides insight into how to design self-assemblies with the performance of enhanced molecule recognition.

  15. Multicomponent click synthesis of new 1,2,3-triazole derivatives of pyrimidine nucleobases: promising acidic corrosion inhibitors for steel.

    PubMed

    González-Olvera, Rodrigo; Espinoza-Vázquez, Araceli; Negrón-Silva, Guillermo E; Palomar-Pardavé, Manuel E; Romero-Romo, Mario A; Santillan, Rosa

    2013-12-06

    A series of new mono-1,2,3-triazole derivatives of pyrimidine nucleobases were synthesized by one-pot copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between N-1-propargyluracil and thymine, sodium azide and several benzyl halides. The desired heterocyclic compounds were obtained in good yields and characterized by NMR, IR, and high resolution mass spectrometry. These compounds were investigated as corrosion inhibitors for steel in 1 M HCl solution, using electrochemical impedance spectroscopy (EIS) technique. The results indicate that these heterocyclic compounds are promising acidic corrosion inhibitors for steel.

  16. Nucleobase-mediated, photocatalytic production of amphiphiles to promote the self-assembly of a simple self-replicating protocell.

    NASA Astrophysics Data System (ADS)

    Monnard, Pierre-Alain; Maurer, Sarah, E.; Albertsen, Anders, N.; Boncella, James, M.; Cape, Jonathan, L.

    replaced by a single nucleobase, 8-oxoguanine, which is tethered to one bipyridine ligand of the metal center. We report here the following major steps towards this chemical protocell: 1) the spontaneous formation of chemical structures consisting of decanoic acid, its precursor, and the simplified NA-ruthenium complexes. 2) the metabolism mediation by a nucleobase to effectively promote the photochemical amphiphile synthesis. 3) the demonstration of reaction selectivity dependent on the nature of the information molecule since only one specific nucleobase that has the required redox potential allows the metabolism to function. Finally, 4) the photochemical formation of amphiphiles can occur efficiently within a preformed membrane, i.e., the protocell compartment. The next step is the integration of short nucleic acid oligomers as opposed to a single nucleobase as the information material to study their photocatalytic activity mediation and polymerization.

  17. FRET study of G-quadruplex forming fluorescent oligonucleotide probes at the lipid monolayer interface

    NASA Astrophysics Data System (ADS)

    Swiatkowska, Angelika; Kosman, Joanna; Juskowiak, Bernard

    2016-01-01

    Spectral properties and G-quadruplex folding ability of fluorescent oligonucleotide probes at the cationic dioctadecyldimethylammonium bromide (DODAB) monolayer interface are reported. Two oligonucleotides, a 19-mer bearing thrombin binding aptamer sequence and a 21-mer with human telomeric sequence, were end-labeled with fluorescent groups (FAM and TAMRA) to give FRET probes F19T and F21T, respectively. The probes exhibited abilities to fold into a quadruplex structure and to bind metal cations (Na+ and K+). Fluorescence spectra of G-quadruplex FRET probes at the monolayer interface are reported for the first time. Investigations included film balance measurements (π-A isotherms) and fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter. The effect of the presence of DODAB monolayer, metal cations and the surface pressure of monolayer on spectral behavior of FRET probes were examined. Adsorption of probe at the cationic monolayer interface resulted in the FRET signal enhancement even in the absence of metal cations. Variation in the monolayer surface pressure exerted rather modest effect on the spectral properties of probes. The fluorescence energy transfer efficiency of monolayer adsorbed probes increased significantly in the presence of sodium or potassium ion in subphase, which indicated that the probes retained their cation binding properties when adsorbed at the monolayer interface.

  18. Sequential data assimilation for single-molecule FRET photon-counting data

    SciTech Connect

    Matsunaga, Yasuhiro; Kidera, Akinori; Sugita, Yuji

    2015-06-07

    Data assimilation is a statistical method designed to improve the quality of numerical simulations in combination with real observations. Here, we develop a sequential data assimilation method that incorporates one-dimensional time-series data of smFRET (single-molecule Förster resonance energy transfer) photon-counting into conformational ensembles of biomolecules derived from “replicated” molecular dynamics (MD) simulations. A particle filter using a large number of “replicated” MD simulations with a likelihood function for smFRET photon-counting data is employed to screen the conformational ensembles that match the experimental data. We examine the performance of the method using emulated smFRET data and coarse-grained (CG) MD simulations of a dye-labeled polyproline-20. The method estimates the dynamics of the end-to-end distance from smFRET data as well as revealing that of latent conformational variables. The particle filter is also able to correct model parameter dependence in CG MD simulations. We discuss the applicability of the method to real experimental data for conformational dynamics of biomolecules.

  19. Sequential data assimilation for single-molecule FRET photon-counting data.

    PubMed

    Matsunaga, Yasuhiro; Kidera, Akinori; Sugita, Yuji

    2015-06-01

    Data assimilation is a statistical method designed to improve the quality of numerical simulations in combination with real observations. Here, we develop a sequential data assimilation method that incorporates one-dimensional time-series data of smFRET (single-molecule Förster resonance energy transfer) photon-counting into conformational ensembles of biomolecules derived from "replicated" molecular dynamics (MD) simulations. A particle filter using a large number of "replicated" MD simulations with a likelihood function for smFRET photon-counting data is employed to screen the conformational ensembles that match the experimental data. We examine the performance of the method using emulated smFRET data and coarse-grained (CG) MD simulations of a dye-labeled polyproline-20. The method estimates the dynamics of the end-to-end distance from smFRET data as well as revealing that of latent conformational variables. The particle filter is also able to correct model parameter dependence in CG MD simulations. We discuss the applicability of the method to real experimental data for conformational dynamics of biomolecules.

  20. Highly Sensitive Homogeneous Immunoassays Based on Construction of Silver Triangular Nanoplates-Quantum Dots FRET System

    NASA Astrophysics Data System (ADS)

    Zeng, Qinghui; Li, Qin; Ji, Wenyu; Bin, Xue; Song, Jie

    2016-05-01

    With growing concerns about health issues worldwide, elegant sensors with high sensitivity and specificity for virus/antigens (Ag) detection are urgent to be developed. Homogeneous immunoassays (HIA) are an important technique with the advantages of small sample volumes requirement and pretreatment-free process. HIA are becoming more favorable for the medical diagnosis and disease surveillance than heterogeneous immunoassays. An important subset of HIA relies on the effect of fluorescence resonance energy transfer (FRET) via a donor-acceptor (D–A) platform, e.g., quantum dots (QDs) donor based FRET system. Being an excellent plasmonic material, silver triangular nanoplates (STNPs) have unique advantages in displaying surface plasmon resonance in the visible to near infrared spectral region, which make them a better acceptor for pairing with QDs in a FRET-based sensing system. However, the reported STNPs generally exhibited broad size distributions, which would greatly restrict their application as HIA acceptor for high detection sensitivity and specificity purpose. In this work, uniform STNPs and red-emitting QDs are firstly applied to construct FRET nanoplatform in the advanced HIA and further be exploited for analyzing virus Ag. The uniform STNPs/QDs nanoplatform based medical sensor provides a straightforward and highly sensitive method for Ag analysis in homogeneous form.

  1. Two New FRET Imaging Measures: Linearly Proportional to and Highly Contrasting the Fraction of Active Molecules

    PubMed Central

    Yamao, Masataka; Aoki, Kazuhiro; Yukinawa, Naoto; Ishii, Shin; Matsuda, Michiyuki; Naoki, Honda

    2016-01-01

    We developed two new FRET imaging measures for intramolecular FRET biosensors, called linearly proportional (LP) and highly contrasting (HC) measures, which can be easily calculated by the fluorescence intensities of donor and acceptor as a ratio between their weighted sums. As an alternative to the conventional ratiometric measure, which non-linearly depends on the fraction of active molecule, we first developed the LP measure, which is linearly proportional to the fraction of active molecules. The LP measure inherently unmixes bleed-through signals and is robust against fluorescence noise. By extending the LP measure, we furthermore designed the HC measure, which provides highly contrasting images of the molecular activity, more than the ratiometric measure. In addition to their advantages, these measures are insensitive to the biosensor expression level, which is a fundamental property of the ratiometric measure. Using artificial data and FRET imaging data, we showed that the LP measure effectively represents the fraction of active molecules and that the HC measure improves visual interpretability by providing high contrast images of molecular activity. Therefore, the LP and HC measures allow us to gain more quantitative and qualitative insights from FRET imaging than the ratiometric measure. PMID:27780260

  2. Tracing the conformational changes in BSA using FRET with environmentally-sensitive squaraine probes

    NASA Astrophysics Data System (ADS)

    Govor, Iryna V.; Tatarets, Anatoliy L.; Obukhova, Olena M.; Terpetschnig, Ewald A.; Gellerman, Gary; Patsenker, Leonid D.

    2016-06-01

    A new potential method of detecting the conformational changes in hydrophobic proteins such as bovine serum albumin (BSA) is introduced. The method is based on the change in the Förster resonance energy transfer (FRET) efficiency between protein-sensitive fluorescent probes. As compared to conventional FRET based methods, in this new approach the donor and acceptor dyes are not covalently linked to protein molecules. Performance of the new method is demonstrated using the protein-sensitive squaraine probes Square-634 (donor) and Square-685 (acceptor) to detect the urea-induced conformational changes of BSA. The FRET efficiency between these probes can be considered a more sensitive parameter to trace protein unfolding as compared to the changes in fluorescence intensity of each of these probes. Addition of urea followed by BSA unfolding causes a noticeable decrease in the emission intensities of these probes (factor of 5.6 for Square-634 and 3.0 for Square-685), and the FRET efficiency changes by a factor of up to 17. Compared to the conventional method the new approach therefore demonstrates to be a more sensitive way to detect the conformational changes in BSA.

  3. An FITC-BODIPY FRET couple: application to selective, ratiometric detection and bioimaging of cysteine.

    PubMed

    Ma, Dong Hee; Kim, Dokyoung; Akisawa, Takuya; Lee, Kyung-Ha; Kim, Kyong-Tai; Ahn, Kyo Han

    2015-04-01

    A novel FRET couple of fluorescein is disclosed, and it was readily constructed by conjugating an amino-BODIPY dye, a new FRET donor, with fluorescein isocyanate. Its potential was demonstrated by a fluorescence sensing system for cysteine, which was prepared by introducing acryloyl groups to the fluorescein moiety. The FRET probe exhibited promising ratiometric response to cysteine with high selectivity and sensitivity in a buffer solution containing acetonitrile at a physiological pH of 7.4, but showed slow reactivity. This slow response was solved by addition of a surfactant, thus allowing ratiometric imaging and determination of the endogenous level of cysteine in cells in HEPES buffer, by confocal fluorescence microscopy. Imaging experiments toward various cells suggested that such aryl acrylate type probes are vulnerable to the ubiquitous esterase activity. For the selected C6 cell line, in which the esterase activity was minimal, the ratiometric quantification of cysteine level was demonstrated. The FRET probe was also applied to determine the level of cysteine in human blood plasma.

  4. Highly Sensitive Homogeneous Immunoassays Based on Construction of Silver Triangular Nanoplates-Quantum Dots FRET System.

    PubMed

    Zeng, Qinghui; Li, Qin; Ji, Wenyu; Bin, Xue; Song, Jie

    2016-01-01

    With growing concerns about health issues worldwide, elegant sensors with high sensitivity and specificity for virus/antigens (Ag) detection are urgent to be developed. Homogeneous immunoassays (HIA) are an important technique with the advantages of small sample volumes requirement and pretreatment-free process. HIA are becoming more favorable for the medical diagnosis and disease surveillance than heterogeneous immunoassays. An important subset of HIA relies on the effect of fluorescence resonance energy transfer (FRET) via a donor-acceptor (D-A) platform, e.g., quantum dots (QDs) donor based FRET system. Being an excellent plasmonic material, silver triangular nanoplates (STNPs) have unique advantages in displaying surface plasmon resonance in the visible to near infrared spectral region, which make them a better acceptor for pairing with QDs in a FRET-based sensing system. However, the reported STNPs generally exhibited broad size distributions, which would greatly restrict their application as HIA acceptor for high detection sensitivity and specificity purpose. In this work, uniform STNPs and red-emitting QDs are firstly applied to construct FRET nanoplatform in the advanced HIA and further be exploited for analyzing virus Ag. The uniform STNPs/QDs nanoplatform based medical sensor provides a straightforward and highly sensitive method for Ag analysis in homogeneous form. PMID:27198713

  5. Sequential data assimilation for single-molecule FRET photon-counting data

    NASA Astrophysics Data System (ADS)

    Matsunaga, Yasuhiro; Kidera, Akinori; Sugita, Yuji

    2015-06-01

    Data assimilation is a statistical method designed to improve the quality of numerical simulations in combination with real observations. Here, we develop a sequential data assimilation method that incorporates one-dimensional time-series data of smFRET (single-molecule Förster resonance energy transfer) photon-counting into conformational ensembles of biomolecules derived from "replicated" molecular dynamics (MD) simulations. A particle filter using a large number of "replicated" MD simulations with a likelihood function for smFRET photon-counting data is employed to screen the conformational ensembles that match the experimental data. We examine the performance of the method using emulated smFRET data and coarse-grained (CG) MD simulations of a dye-labeled polyproline-20. The method estimates the dynamics of the end-to-end distance from smFRET data as well as revealing that of latent conformational variables. The particle filter is also able to correct model parameter dependence in CG MD simulations. We discuss the applicability of the method to real experimental data for conformational dynamics of biomolecules.

  6. Paramagnetic Nanoparticles as a Platform for FRET-Based Sarcosine Picomolar Detection

    PubMed Central

    Heger, Zbynek; Cernei, Natalia; Krizkova, Sona; Masarik, Michal; Kopel, Pavel; Hodek, Petr; Zitka, Ondrej; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Herein, we describe an ultrasensitive specific biosensing system for detection of sarcosine as a potential biomarker of prostate carcinoma based on Förster resonance energy transfer (FRET). The FRET biosensor employs anti-sarcosine antibodies immobilized on paramagnetic nanoparticles surface for specific antigen binding. Successful binding of sarcosine leads to assembly of a sandwich construct composed of anti-sarcosine antibodies keeping the Förster distance (Ro) of FRET pair in required proximity. The detection is based on spectral overlap between gold-functionalized green fluorescent protein and antibodies@quantum dots bioconjugate (λex 400 nm). The saturation curve of sarcosine based on FRET efficiency (F604/F510 ratio) was tested within linear dynamic range from 5 to 50 nM with detection limit down to 50 pM. Assembled biosensor was then successfully employed for sarcosine quantification in prostatic cell lines (PC3, 22Rv1, PNT1A), and urinary samples of prostate adenocarcinoma patients. PMID:25746688

  7. Evaluation of Ti-48Al-2Cr-2Nb Under Fretting Conditions

    NASA Technical Reports Server (NTRS)

    Miyoshi, Kazuhisa; Lerch, Bradley A.; Draper, Susan L.; Raj, Sai V.

    2001-01-01

    The fretting behavior of Ti-48Al-2Cr-2Nb (y-TiAl) in contact with the nickel-base superalloy 718 was examined in air at temperatures from 296 to 823 K (23 to 550 C). The interfacial adhesive bonds between Ti-48Al-2Cr-2Nb and superalloy 718 were generally stronger than the cohesive bonds within Ti-48Al-2Cr-2Nb. The failed Ti-48Al-2Cr-2Nb debris subsequently transferred to the superalloy 718. In reference experiments conducted with Ti-6Al-4V against superalloy 718 under identical fretting conditions, the degree of transfer was greater for Ti-6A1-4V than for Ti-48Al-2Cr-2Nb. Wear of Ti-48Al-2Cr-2Nb generally decreased with increasing fretting frequency. The increasing rate of oxidation at elevated temperatures led to a drop in wear at 473 K. However, fretting wear increased as the temperature was increased from 473 to 823 K. At 723 and 823 K, oxide film disruption generated cracks, loose wear debris, and pits on the Ti-48Al-2Cr-2Nb wear surface. Both increasing slip amplitude and increasing load tended to produce more metallic wear debris, causing severe abrasive wear in the contacting metals.

  8. Real-time submillisecond single-molecule FRET dynamics of freely diffusing molecules with liposome tethering

    NASA Astrophysics Data System (ADS)

    Kim, Jae-Yeol; Kim, Cheolhee; Lee, Nam Ki

    2015-04-01

    Single-molecule fluorescence resonance energy transfer (smFRET) is one of the powerful techniques for deciphering the dynamics of unsynchronized biomolecules. However, smFRET is limited in its temporal resolution for observing dynamics. Here, we report a novel method for observing real-time dynamics with submillisecond resolution by tethering molecules to freely diffusing 100-nm-sized liposomes. The observation time for a diffusing molecule is extended to 100 ms with a submillisecond resolution, which allows for direct analysis of the transition states from the FRET time trace using hidden Markov modelling. We measure transition rates of up to 1,500 s-1 between two conformers of a Holliday junction. The rapid diffusional migration of Deinococcus radiodurans single-stranded DNA-binding protein (SSB) on single-stranded DNA is resolved by FRET, faster than that of Escherichia coli SSB by an order of magnitude. Our approach is a powerful method for studying the dynamics and movements of biomolecules at submillisecond resolution.

  9. Development of naturally selected and molecularly engineered intrachain and competitive FRET-aptamers and aptamer beacons.

    PubMed

    Bruno, John G; Zuniga, Miguel A; Carrillo, Maria P; Phillips, Taylor

    2011-08-01

    Several different approaches have been taken to development of homogeneous fluorescent aptamer assays including end-labeled beacons and signaling aptamers which are intrinsically quenched by nucleotides. Two new strategies dubbed "intrachain" and "competitive" FRET-aptamer assays are summarized in this review. Intrachain and competitive FRET-aptamers can be engineered on the molecular level through a series exploratory experiments involving prior knowledge of aptamer secondary or tertiary structures and hypotheses about aptamer conformational changes. However, there is an intrinsic risk of altering aptamer affinity or specificity associated with chemical modifications of an aptamer. Natural selection methods for FRET-aptamers have also been devised to potentially obviate the chemical modification problem. The naturally selected aptamers are subjected to fluorophore (F)- and or quencher (Q)-conjugated nucleotide triphosphate (NTP) incorporation by polymerase chain reaction (PCR) with permissive polymerases such as Deep Vent exo-, but still demonstrate sensitive and specific assay performance despite modified bases, because they are ultimately selected after decoration with F and Q. This paper summarizes work in this area and presents some new examples of the engineered and naturally selected FRET-aptamers for detection of vitamin D.

  10. Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Periasamy, Ammasi; Kay, Steve A.; Day, Richard N.

    1997-05-01

    Fluorescence resonance energy transfer (FRET) imaging microscopy is a unique tool to visualize the spatiotemporal dynamics of protein interactions in living cells. Genetic vectors that encode protein fusions with green fluorescent protein (GFP) provide a method for imaging protein localization in living cells. We used FRET to study dimerization of the pituitary-specific transcription factor Pit-1 fused to GFP and BFP. A fusion protein containing GFP separated from BFP by 29 amino acids served as a positive control for FRET. Transcriptional activity of the GFP- and BFP-Pit-1 fusion proteins was demonstrated by their ability to activate the prolactin gene promoter. Using optimized excitation and emission filters, cells expressing the fluorescently-tagged Pit-1 proteins were imaged with a back- thinned, back-illuminated CCD chip that has about 50% quantum efficiency in the blue range. 2D FRET images acquired at the focal plane demonstrated Pit-1 protein associations in the nucleus of living cells. The significance of 2- and 3-D energy transfer imaging from these living cells is discussed.

  11. Corrosion and Fretting of a Modular Hip System: A Retrieval Analysis of 60 Rejuvenate Stems.

    PubMed

    De Martino, Ivan; Assini, Joseph B; Elpers, Marcella E; Wright, Timothy M; Westrich, Geoffrey H

    2015-08-01

    Femoral stems with dual-taper modularity were introduced to allow independent control of length, offset, and version. Corrosion and fretting related to micromotion at the neck-stem junction are thought to stimulate an adverse local tissue reaction (ALTR). Analysis of 60 consecutively retrieved modular-neck stem implants (Rejuvenate, Stryker) revised primarily for ALTR was done to determine the variables influencing corrosion and fretting patterns at the neck-stem interface. Taper damage evaluation was performed with stereomicrocopic analysis with two observers. Evidence of fretting and corrosion was seen at the neck-stem taper in all implants, including three implants revised for periprosthetic fractures within four weeks of the index surgery indicating that this process starts early. Femoral stems paired with the long overall neck lengths had significantly higher corrosion scores. Correlation of the corrosion severity at particular locations with the length of implantation suggests that the neck-stem junction experiences cyclic cantilever bending in vivo. The positive correlation between the length of implantation and fretting/corrosion scores bodes poorly for patients who still have this implant. Scanning electron microscopy on a subset of specimens was also performed to evaluate the black corrosion material. We strongly urge frequent follow-up exams for every patient with this particular modular hip stem.

  12. Dual FRET molecular beacons for mRNA detection in living cells.

    PubMed

    Santangelo, Philip J; Nix, Brent; Tsourkas, Andrew; Bao, Gang

    2004-01-01

    The ability to visualize in real-time the expression level and localization of specific endogenous RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we demonstrate such a capability using a pair of molecular beacons, one with a donor and the other with an acceptor fluorophore that hybridize to adjacent regions on the same mRNA target, resulting in fluorescence resonance energy transfer (FRET). Detection of the FRET signal significantly reduced false positives, leading to sensitive imaging of K-ras and survivin mRNAs in live HDF and MIAPaCa-2 cells. FRET detection gave a ratio of 2.25 of K-ras mRNA expression in stimulated and unstimulated HDF, comparable to the ratio of 1.95 using RT-PCR, and in contrast to the single-beacon result of 1.2. We further revealed intriguing details of K-ras and survivin mRNA localization in living cells. The dual FRET molecular beacons approach provides a novel technique for sensitive RNA detection and quantification in living cells.

  13. Highly Sensitive Homogeneous Immunoassays Based on Construction of Silver Triangular Nanoplates-Quantum Dots FRET System

    PubMed Central

    Zeng, Qinghui; Li, Qin; Ji, Wenyu; Bin, Xue; Song, Jie

    2016-01-01

    With growing concerns about health issues worldwide, elegant sensors with high sensitivity and specificity for virus/antigens (Ag) detection are urgent to be developed. Homogeneous immunoassays (HIA) are an important technique with the advantages of small sample volumes requirement and pretreatment-free process. HIA are becoming more favorable for the medical diagnosis and disease surveillance than heterogeneous immunoassays. An important subset of HIA relies on the effect of fluorescence resonance energy transfer (FRET) via a donor-acceptor (D–A) platform, e.g., quantum dots (QDs) donor based FRET system. Being an excellent plasmonic material, silver triangular nanoplates (STNPs) have unique advantages in displaying surface plasmon resonance in the visible to near infrared spectral region, which make them a better acceptor for pairing with QDs in a FRET-based sensing system. However, the reported STNPs generally exhibited broad size distributions, which would greatly restrict their application as HIA acceptor for high detection sensitivity and specificity purpose. In this work, uniform STNPs and red-emitting QDs are firstly applied to construct FRET nanoplatform in the advanced HIA and further be exploited for analyzing virus Ag. The uniform STNPs/QDs nanoplatform based medical sensor provides a straightforward and highly sensitive method for Ag analysis in homogeneous form. PMID:27198713

  14. Solution-Based Single-Molecule FRET Studies of K(+) Channel Gating in a Lipid Bilayer.

    PubMed

    Sadler, Emma E; Kapanidis, Achillefs N; Tucker, Stephen J

    2016-06-21

    Ion channels are dynamic multimeric proteins that often undergo multiple unsynchronized structural movements as they switch between their open and closed states. Such structural changes are difficult to measure within the context of a native lipid bilayer and have often been monitored via macroscopic changes in Förster resonance energy transfer (FRET) between probes attached to different parts of the protein. However, the resolution of this approach is limited by ensemble averaging of structurally heterogeneous subpopulations. These problems can be overcome by measurement of FRET in single molecules, but this presents many challenges, in particular the ability to control labeling of subunits within a multimeric protein with acceptor and donor fluorophores, as well as the requirement to image large numbers of individual molecules in a membrane environment. To address these challenges, we randomly labeled tetrameric KirBac1.1 potassium channels, reconstituted them into lipid nanodiscs, and performed single-molecule FRET confocal microscopy with alternating-laser excitation as the channels diffused in solution. These solution-based single-molecule FRET measurements of a multimeric ion channel in a lipid bilayer have allowed us to probe the structural changes that occur upon channel activation and inhibition. Our results provide direct evidence of the twist-to-shrink movement of the helix bundle crossing during channel gating and demonstrate how this method might be applied to real-time structural studies of ion channel gating. PMID:27332124

  15. Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching.

    PubMed

    Rodighiero, Simona; Bazzini, Claudia; Ritter, Markus; Fürst, Johannes; Botta, Guido; Meyer, Giuliano; Paulmichl, Markus

    2008-01-01

    Fluorescence resonance energy transfer (FRET) is a technique used for the study of functional interactions between molecules. The intimate vicinity between two fluorescent molecules (FRET-pair; donor and acceptor) allows for an energy transfer, which can be directly calculated as the so called FRET efficiency. This technique is used in fixed as well as living cells. Here we show first, measured by the FRET technique, that the ICln ion channel is transposed from the cytosol towards the cellular membrane in HEK cells after swelling, and second, that the calculation of the FRET efficiency by de-quenching the donor cyan-fluorescent-protein (CFP) emission due to acceptor-photobleaching leads to erroneous estimate of the FRET efficiency in fixed, mounted and sealed specimens. The acceptor photobleaching leads to a modification of the donor cyan-fluorescent-protein, which shows then a strong emission, thus mimicking functional interaction between CFP (donor) and yellow-fluorescent-protein (YFP; acceptor). Moreover, the procedure of acceptor photobleaching masks physiological (non random) interaction between molecules within the fixed, mounted and sealed cell. We show that no artifactual CFP modifications arise when using the acceptor photobleaching technique under in vivo conditions, and we offer strategies to minimize erroneous FRET efficiency calculations if cells need to be fixed. PMID:18453757

  16. A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy.

    PubMed

    Tao, Wen; Rubart, Michael; Ryan, Jennifer; Xiao, Xiao; Qiao, Chunping; Hato, Takashi; Davidson, Michael W; Dunn, Kenneth W; Day, Richard N

    2015-12-01

    The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes. PMID:26333599

  17. G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay.

    PubMed

    Semack, Ansley; Malik, Rabia U; Sivaramakrishnan, Sivaraj

    2016-01-01

    Fӧrster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between Gα subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the β2-adrenergic receptor and the Gαs C-terminus peptide upon treatment with 100 µM isoproterenol hydrochloride as previously characterized(1). Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a Gα C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution. PMID:27684955

  18. Correlative Förster Resonance Electron Transfer-Proximity Ligation Assay (FRET-PLA) Technique for Studying Interactions Involving Membrane Proteins.

    PubMed

    Ivanusic, Daniel; Denner, Joachim; Bannert, Norbert

    2016-01-01

    This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc. PMID:27479505

  19. Correlative Förster Resonance Electron Transfer-Proximity Ligation Assay (FRET-PLA) Technique for Studying Interactions Involving Membrane Proteins.

    PubMed

    Ivanusic, Daniel; Denner, Joachim; Bannert, Norbert

    2016-08-01

    This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc.

  20. Characterization of Membrane Protein Interactions in Plasma Membrane Derived Vesicles with Quantitative Imaging FRET

    PubMed Central

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2016-01-01

    CONSPECTUS Here we describe an experimental tool, termed Quantitative Imaging Förster Resonance Energy Transfer (QI-FRET), which enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles which bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), an RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor

  1. Dispersion interactions between urea and nucleobases contribute to the destabilization of RNA by urea in aqueous solution.

    PubMed

    Kasavajhala, Koushik; Bikkina, Swetha; Patil, Indrajit; MacKerell, Alexander D; Priyakumar, U Deva

    2015-03-01

    Urea has long been used to investigate protein folding and, more recently, RNA folding. Studies have proposed that urea denatures RNA by participating in stacking interactions and hydrogen bonds with nucleic acid bases. In this study, the ability of urea to form unconventional stacking interactions with RNA bases is investigated using ab initio calculations (RI-MP2 and CCSD(T) methods with the aug-cc-pVDZ basis set). A total of 29 stable nucleobase-urea stacked complexes are identified in which the intermolecular interaction energies (up to -14 kcal/mol) are dominated by dispersion effects. Natural bond orbital (NBO) and atoms in molecules (AIM) calculations further confirm strong interactions between urea and nucleobases. Calculations on model systems with multiple urea and water molecules interacting with a guanine base lead to a hypothesis that urea molecules along with water are able to form cage-like structures capable of trapping nucleic acid bases in extrahelical states by forming both hydrogen-bonded and dispersion interactions, thereby contributing to the unfolding of RNA in the presence of urea in aqueous solution.

  2. Fast Simultaneous Determination of 13 Nucleosides and Nucleobases in Cordyceps sinensis by UHPLC-ESI-MS/MS.

    PubMed

    Zong, Shi-Yu; Han, Han; Wang, Bing; Li, Ning; Dong, Tina Ting-Xia; Zhang, Tong; Tsim, Karl W K

    2015-01-01

    A reliable ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the fast simultaneous determination of 13 nucleosides and nucleobases in Cordyceps sinensis (C. sinensis) with 2-chloroadenosine as internal standard was developed and validated. Samples were ultrasonically extracted in an ice bath thrice, and the optimum analyte separation was performed on an ACQUITY UPLC(TM) HSS C18 column (100 mm × 2.1 mm, 1.8 μm) with gradient elution. All targeted analytes were separated in 5.5 min. Furthermore, all calibration curves showed good linear regression (r > 0.9970) within the test ranges, and the limits of quantitation and detection of the 13 analytes were less than 150 and 75 ng/mL, respectively. The relative standard deviations (RSDs) of intra- and inter-day precisions were <6.23%. Recoveries of the quantified analytes ranged within 85.3%-117.3%, with RSD < 6.18%. The developed UHPLC-ESI-MS/MS method was successfully applied to determine nucleosides and nucleobases in 11 batches of C. sinensis samples from different regions in China. The range for the total content in the analyzed samples was 1329-2057 µg/g. PMID:26690105

  3. A Novel [15N] Glutamine Flux using LC-MS/MS-SRM for Determination of Nucleosides and Nucleobases

    PubMed Central

    Jin, Feng; Bhowmik, Salil Kumar; Putluri, Vasanta; Gu, Franklin; Gohlke, Jie; Von Rundstedt, Friedrich Carl; Dasgupta, Subhamoy; Krishnapuram, Rashmi; O’Malley, Bert W.; Sreekumar, Arun; Putluri, Nagireddy

    2016-01-01

    The growth of cancer cells relies more on increased proliferation and autonomy compared to non-malignant cells. The rate of de novo nucleotide biosynthesis correlates with cell proliferation rates. In part, glutamine is needed to sustain high rates of cellular proliferation as a key nitrogen donor in purine and pyrimidine nucleotide biosynthesis. In addition, glutamine serves as an essential substrate for key enzymes involved in the de novo synthesis of purine and pyrimidine nucleotides. Here, we developed a novel liquid chromatography (LC-MS) to quantify glutamine-derived [15N] nitrogen flux into nucleosides and nucleobases (purines and pyrimidines). For this, DNA from 5637 bladder cancer cell line cultured in 15N labelled glutamine and then enzymatically hydrolyzed by sequential digestion. Subsequently, DNA hydrolysates were separated by LC-MS and Selected Reaction Monitoring (SRM) was employed to identify the nucleobases and nucleosides. Thus, high sensitivity and reproducibility of the method make it a valuable tool to identify the nitrogen flux primarily derived from glutamine and can be further adaptable for high throughput analysis of large set of DNA in a clinical setting. PMID:27158554

  4. Strikingly different effects of hydrogen bonding on the photodynamics of individual nucleobases in DNA: comparison of guanine and cytosine.

    PubMed

    Zelený, Tomáš; Ruckenbauer, Matthias; Aquino, Adelia J A; Müller, Thomas; Lankaš, Filip; Dršata, Tomáš; Hase, William L; Nachtigallova, Dana; Lischka, Hans

    2012-08-22

    Ab initio surface hopping dynamics calculations were performed to study the photophysical behavior of cytosine and guanine embedded in DNA using a quantum mechanical/molecular mechanics (QM/MM) approach. It was found that the decay rates of photo excited cytosine and guanine were affected in a completely different way by the hydrogen bonding to the DNA environment. In case of cytosine, the geometrical restrictions exerted by the hydrogen bonds did not influence the relaxation time of cytosine significantly due to the generally small cytosine ring puckering required to access the crossing region between excited and ground state. On the contrary, the presence of hydrogen bonds significantly altered the photodynamics of guanine. The analysis of the dynamics indicates that the major contribution to the lifetime changes comes from the interstrand hydrogen bonds. These bonds considerably restricted the out-of-plane motions of the NH(2) group of guanine which are necessary for the ultrafast decay to the ground state. As a result, only a negligible amount of trajectories decayed into the ground state for guanine embedded in DNA within the simulation time of 0.5 ps, while for comparison, the isolated guanine relaxed to the ground state with a lifetime of about 0.22 ps. These examples show that, in addition to phenomena related to electronic interactions between nucleobases, there also exist relatively simple mechanisms in DNA by which the lifetime of a nucleobase is significantly enhanced as compared to the gas phase. PMID:22845192

  5. Characterization of nucleosides and nucleobases in natural Cordyceps by HILIC-ESI/TOF/MS and HILIC-ESI/MS.

    PubMed

    Zhao, Heng-Qiang; Wang, Xiao; Li, Hong-Mei; Yang, Bin; Yang, Hong-Jun; Huang, Luqi

    2013-08-15

    A method combining hydrophilic interaction chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS) was developed for the characterization and determination of natural Cordyceps. Separation was achieved on a Waters Xbridge Amide column with gradient elution. Identification of 15 target nucleosides and nucleobases was based on retention time, UV spectra and mass measurements of the protonated molecules ([M+H]⁺) and main fragment ions (ESI-TOF/MS). Eight non-target compounds were tentatively identified by ESI-TOF/MS. The 15 target compounds were quantified by HILIC-ESI-MS/MS using time-programmed selective ion monitoring or multiple reaction monitoring in positive-ion mode under optimized mass conditions. This technique showed good linearity, repeatability and recovery. This approach was also successfully implemented in the analysis of nucleosides and nucleobases in 12 batches of natural Cordyceps samples that were collected from different regions in China. The developed HILIC-ESI-MS method exhibited clear advantages in identifying and determining highly polar bioactive components in Cordyceps, as well as their quality control.

  6. Fast Simultaneous Determination of 13 Nucleosides and Nucleobases in Cordyceps sinensis by UHPLC-ESI-MS/MS.

    PubMed

    Zong, Shi-Yu; Han, Han; Wang, Bing; Li, Ning; Dong, Tina Ting-Xia; Zhang, Tong; Tsim, Karl W K

    2015-12-04

    A reliable ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the fast simultaneous determination of 13 nucleosides and nucleobases in Cordyceps sinensis (C. sinensis) with 2-chloroadenosine as internal standard was developed and validated. Samples were ultrasonically extracted in an ice bath thrice, and the optimum analyte separation was performed on an ACQUITY UPLC(TM) HSS C18 column (100 mm × 2.1 mm, 1.8 μm) with gradient elution. All targeted analytes were separated in 5.5 min. Furthermore, all calibration curves showed good linear regression (r > 0.9970) within the test ranges, and the limits of quantitation and detection of the 13 analytes were less than 150 and 75 ng/mL, respectively. The relative standard deviations (RSDs) of intra- and inter-day precisions were <6.23%. Recoveries of the quantified analytes ranged within 85.3%-117.3%, with RSD < 6.18%. The developed UHPLC-ESI-MS/MS method was successfully applied to determine nucleosides and nucleobases in 11 batches of C. sinensis samples from different regions in China. The range for the total content in the analyzed samples was 1329-2057 µg/g.

  7. Phosphonate analogue substrates for enolase.

    PubMed

    Anderson, V E; Cleland, W W

    1990-11-20

    Phosphonate analogues in which the bridge between C-2 and phosphorus is a CH2 group are slow substrates for yeast enolase. The pH variation of the kinetic parameters for the methylene analogue of 2-phosphoglycerate suggests that the substrate binds as a dianion and that Mg2+ can bind subsequently only if a metal ligand and the catalytic base are unprotonated. Primary deuterium isotope effects of 4-8 on V/KMg, but ones of only 1.15-1.32 on V for dehydration, show that proton removal to give the carbanion intermediate largely limits V/KMg and that a slow step follows which largely limits V (presumably carbanion breakdown). Since there is a D2O solvent isotope effect on V for the reverse reaction of 5, but not an appreciable one on the forward reaction, it appears that the slow rates with phosphonate analogues result from the fact that the carbanion intermediate is more stable than that formed from the normal substrates, and its reaction in both directions limits V. Increased stability as a result of replacement of oxygen by carbon at C-2 of the carbanion is the expected chemical behavior. PMID:2271661

  8. Substrate analogues for isoprenoid enzymes

    SciTech Connect

    Stremler, K.E.

    1987-01-01

    Diphosphonate analogues of geranyl diphosphate, resistant to degradation by phosphatases, were found to be alternate substrates for the reaction with farnesyl diphosphate synthetase isolated from avian liver. The difluoromethane analogue was shown to be the better alternate substrate, in agreement with solvolysis results which indicate that the electronegativity of the difluoromethylene unit more closely approximates that of the normal bridging oxygen. The usefulness of the C/sub 10/ difluoro analogue, for detecting low levels of isoprenoid enzymes in the presence of high levels of phosphatase activity, was demonstrated with a cell-free preparation from lemon peel. A series of C/sub 5/ through C/sub 15/ homoallylic and allylic diphosphonates, as well as two 5'-nucleotide diphosphonates, was prepared in high overall yield using the activation-displacement sequence. Radiolabeled samples of several of the allylic diphosphonates were prepared with tritium located at C1. A series of geraniols, stereospecifically deuterated at C1, was prepared. The enantiomeric purities and absolute configurations were determined by derivatization as the mandelate esters for analysis by /sup 1/H NMR. The stereochemistry of the activation-displacement sequence was examined using C1-deuterated substrates.

  9. Policy issues in space analogues

    NASA Astrophysics Data System (ADS)

    Auger, Robin N.; Facktor, Debra D.

    Space mission planning is increasingly focusing on destinations beyond Earth orbit. Advancements in technology will inevitably be required to enable long-duration human spaceflight missions, and breakthroughs in the policy arena will also be needed to achieve success in such missions. By exploring how policy issues have been addressed in analogous extreme environments, policymakers can develop a framework for addressing these issues as they apply to long-term human spaceflight. Policy issues that need to be addressed include: crew selection, training, organization, and activities, medical testing, illness, injury, and death; communication; legal accountability and liability; mission safety and risk management; and environmental contamination. This paper outlines the approach of a study underway by The George Washington University and ANSER to examine how these policy issues have been addressed in several analogues and how the experiences of these analogues can help formulate policies for long-duration human spaceflight missions. Analogues being studied include Antarctic bases, submarine voyages, undersea stations, Biosphere 2, and the U.S. Skylab and Russian Mir space stations.

  10. A long-wavelength quantum dot-concentric FRET configuration: characterization and application in a multiplexed hybridization assay.

    PubMed

    Li, Jia Jun; Algar, W Russ

    2016-06-21

    Quantum dot-based concentric Förster resonance energy transfer (cFRET) is a promising modality for the development of multifunctional fluorescent probes for bioanalysis and bioimaging. To date, the scope of cFRET has been largely limited to a prototypical configuration with a particular combination of quantum dot (QD) and fluorescent dyes linked through peptides. Expansion of the scope of cFRET is critical for its further development. Here, we expand the scope of cFRET in two capacities. First, we design and characterize a new long-wavelength cFRET configuration that combines red- and deep-red fluorescent dyes, Alexa Fluor 633 and Alexa Fluor 680, with an orange-emitting QD. Sequential and competitive energy transfer pathways are characterized through a rate analysis, where the balance of these rates more strongly favours competitive energy transfer in the new long-wavelength configuration versus sequential energy transfer in the previous prototypical configuration. Although the new cFRET configuration is more susceptible to photobleaching, its superior brightness and longer-wavelength excitation and emission provide an order of magnitude higher signal-to-background ratios in biological matrices (e.g., serum, blood) than the previous prototypical configuration. Second, we demonstrate that an oligonucleotide-linked, long-wavelength cFRET configuration has energy transfer similar to an analogous peptide-linked configuration, where the oligonucleotide-linked cFRET configuration can be combined with toehold-mediated strand displacement for the multiplexed detection of unlabeled nucleic acid targets as a single vector. Overall, this work establishes the general applicability of cFRET and introduces new strategies for its bioanalytical application. PMID:27048838

  11. Preliminary Study on Fatigue Strengths of Fretted Ti-48Al-2Cr-2Nb

    NASA Technical Reports Server (NTRS)

    Miyoshi, Kazuhisa; Lerch, Bradley A.; Draper, Susan L.

    2002-01-01

    The fatigue behavior (stress-life curve) of gamma titanium aluminide (Ti-48Al-2Cr-2Nb, atomic percent) was examined by conducting two tests: first, a fretting wear test with a fatigue specimen in contact with a typical nickel-based superalloy contact pad in air at temperatures of 296 and 823 K and second, a high-cycle fatigue test of the prefretted Ti-48Al-2Cr-2Nb fatigue specimen at 923 K. Reference high-cycle fatigue tests were also conducted with unfretted Ti-48Al-2Cr-2Nb specimens at 923 K. All Ti-48Al-2Cr-2Nb fatigue specimens were machined from cast slabs. The results indicate that the stress-life results for the fretted Ti-48Al-2Cr-2Nb specimens exhibited a behavior similar to those of the unfretted Ti-48Al-2Cr-2Nb specimens. The values of maximum stress and life for the fretted specimens were almost the same as those for the unfretted specimens. The resultant stress-life curve for the unfretted fatigue specimens was very flat. The flat appearance in the stress-life curve of the unfretted specimens is attributed to the presence of a high density of casting pores. The fatigue strengths of both the fretted and unfretted specimens can be significantly affected by the presence of this porosity, which can decrease the fatigue life of Ti-48Al-2Cr-2Nb. The presence of the porosity made discerning the effect of fretting damage on fatigue strength and life of the specimens difficult.

  12. FRET imaging of diatoms expressing a biosilica-localized ribose sensor.

    PubMed

    Marshall, Kathryn E; Robinson, Errol W; Hengel, Shawna M; Paša-Tolić, Ljiljana; Roesijadi, Guritno

    2012-01-01

    Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation.

  13. Temporal Data Set Reduction Based on D-Optimality for Quantitative FLIM-FRET Imaging

    PubMed Central

    Omer, Travis; Intes, Xavier; Hahn, Juergen

    2015-01-01

    Fluorescence lifetime imaging (FLIM) when paired with Förster resonance energy transfer (FLIM-FRET) enables the monitoring of nanoscale interactions in living biological samples. FLIM-FRET model-based estimation methods allow the quantitative retrieval of parameters such as the quenched (interacting) and unquenched (non-interacting) fractional populations of the donor fluorophore and/or the distance of the interactions. The quantitative accuracy of such model-based approaches is dependent on multiple factors such as signal-to-noise ratio and number of temporal points acquired when sampling the fluorescence decays. For high-throughput or in vivo applications of FLIM-FRET, it is desirable to acquire a limited number of temporal points for fast acquisition times. Yet, it is critical to acquire temporal data sets with sufficient information content to allow for accurate FLIM-FRET parameter estimation. Herein, an optimal experimental design approach based upon sensitivity analysis is presented in order to identify the time points that provide the best quantitative estimates of the parameters for a determined number of temporal sampling points. More specifically, the D-optimality criterion is employed to identify, within a sparse temporal data set, the set of time points leading to optimal estimations of the quenched fractional population of the donor fluorophore. Overall, a reduced set of 10 time points (compared to a typical complete set of 90 time points) was identified to have minimal impact on parameter estimation accuracy (≈5%), with in silico and in vivo experiment validations. This reduction of the number of needed time points by almost an order of magnitude allows the use of FLIM-FRET for certain high-throughput applications which would be infeasible if the entire number of time sampling points were used. PMID:26658308

  14. FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

    SciTech Connect

    Marshall, Kathryn E.; Robinson, E. W.; Hengel, Shawna M.; Pasa-Tolic, Ljiljana; Roesijadi, Guritno

    2012-03-21

    Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification that enables localization of recombinant proteins in the biosilica cell wall. Our objective was to functionalize diatom biosilica with a reagent-less biosensor with FRET-based imaging capabilities for signaling. The design of the fusion protein conferring these properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the recombinant chimeric protein was first confirmed in E. coli-expressed proteins, prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of CRY showed 95% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica targeting exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 {mu}M and 142.8 {mu}M, respectively. The addition of the silaffin tag for biosilica localization did not influence the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated biosilica in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand binding and conformational change for FRET-signal generation.

  15. Intracellular cascade FRET for temperature imaging of living cells with polymeric ratiometric fluorescent thermometers.

    PubMed

    Hu, Xianglong; Li, Yang; Liu, Tao; Zhang, Guoying; Liu, Shiyong

    2015-07-22

    Intracellular temperature plays a prominent role in cellular functions and biochemical activities inside living cells, but effective intracellular temperature sensing and imaging is still in its infancy. Herein, thermoresponsive double hydrophilic block copolymers (DHBCs)-based fluorescent thermometers were fabricated to investigate their application in intracellular temperature imaging. Blue-emitting coumarin monomer, CMA, green-emitting 7-nitro-2,1,3-benzoxadiazole (NBD) monomer, NBDAE, and red-emitting rhodamine B monomer, RhBEA, were copolymerized separately with N-isopropylacrylamide (NIPAM) to afford dye-labeled PEG-b-P(NIPAM-co-CMA), PEG-b-P(NIPAM-co-NBDAE), and PEG-b-P(NIPAM-co-RhBEA). Because of the favorable fluorescence resonance energy transfer (FRET) potentials between CMA and NBDAE, NBDAE and RhBEA, as well as the slight tendency between CMA and RhBEA fluorophore pairs, three polymeric thermometers based on traditional one-step FRET were fabricated by facile mixing two of these three fluorescent DHBCs, whereas exhibiting limited advantages. Thus, two-step cascade FRET among three polymeric fluorophores was further interrogated, in which NBD acted as a bridging dye by transferring energy from CMA to RhBEA. Through the delicate optimization of the molar contents of three polymeric components, a ∼8.4-fold ratio change occurred in the temperature range of 20-44 °C, and the detection sensitivity improved significantly, reached as low as ∼0.4 °C, which definitely outperformed other one-step FRET thermometers in the intracellular temperature imaging of living cells. To our knowledge, this work represents the first example of polymeric ratiometric thermometer employing thermoresponsive polymer-based cascade FRET mechanism.

  16. Diagnosis of canine leptospirosis by a highly sensitive FRET-PCR targeting the lig genes.

    PubMed

    Xu, Chuanling; Loftis, Amanda; Ahluwalia, Sudhir K; Gao, Dongya; Verma, Ashutosh; Wang, Chengming; Kaltenboeck, Bernhard

    2014-01-01

    Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-PCR to detect common pathogenic leptospires in dogs, including Leptospira interrogans serovars Autumnalis, Canicola, Copenhageni (Icterohaemorrhagiae serogroup) and Pomona, and Leptospira kirschneri serovar Grippotyphosa. This PCR targets the lig genes, exclusively found in the pathogenic Leptospira species but not in saprophytic species (L. biflexa). A robust, high-stringency step-down real-time platform was coupled to the highly specific detection of leptospiral DNA by fluorescently labeled FRET probes. This enabled the detection of a single copy of the lig gene in a PCR containing DNA from up to 50 µL canine blood or 400 µL urine. Sensitivity determination by use of limiting serial dilutions of extracted leptospiral DNA indicated that the lig FRET-PCR we established was almost 100-fold more sensitive than the widely accepted lipL32 SYBR assay and 10-fold more sensitive than a 16S rRNA TaqMan assay. Application of this method to 207 dogs with potential leptospiral infection enabled us to diagnose three cases of canine leptospirosis characterized by low amounts of leptospiral DNA in body fluids. Detection of canine leptospirosis with the lig FRET-PCR was more sensitive with the lig FRET-PCR than with the 16S rRNA TaqMan PCR, which detected only 2 of the 3 cases, and the lipL32 SYBR PCR, which detected none of the 3 dogs with leptospirosis. PMID:24586833

  17. Parallel multispot smFRET analysis using an 8-pixel SPAD array

    PubMed Central

    Ingargiola, A.; Colyer, R. A.; Kim, D.; Panzeri, F.; Lin, R.; Gulinatti, A.; Rech, I.; Ghioni, M.; Weiss, S.; Michalet, X.

    2012-01-01

    Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules. PMID:24382989

  18. Connectivity patterns and rotamer states of nucleobases determine acid-base properties of metalated purine quartets.

    PubMed

    Lüth, Marc Sven; Freisinger, Eva; Kampf, Gunnar; Garijo Anorbe, Marta; Griesser, Rolf; Operschall, Bert P; Sigel, Helmut; Lippert, Bernhard

    2015-07-01

    Potentiometric pH titrations and pD dependent (1)H NMR spectroscopy have been applied to study the acidification of the exocyclic amino group of adenine (A) model nucleobases (N9 position blocked by alkyl groups) when carrying trans-a2Pt(II) (with a=NH3 or CH3NH2) entities both at N1 and N7 positions. As demonstrated, in trinuclear complexes containing central A-Pt-A units, it depends on the connectivity pattern of the adenine bases (N7/N7 or N1/N1) and their rotamer states (head-head or head-tail), how large the acidifying effect is. Specifically, a series of trinuclear complexes with (A-N7)-Pt-(N7-A) and (A-N1)-Pt-(N1-A) cross-linking patterns and terminal 9-alkylguanine ligands (9MeGH, 9EtGH) have been analyzed in this respect, and it is shown that, for example, the 9MeA ligands in trans-,trans-,trans-[Pt(NH3)2(N7-9MeA-N1)2{Pt(NH3)2(9EtGH-N7)}2](ClO4)6·6H2O (4a) and trans-,trans-,trans-[Pt(NH3)2(N7-9EtA-N1)2{Pt(CH3NH2)2(9-MeGH-N7)}2](ClO4)6·3H2O (4b) are more acidic, by ca. 1.3 units (first pKa), than the linkage isomer trans-,trans-,trans-[Pt(CH3NH2)2(N1-9MeA-N7)2{Pt(NH3)2(9MeGH-N7)}2](NO3)6·6.25H2O (1b). Overall, acidifications in these types of complexes amount to 7-9 units, bringing the pKa values of such adenine ligands in the best case close to the physiological pH range. Comparison with pKa values of related trinuclear Pt(II) complexes having different co-ligands at the Pt ions, confirms this picture and supports our earlier proposal that the close proximity of the exocyclic amino groups in a head-head arrangement of (A-N7)-Pt-(N7-A), and the stabilization of the resulting N6H(-)⋯H2N6 unit, is key to this difference.

  19. Connectivity patterns and rotamer states of nucleobases determine acid-base properties of metalated purine quartets.

    PubMed

    Lüth, Marc Sven; Freisinger, Eva; Kampf, Gunnar; Garijo Anorbe, Marta; Griesser, Rolf; Operschall, Bert P; Sigel, Helmut; Lippert, Bernhard

    2015-07-01

    Potentiometric pH titrations and pD dependent (1)H NMR spectroscopy have been applied to study the acidification of the exocyclic amino group of adenine (A) model nucleobases (N9 position blocked by alkyl groups) when carrying trans-a2Pt(II) (with a=NH3 or CH3NH2) entities both at N1 and N7 positions. As demonstrated, in trinuclear complexes containing central A-Pt-A units, it depends on the connectivity pattern of the adenine bases (N7/N7 or N1/N1) and their rotamer states (head-head or head-tail), how large the acidifying effect is. Specifically, a series of trinuclear complexes with (A-N7)-Pt-(N7-A) and (A-N1)-Pt-(N1-A) cross-linking patterns and terminal 9-alkylguanine ligands (9MeGH, 9EtGH) have been analyzed in this respect, and it is shown that, for example, the 9MeA ligands in trans-,trans-,trans-[Pt(NH3)2(N7-9MeA-N1)2{Pt(NH3)2(9EtGH-N7)}2](ClO4)6·6H2O (4a) and trans-,trans-,trans-[Pt(NH3)2(N7-9EtA-N1)2{Pt(CH3NH2)2(9-MeGH-N7)}2](ClO4)6·3H2O (4b) are more acidic, by ca. 1.3 units (first pKa), than the linkage isomer trans-,trans-,trans-[Pt(CH3NH2)2(N1-9MeA-N7)2{Pt(NH3)2(9MeGH-N7)}2](NO3)6·6.25H2O (1b). Overall, acidifications in these types of complexes amount to 7-9 units, bringing the pKa values of such adenine ligands in the best case close to the physiological pH range. Comparison with pKa values of related trinuclear Pt(II) complexes having different co-ligands at the Pt ions, confirms this picture and supports our earlier proposal that the close proximity of the exocyclic amino groups in a head-head arrangement of (A-N7)-Pt-(N7-A), and the stabilization of the resulting N6H(-)⋯H2N6 unit, is key to this difference. PMID:25773716

  20. Dideoxy nucleoside triphosphate (ddNTP) analogues: Synthesis and polymerase substrate activities of pyrrolidinyl nucleoside triphosphates (prNTPs).

    PubMed

    Gade, Chandrasekhar Reddy; Dixit, Manjusha; Sharma, Nagendra K

    2016-09-15

    The dideoxynucleoside triphosphates (ddNTPs) terminate the bio-polymerization of DNA and become essential chemical component of DNA sequencing technology which is now basic tool for molecular biology research. In this method the radiolabeled or fluorescent dye labeled ddNTP analogues are being used for DNA sequencing by detection of the terminated DNA fragment after single labeled ddNTP incorporation into DNA under PCR conditions. This report describes the syntheses of rationally designed novel amino-functionalized ddNTP analogue such as Pyrrolidine nucleoside triphosphates (prNTPs), and their polymerase activities with DNA polymerase by LC-MS and Gel-electrophoretic techniques. The Mass and PAGE analyses strongly support the incorporation of prNTPs into DNA oligonucleotide with Therminator DNA polymerase as like control substrate ddNTP. As resultant the DNA oligonucleotide are functionalized as amine group by prNTP incorporation with polymerase. Hence prNTPs provide opportunities to prepare demandable conjugated DNA with other biomolecules/dyes/fluorescence molecule without modifying nucleobase structure. PMID:27377861

  1. Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design

    PubMed Central

    Foller Larsen, Anders; Dumat, Blaise; Wranne, Moa S.; Lawson, Christopher P.; Preus, Søren; Bood, Mattias; Gradén, Henrik; Marcus Wilhelmsson, L.; Grøtli, Morten

    2015-01-01

    Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (εΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs. PMID:26227585

  2. Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design

    NASA Astrophysics Data System (ADS)

    Foller Larsen, Anders; Dumat, Blaise; Wranne, Moa S.; Lawson, Christopher P.; Preus, Søren; Bood, Mattias; Gradén, Henrik; Marcus Wilhelmsson, L.; Grøtli, Morten

    2015-07-01

    Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (ɛΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.

  3. HILIC-UPLC-MS/MS combined with hierarchical clustering analysis to rapidly analyze and evaluate nucleobases and nucleosides in Ginkgo biloba leaves.

    PubMed

    Yao, Xin; Zhou, Guisheng; Tang, Yuping; Guo, Sheng; Qian, Dawei; Duan, Jin-Ao

    2015-02-01

    Ginkgo biloba leaf extract has been widely used in dietary supplements and more recently in some foods and beverages. In addition to the well-known flavonol glycosides and terpene lactones, G. biloba leaves are also rich in nucleobases and nucleosides. To determine the content of nucleobases and nucleosides in G. biloba leaves at trace levels, a reliable method has been established by using hydrophilic interaction ultra performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HILIC-UPLC-TQ-MS/MS) working in multiple reaction monitoring mode. Eleven nucleobases and nucleosides were simultaneously determined in seven min. The proposed method was fully validated in terms of linearity, sensitivity, and repeatability, as well as recovery. Furthermore, hierarchical clustering analysis (HCA) was performed to evaluate and classify the samples according to the contents of the eleven chemical constituents. The established approach could be helpful for evaluation of the potential values as dietary supplements and the quality control of G. biloba leaves, which might also be utilized for the investigation of other medicinal herbs containing nucleobases and nucleosides.

  4. Theoretical Study of the Photophysics of 8-Vinylguanine, an Isomorphic Fluorescent Analogue of Guanine.

    PubMed

    Kochman, Michał A; Pola, Martina; Miller, R J Dwayne

    2016-08-11

    Paving the way for the application of the algebraic-diagrammatic construction scheme of second-order (ADC(2)) to systems based on the guanine chromophore, we demonstrate the this excited-state electronic structure method provides a realistic description of the photochemistry of 9H-guanine, in close agreement with the benchmark provided by the CASPT2 method. We then proceed to apply the ADC(2) method to the photochemistry of 8-vinylguanine (8vG), a minimally modified analogue of guanine which, unlike the naturally occurring nucleobase, displays intense fluorescence, indicative of a much longer-lived excited electronic state. The emissive electronic state of 8vG is identified as an ππ*-type intramolecular charge transfer (ICT) state, in which a charge of roughly -0.2 e is transferred from the guanine moiety onto the vinyl substituent. The main radiationless deactivation pathway competing with fluorescence is predicted to involve the molecule leaving the minimum on the ICT ππ* state, and reaching a region of the S1 adiabatic state where it resembles the La ππ* state of unmodified 9H-guanine. The topology of the La ππ* region of the S1 state favors subsequent internal conversion at a crossing seam with the ground electronic state. The sensitivity of this process to environment polarity may explain the experimentally observed fluorescence quenching of 8vG upon incorporation in single- and double-stranded DNA. PMID:27427772

  5. Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

    NASA Astrophysics Data System (ADS)

    Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

    2015-10-01

    DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR

  6. Controlled synthesis of Pt nanoparticles array through electroreduction of cisplatin bound at nucleobases terminated surface and application into H2O2 sensing.

    PubMed

    Ji, Shujun; Guo, Qingqing; Yue, Qiaoli; Wang, Lei; Wang, Huaisheng; Zhao, Jinsheng; Dong, Ruixin; Liu, Jifeng; Jia, Jianbo

    2011-01-15

    Fabrication of sub-monolayer array of Pt nanoparticles (PtNPs) assembled at nucleobases terminated layers and their application into H(2)O(2) and glucose sensing were reported. To prepare such a PtNPs assembly, 3-mercaptopropionic acid (MPA), Zr(4+), nucleotide-5'-monophosphate (NTMP including guanosine, adenosine, cytidine, uridine-5'-monophosphate, and abbreviations were GMP, AMP, CMP, UMP, respectively) were adsorbed onto Au substrate sequentially to form nucleobases terminated surface and Zr(4+) acted as binder to link carboxylic and phosphoric groups (NTMP/Zr(4+)/MPA/Au). Complexation of cisplatin, cis-Pt(NH(3))(2)Cl(2), with terminated nucleobases and following electrochemical reduction of surface-bound cisplatin gave PtNPs attached surface. Different PtNPs coverage or particle density was obtained depending on the NTMP used and decreased in the order: PtNPs/GMP/Zr(4+)/MPA/Au>PtNPs/AMP/Zr(4+)/MPA/Au>PtNPs/CMP/Zr(4+)/MPA/Au>PtNPs/UMP/Zr(4+)/MPA/Au. The surface loading of Pt was between 160 and 16 ng/cm(2). The as prepared PtNPs can be used as electrocatalysts for H(2)O(2) sensing (detection limit of H(2)O(2)<100 nM) and the sensitivity increased with decreasing PtNPs density. After adsorption of glucose oxidase, the modified electrode can be used as enzymatic electrode for glucose sensing and a detection limit of 38.5 μM was achieved. This study provided an example of fabricating PtNP arrays utilising surface complexation of cisplatin with nucleobases. The advantage of this method is that the NP density can be controlled through changing nucleobases or Pt complexes used to obtain suitable kinetics of the complexation reactions. Additionally, the PtNPs sub-monolayer as prepared has high sensitivity for H(2)O(2) sensing even at a very low loading of Pt.

  7. Determination of the nucleosides and nucleobases in Tuber samples by dispersive solid-phase extraction combined with liquid chromatography-mass spectrometry.

    PubMed

    Liu, Ping; Li, Yuan-Yuan; Li, Hong-Mei; Wan, Duan-Ji; Tang, Ya-Jie

    2011-02-21

    A simple, fast and inexpensive method based on dispersive solid phase extraction (DSPE) combined with LC-MS was developed for simultaneous determination of 7 nucleosides and nucleobases (i.e., adenine, hypoxanthine, uridine, adenosine, guanine, guanosine, and inosine) in Tuber fruiting-bodies and fermentation mycelia. The DSPE procedure was firstly introduced to remove the protein interference from sample solutions, and D3520 macroporous resin was chosen as the DSPE sorbent because of its high removal capability on protein interferences, but low adsorption rate on analytes. Besides, key parameters on DSPE procedure (i.e., macroporous resin type, macroporous resin amount, methanol concentration, and vortex time) were optimized, and the protein removal efficacy could achieve about 95% after the process optimization. Though the method validation test, the DSPE-LC-MS method was confirmed to be precise, accurate and sensitive, and the column blinding problem was solved successfully. By using this established method, the total amount of nucleosides and nucleobases in the fermentation mycelia was determined to range from 4881.5 to 12,592.9μgg⁻¹, which was about 2-25 times higher than the fruiting-bodies (from 498.1 to 2274.1μgg⁻¹). The formulation of nucleosides and nucleobases in the fermentation mycelia maintained relatively constant, while the formulation in Tuber fruiting-bodies varied significantly with their species. Hierarchical cluster analysis (HCA) showed the formulation similarity of nucleosides and nucleobases between Tuber fermentation mycelia and the fruiting-bodies of Tuber indicum and Tuber himalayense. From the viewpoint of nucleosides and nucleobases, this work confirms the potentiality of Tuber fermentation mycelia as the alternative resource for its fruiting-bodies.

  8. Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

    PubMed Central

    Warren, Sean C.; Margineanu, Anca; Katan, Matilda; Dunsby, Chris; French, Paul M. W.

    2015-01-01

    Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. PMID:26133241

  9. Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells.

    PubMed

    Warren, Sean C; Margineanu, Anca; Katan, Matilda; Dunsby, Chris; French, Paul M W

    2015-06-30

    Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3'-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3'-phosphoinositide accumulation.

  10. Novel multistep BRET-FRET energy transfer using nanoconjugates of firefly proteins, quantum dots, and red fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Alam, Rabeka; Zylstra, Joshua; Fontaine, Danielle M.; Branchini, Bruce R.; Maye, Mathew M.

    2013-05-01

    Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated.Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated. Electronic supplementary information (ESI) available: Experimental details, Fig. S1 and Table S1-S4. See DOI: 10.1039/c3nr01842c

  11. Triazolyl-donor-acceptor chromophore-decorated unnatural amino acids and peptides: FRET events in a β-turn conformation.

    PubMed

    Bag, Subhendu Sekhar; Jana, Subhashis; Yashmeen, Afsana; Senthilkumar, K; Bag, Raghunath

    2014-01-14

    The β-turn conformation and FRET process were established in the designed tripeptide containing fluorescent triazolyl donor and acceptor-decorated unnatural amino acids separated by a natural alanine.

  12. Application of the FRET method for monitoring the dynamics of caspase-3 activation during apoptosis in living cells

    NASA Astrophysics Data System (ADS)

    Chen, Tongsheng; Xing, Da

    2005-01-01

    Activation of caspase-3 is a central event in apoptosis. A fluorescence techniques, fluorescence resonance energy transfer (FRET), was used to study the dynamic of caspase-3 activation during apoptosis induced by tumor necrosis factor TNF-α in living cells. The FRET probe consists a CFP (cyan fluorescent protein) and a Venus (YFP mutant, yellow fluorescent protein) with a specialized linker containing the caspase-3 cleavage sequence: DEVD (Luo et al., 2001). Human lung adenocarcinoma cell line (ASTC-a-1) were stably expressed with the FRET probe and then were treated by TNF-α, respectively. Experimental results showed that FRET could monitor more insensitively the dynamic of caspase-3 activation in real-time in vivo, and this technique will be highly useful for correlating the caspase-3 activation with other apoptotic events and for rapid-screening of potential drugs that may target the apoptotic process.

  13. FUNCTION GENERATOR FOR ANALOGUE COMPUTERS

    DOEpatents

    Skramstad, H.K.; Wright, J.H.; Taback, L.

    1961-12-12

    An improved analogue computer is designed which can be used to determine the final ground position of radioactive fallout particles in an atomic cloud. The computer determines the fallout pattern on the basis of known wind velocity and direction at various altitudes, and intensity of radioactivity in the mushroom cloud as a function of particle size and initial height in the cloud. The output is then displayed on a cathode-ray tube so that the average or total luminance of the tube screen at any point represents the intensity of radioactive fallout at the geographical location represented by that point. (AEC)

  14. The Canadian Analogue Research Network (CARN): Opportunities for Terrestrial Analogue Studies in Canada and Abroad

    NASA Astrophysics Data System (ADS)

    Hipkin, V.; Osinski, G. R.; Berinstain, A.; Léveillé, R.

    2007-03-01

    We will present an overview of the Canadian Analogue Research Network (CARN), including a description of the various analogue sites in CARN, potential new sites, and a discussion regarding how CARN is applicable to the global exploration strategy.

  15. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting.

    PubMed

    Bajar, Bryce T; Wang, Emily S; Lam, Amy J; Kim, Bongjae B; Jacobs, Conor L; Howe, Elizabeth S; Davidson, Michael W; Lin, Michael Z; Chu, Jun

    2016-01-01

    Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude. PMID:26879144

  16. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting

    PubMed Central

    Bajar, Bryce T.; Wang, Emily S.; Lam, Amy J.; Kim, Bongjae B.; Jacobs, Conor L.; Howe, Elizabeth S.; Davidson, Michael W.; Lin, Michael Z.; Chu, Jun

    2016-01-01

    Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude. PMID:26879144

  17. FRET-based nanoscale point-to-point and broadcast communications with multi-exciton transmission and channel routing.

    PubMed

    Kuscu, Murat; Akan, Ozgur B

    2014-09-01

    Nanoscale communication based on Förster Resonance Energy Transfer (FRET) enables nanoscale single molecular devices to communicate with each other utilizing excitons generated on fluorescent molecules as information carriers. Based on the point-to-point single-exciton FRET-based nanocommunication model, we investigate the multiple-exciton case for point-to-point and broadcast communications following an information theoretical approach and conducting simulations through Monte Carlo approach. We demonstrate that the multi-exciton transmission significantly improves the channel reliability and the range of the communication up to tens of nanometers for immobile nanonodes providing high data transmission rates. Furthermore, our analyses indicate that multi-exciton transmission enables broadcasting of information from a transmitter nanonode to many receiver nanonodes pointing out the potential of FRET-based communication to extend over nanonetworks. In this study, we also propose electrically and chemically controllable routing mechanisms exploiting the strong dependence of FRET rate on spectral and spatial characteristics of fluorescent molecules. We show that the proposed routing mechanisms enable the remote control of information flow in FRET-based nanonetworks. The high transmission rates obtained by multi-exciton scheme for point-to-point and broadcast communications, as well as the routing opportunities make FRET-based communication promising for future molecular computers.

  18. Terbium to Quantum Dot FRET Bioconjugates for Clinical Diagnostics: Influence of Human Plasma on Optical and Assembly Properties

    PubMed Central

    Morgner, Frank; Stufler, Stefan; Geißler, Daniel; Medintz, Igor L.; Algar, W. Russ; Susumu, Kimihiro; Stewart, Michael H.; Blanco-Canosa, Juan B.; Dawson, Philip E.; Hildebrandt, Niko

    2011-01-01

    Förster resonance energy transfer (FRET) from luminescent terbium complexes (LTC) as donors to semiconductor quantum dots (QDs) as acceptors allows extraordinary large FRET efficiencies due to the long Förster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma. PMID:22163719

  19. Evaluation of quantum dot-based concentric FRET configurations with a fluorescent dye and dark quencher for multiplexed bioanalyses

    NASA Astrophysics Data System (ADS)

    Conroy, Erin M.; Algar, W. Russ

    2014-03-01

    Semiconductor quantum dots (QDs) continue to emerge as a highly advantageous platform for bioanalysis. Their unique physical and optical properties are especially well suited for Förster resonance energy transfer (FRET)-based bioprobes. Concentric FRET configurations are a recent development in this area of research and are best described as QD bioconjugates where multiple energy transfer pathways have been assembled around the central QD. Concentric FRET configurations permit multiplexed bioanalysis using one type of QD vector, but require more sophisticated analyses than conventional FRET pairs. In this paper, we describe the design and characterization of a new concentric FRET configuration that assembles both a fluorescent dye, Alexa Fluor 555 or Alexa Fluor 647, and a dark quencher, QSY9, at different ratios around a central CdSeS/ZnS QD. It was found that the magnitudes of the total photoluminescence (PL) intensity and either the A555/QD or A647/QD PL ratio can be related to the number of QSY9 and A555 or A647 per QD. The trends in these parameters with changes in the number of each dye molecule per QD have both similarities and differences between configurations with A555 and A647. In each case, a system of equations can be defined to permit calculation of the number of each dye molecule per QD from PL measurements. Both of these dark quencher-based concentric FRET configurations are therefore good candidates for quantitative, multiplexed bioanalysis.

  20. The Valles natural analogue project

    SciTech Connect

    Stockman, H.; Krumhansl, J.; Ho, C.; McConnell, V.

    1994-12-01

    The contact between an obsidian flow and a steep-walled tuff canyon was examined as an analogue for a highlevel waste repository. The analogue site is located in the Valles Caldera in New Mexico, where a massive obsidian flow filled a paleocanyon in the Battleship Rock tuff. The obsidian flow provided a heat source, analogous to waste panels or an igneous intrusion in a repository, and caused evaporation and migration of water. The tuff and obsidian samples were analyzed for major and trace elements and mineralogy by INAA, XRF, X-ray diffraction; and scanning electron microscopy and electron microprobe. Samples were also analyzed for D/H and {sup 39}Ar/{sup 4O} isotopic composition. Overall,the effects of the heating event seem to have been slight and limited to the tuff nearest the contact. There is some evidence of devitrification and migration of volatiles in the tuff within 10 meters of the contact, but variations in major and trace element chemistry are small and difficult to distinguish from the natural (pre-heating) variability of the rocks.

  1. GAS PHASE SYNTHESIS OF (ISO)QUINOLINE AND ITS ROLE IN THE FORMATION OF NUCLEOBASES IN THE INTERSTELLAR MEDIUM

    SciTech Connect

    Parker, Dorian S. N.; Kaiser, Ralf I.; Kostko, Oleg; Troy, Tyler P.; Ahmed, Musahid; Mebel, Alexander M.; Tielens, Alexander G. G. M.

    2015-04-20

    Nitrogen-substituted polycyclic aromatic hydrocarbons (NPAHs) have been proposed to play a key role in the astrochemical evolution of the interstellar medium, yet the formation mechanisms of even their simplest prototypes—quinoline and isoquinoline—remain elusive. Here, we reveal a novel concept that under high temperature conditions representing circumstellar envelopes of carbon stars, (iso)quinoline can be synthesized via the reaction of pyridyl radicals with two acetylene molecules. The facile gas phase formation of (iso)quinoline in circumstellar envelopes defines a hitherto elusive reaction class synthesizing aromatic structures with embedded nitrogen atoms that are essential building blocks in contemporary biological-structural motifs. Once ejected from circumstellar shells and incorporated into icy interstellar grains in cold molecular clouds, these NPAHs can be functionalized by photo processing forming nucleobase-type structures as sampled in the Murchison meteorite.

  2. Synthesis and evaluation of the biostability and cell compatibility of novel conjugates of nucleobase, peptidic epitope, and saccharide

    PubMed Central

    Yuan, Dan; Du, Xuewen; Shi, Junfeng; Zhou, Ning; Baoum, Abdulgader Ahmed; Al Footy, Khalid Omar; Badahdah, Khadija Omar

    2015-01-01

    Summary This article reports the synthesis of a new class of conjugates containing a nucleobase, a peptidic epitope, and a saccharide and the evalution of their gelation, biostability, and cell compatibility. We demonstrate a facile synthetic process, based on solid-phase peptide synthesis of nucleopeptides, to connect a saccharide with the nucleopeptides for producing the target conjugates. All the conjugates themselves (1–8) display excellent solubility in water without forming hydrogels. However, a mixture of 5 and 8 self-assembles to form nanofibers and results in a supramolecular hydrogel. The proteolytic stabilities of the conjugates depend on the functional peptidic epitopes. We found that TTPV is proteolytic resistant and LGFNI is susceptible to proteolysis. In addition, all the conjugates are compatible to the mammalian cells tested. PMID:26425189

  3. Theoretical study of physisorption of nucleobases on boron nitride nanotubes: a new class of hybrid nano-biomaterials.

    PubMed

    Mukhopadhyay, Saikat; Gowtham, S; Scheicher, Ralph H; Pandey, Ravindra; Karna, Shashi P

    2010-04-23

    We investigate the adsorption of the nucleic acid bases-adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U)-on the outer wall of a high curvature semiconducting single-walled boron nitride nanotube (BNNT) by first-principles density functional theory calculations. The calculated binding energy shows the order: G > A approximately C approximately T approximately U, implying that the interaction strength of the high curvature BNNT with the nucleobases, G being an exception, is nearly the same. A higher binding energy for the G-BNNT conjugate appears to result from hybridization of the molecular orbitals of G and the BNNT. A smaller energy gap predicted for the G-BNNT conjugate relative to that of the pristine BNNT may be useful in the application of this class of biofunctional materials to the design of next-generation sensing devices. PMID:20351402

  4. Theoretical study of physisorption of nucleobases on boron nitride nanotubes: a new class of hybrid nano-biomaterials

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, Saikat; Gowtham, S.; Scheicher, Ralph H.; Pandey, Ravindra; Karna, Shashi P.

    2010-04-01

    We investigate the adsorption of the nucleic acid bases—adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U)—on the outer wall of a high curvature semiconducting single-walled boron nitride nanotube (BNNT) by first-principles density functional theory calculations. The calculated binding energy shows the order: G > A≈C≈T≈U, implying that the interaction strength of the high curvature BNNT with the nucleobases, G being an exception, is nearly the same. A higher binding energy for the G-BNNT conjugate appears to result from hybridization of the molecular orbitals of G and the BNNT. A smaller energy gap predicted for the G-BNNT conjugate relative to that of the pristine BNNT may be useful in the application of this class of biofunctional materials to the design of next-generation sensing devices.

  5. Contents Changes of Triterpenic Acids, Nucleosides, Nucleobases, and Saccharides in Jujube (Ziziphus jujuba) Fruit During the Drying and Steaming Process.

    PubMed

    Guo, Sheng; Duan, Jin-Ao; Zhang, Ying; Qian, Dawei; Tang, Yuping; Zhu, Zhenhua; Wang, Hanqing

    2015-12-12

    Chinese jujube (Ziziphus jujuba), a medicinal and edible plant, is widely consumed in Asian countries owing to the remarkable health activities of its fruits. To facilitate selection of the suitable processing method for jujube fruits, in this study their contents of triterpenic acids, nucleosides, nucleobases and saccharides after drying and steaming treatment were determined using ultra-high performance liquid chromatography and high performance liquid chromatography coupled with evaporative light scattering detector methods. The results showed that except for sucrose, the content levels of most analytes were increasing in the jujube fruits during drying treatment at 45 °C. The levels of cyclic nucleotides such as adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, were significantly decreased after the fruits were steamed. Therefore, owing to the bioactivities of these components for human health, the dried fruits would be the better choice as medicinal material or functional food, and dried jujube fruit should not be further steamed.

  6. Universal limiting shape of worn profile under multiple-mode fretting conditions: theory and experimental evidence

    PubMed Central

    Dmitriev, Andrey I.; Voll, Lars B.; Psakhie, Sergey G.; Popov, Valentin L.

    2016-01-01

    We consider multiple-mode fretting wear in a frictional contact of elastic bodies subjected to a small-amplitude oscillation, which may include in-plane and out-of-plane translation, torsion and tilting (“periodic rolling”). While the detailed kinetics of wear depends on the particular loading history and wear mechanism, the final worn shape, under some additional conditions, occurs to be universal for all types and loading and wear mechanisms. This universal form is determined solely by the radius of the permanent stick region and the maximum indentation depth during the loading cycle. We provide experimental evidence for the correctness of the theoretically predicted limiting shape. The existence of the universal limiting shape can be used for designing joints which are resistant to fretting wear. PMID:26979092

  7. FRET-based dimeric aptamer probe for selective and sensitive Lup an 1 allergen detection.

    PubMed

    Mairal, T; Nadal, P; Svobodova, M; O'Sullivan, C K

    2014-04-15

    A sensitive method for the rapid and sensitive detection of the anaphylactic food allergen Lup an 1 (β-conglutin) exploiting fluorescence resonance energy transfer (FRET) has been developed. A high affinity dimeric form of a truncated 11-mer aptamer against β-conglutin was used, with each monomeric aptamer being flanked by donor/acceptor moieties. The dimeric form in the absence of target yields fluorescence emission due to the FRET from the excited fluorophore to the proximal second fluorophore. However, upon addition of β-conglutin, the specific interaction induces a change in the bi-aptameric structure resulting in an increase in fluorescence emission. The method is highly specific and sensitive, with a detection limit of 150 pM, providing an effective tool for the direct detection of the toxic β-conglutin subunit in foodstuffs in just 1 min at room temperature. PMID:24280051

  8. Selected fretting-wear-resistant coatings for Ti-6 pct Al-4 pct V alloy

    NASA Technical Reports Server (NTRS)

    Bill, R. C.

    1985-01-01

    The ability of several wear-resistant coatings to reduce fretting in the Ti-6Al-4V alloy is investigated. The experimental apparatus and procedures for evaluating fretting in uncoated Ti-6Al-4V alloy and in the alloy with plasma-sprayed coatings, polymer-bonded coating, and surface treatments are described. The wear volume and wear rate for the alloys are measured and compared. It is concluded that Al2O3 with 13 percent TiO2, preoxidation and nitride surface treatments, and MoS2 sputtering result in wear-resistant surfaces; however, the polyimide coating is the most wear resistant coating in both dry and moist air, and it causes the least wear to the uncoated alloy surface.

  9. Investigation of pH and temperature effects on FRET systems for glucose sensing

    NASA Astrophysics Data System (ADS)

    Meledeo, Michael A.; Ibey, Bennett L.; O'Neal, D. P.; Pishko, Michael V.; Cote, Gerard L.

    2002-05-01

    Glucose monitoring is of critical importance in the life of Type I and many Type II diabetics. This research furthers work toward a minimally invasive implantable glucose sensor based on fluorescence detection. Current experimental models use heterogeneous fluorescence resonance energy transfer (FRET) systems for sensing; ideally, the response of one fluorophore bound to a large polysaccharide is enhanced greatly in the presence of glucose while the other fluorophore bound to a glucose sensitive protein is diminished or unaffected. Many fluorophores are affected by environmental factors such as pH and temperature. FRET experiments using two fluorophores, tetramethylrodamine isothiocyanate (TRITC) and fluoroscein isothiocyanate (FITC), are performed evaluating the effects of fluctuations over the range of pH 4-8 and temperature 25-45 degree(s)C for various concentrations of glucose in a flow cell. TRITC is bound to the lectin Concanavalin A (Con A), and FITC is bound to dextran molecules of varying sizes.

  10. Universal limiting shape of worn profile under multiple-mode fretting conditions: theory and experimental evidence

    NASA Astrophysics Data System (ADS)

    Dmitriev, Andrey I.; Voll, Lars B.; Psakhie, Sergey G.; Popov, Valentin L.

    2016-03-01

    We consider multiple-mode fretting wear in a frictional contact of elastic bodies subjected to a small-amplitude oscillation, which may include in-plane and out-of-plane translation, torsion and tilting (“periodic rolling”). While the detailed kinetics of wear depends on the particular loading history and wear mechanism, the final worn shape, under some additional conditions, occurs to be universal for all types and loading and wear mechanisms. This universal form is determined solely by the radius of the permanent stick region and the maximum indentation depth during the loading cycle. We provide experimental evidence for the correctness of the theoretically predicted limiting shape. The existence of the universal limiting shape can be used for designing joints which are resistant to fretting wear.

  11. The character of fracture of iron based thermal coating during fretting

    NASA Astrophysics Data System (ADS)

    Kovalevskaya, Zh G.; Kovalevskiy, E. A.; Khimich, M. A.

    2016-04-01

    The character of destruction of thermal coatings during fretting has been investigated. An iron based plasma coating has been tested with oscillation amplitude from 30 to 200 microns. The tests were conducted in air. It has been determined that the main factor influencing the rate of the wear of the coating during fretting corrosion is the size of the coating area involved into the wear process. The coating exhibits high wear resistance when the amplitude of the oscillation is commensurate with the size of the sprayed particles. During destruction of the coating the leading role belongs to fatigue-oxidation processes. The wear of the coating acquires a catastrophic character when coating macro defects - pores and interlayer boundaries - are involved into the wear process.

  12. On modeling biomolecular-surface nonbonded interactions: application to nucleobase adsorption on single-wall carbon nanotube surfaces.

    PubMed

    Akdim, B; Pachter, R; Day, P N; Kim, S S; Naik, R R

    2012-04-27

    In this work we explored the selectivity of single nucleobases towards adsorption on chiral single-wall carbon nanotubes (SWCNTs) by density functional theory calculations. Specifically, the adsorption of molecular models of guanine (G), adenine (A), thymine (T), and cytosine (C), as well as of AT and GC Watson-Crick (WC) base pairs on chiral SWCNT C(6, 5), C(9, 1) and C(8, 3) model structures, was analyzed in detail. The importance of correcting the exchange-correlation functional for London dispersion was clearly demonstrated, yet limitations in modeling such interactions by considering the SWCNT as a molecular model may mask subtle effects in a molecular-macroscopic material system. The trend in the calculated adsorption energies of the nucleobases on same diameter C(6, 5) and C(9, 1) SWCNT surfaces, i.e., G > A > T > C, was consistent with related computations and experimental work on graphitic surfaces, however contradicting experimental data on the adsorption of single-strand short homo-oligonucleotides on SWCNTs that demonstrated a trend of G > C > A > T (Albertorio et al 2009 Nanotechnology 20 395101). A possible role of electrostatic interactions in this case was partially captured by applying the effective fragment potential method, emphasizing that the interplay of the various contributions in modeling nonbonded interactions is complicated by theoretical limitations. Finally, because the calculated adsorption energies for Watson-Crick base pairs have shown little effect upon adsorption of the base pair farther from the surface, the results on SWCNT sorting by salmon genomic DNA could be indicative of partial unfolding of the double helix upon adsorption on the SWCNT surface.

  13. Accuracy of maximum likelihood estimates of a two-state model in single-molecule FRET

    SciTech Connect

    Gopich, Irina V.

    2015-01-21

    Photon sequences from single-molecule Förster resonance energy transfer (FRET) experiments can be analyzed using a maximum likelihood method. Parameters of the underlying kinetic model (FRET efficiencies of the states and transition rates between conformational states) are obtained by maximizing the appropriate likelihood function. In addition, the errors (uncertainties) of the extracted parameters can be obtained from the curvature of the likelihood function at the maximum. We study the standard deviations of the parameters of a two-state model obtained from photon sequences with recorded colors and arrival times. The standard deviations can be obtained analytically in a special case when the FRET efficiencies of the states are 0 and 1 and in the limiting cases of fast and slow conformational dynamics. These results are compared with the results of numerical simulations. The accuracy and, therefore, the ability to predict model parameters depend on how fast the transition rates are compared to the photon count rate. In the limit of slow transitions, the key parameters that determine the accuracy are the number of transitions between the states and the number of independent photon sequences. In the fast transition limit, the accuracy is determined by the small fraction of photons that are correlated with their neighbors. The relative standard deviation of the relaxation rate has a “chevron” shape as a function of the transition rate in the log-log scale. The location of the minimum of this function dramatically depends on how well the FRET efficiencies of the states are separated.

  14. Miniaturized FRET assays and microfluidics: key components for ultra-high-throughput screening.

    PubMed

    Mere; Bennett; Coassin; England; Hamman; Rink; Zimmerman; Negulescu

    1999-08-01

    Assay miniaturization applicable across a wide range of target classes, along with automation and process integration, are well-recognized goals for ultra-high-throughput screening on an industrial scale. This report summarizes the implementation of fluorescence resonance energy transfer (FRET)-based biochemical and cell-based assays in 3456-well NanoWelltrade mark assay plates using key components of Aurora's ultra-high-throughput screening system.

  15. Handheld Fluorescence Resonance Energy Transfer (FRET)-Aptamer Sensor for Bone Markers

    NASA Technical Reports Server (NTRS)

    Bruno, John G.

    2015-01-01

    Astronauts lose significant bone mass during lengthy space flights. NASA wishes to monitor this bone loss in order to develop nutritional and exercise countermeasures. Operational Technologies Corporation (OpTech) has developed a handheld device that quantifies bone loss in a spacecraft environment. The innovation works by adding fluorescent dyes and quenchers to aptamers to enable pushbutton, one-step bind-and-detect FRET assays that can be freeze-dried, rehydrated with body fluids, and used to quantify bone loss.

  16. Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors

    NASA Astrophysics Data System (ADS)

    Okumoto, Sakiko; Looger, Loren L.; Micheva, Kristina D.; Reimer, Richard J.; Smith, Stephen J.; Frommer, Wolf B.

    2005-06-01

    Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery. aspartate | hippocampal neuron | neurotransmitter | secretion | transport

  17. Compact quantum dot-antibody conjugates for FRET immunoassays with subnanomolar detection limits.

    PubMed

    Mattera, Lucia; Bhuckory, Shashi; Wegner, K David; Qiu, Xue; Agnese, Fabio; Lincheneau, Christophe; Senden, Tim; Djurado, David; Charbonnière, Loïc J; Hildebrandt, Niko; Reiss, Peter

    2016-06-01

    A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter <13 nm, PL quantum yield up to 66% at 705 nm, and colloidal stability of several months in various buffers. They were applied as FRET acceptors in homogeneous, time-gated immunoassays using Tb-antibodies as FRET donors, both coupled by an immunological sandwich complex between the two antibodies and a PSA (prostate specific antigen) biomarker. The advantages of the compact surface coating for FRET could be demonstrated by an 6.2 and 2.5 fold improvement of the limit of detection (LOD) for PSA compared to commercially available hydrophilic QDs emitting at 605 and 705 nm, respectively. While the commercial QDs contain identical inorganic cores responsible for their fluorescence, they are coated with a comparably thick amphiphilic polymer layer leading to much larger hydrodynamic diameters (>26 nm without biomolecules). The LODs of 0.8 and 3.7 ng mL(-1) obtained in 50 μL serum samples are below the clinical cut-off level of PSA (4 ng mL(-1)) and demonstrate their direct applicability in clinical diagnostics. PMID:27188210

  18. Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

    PubMed Central

    Hukasova, Elvira; Silva Cascales, Helena; Kumar, Shravan R.; Lindqvist, Arne

    2012-01-01

    Förster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14. Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing. PMID:22314640

  19. Kinetics of Precursor Interactions with the Bacterial Tat Translocase Detected by Real-time FRET*

    PubMed Central

    Whitaker, Neal; Bageshwar, Umesh K.; Musser, Siegfried M.

    2012-01-01

    The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time. PMID:22315217

  20. G-Tetraplex-Induced FRET within Telomeric Repeat Sequences Using (Py) A-(Per) A as Energy Donor-Acceptor Pair.

    PubMed

    Kundu, Rajen

    2016-01-01

    G-tetraplex induced fluorescence resonance energy transfer (FRET) within telomeric repeat sequences has been studied using a nucleoside-tethered FRET pair embedded in the human telomeric G-quadruplex forming sequence (5'-A GGG TT(Py) A GGG TT(Per) A GGG TTA GGG-3', Py=pyrene, Per=perylene). Conformational change from a single strand to an anti-parallel G-quadruplex leads to FRET from energy donor ((Py) A) to acceptor ((Per) A). The distance between the FRET donor/acceptor partners was controlled by changing the number of G-quartet spacer units. The FRET efficiency decreases with increase in G-quartet units. Overall findings indicate that this could be further used for the development of FRET-based sensing and measurement techniques. PMID:26490798

  1. Fluorescence anisotropy and FRET studies of G-quadruplex formation in presence of different cations

    NASA Astrophysics Data System (ADS)

    Juskowiak, Bernard; Galezowska, Elzbieta; Zawadzka, Anna; Gluszynska, Agata; Takenaka, Shigeori

    2006-07-01

    Results of the steady-state fluorescence, anisotropy and FRET measurements of G-quadruplex formation in the presence of selected cations (Li +, Na +, K +, NEt 4+ and Mg 2+) are reported. Three different fluorescent oligonucleotides with human telomeric sequence labeled with fluorescein (FAM) and tetramethylrhodamine (TAMRA) were investigated: a dual-labeled 21-mer denoted as PSO (Potassium Sensing Oligonucleotide) and two 5'- and 3'- single-labeled probes, FAM-21 and 21-TAMRA, respectively. The fluorescence signal of FAM-21 increased significantly for all systems and the fluorescence enhancement was comparable in magnitude for monovalent cations but it was more pronounced for Mg 2+ cation. This phenomenon was attributed to the protolytic equilibria of FAM affected by the variation in ionic strength. On the other hand, fluorescence of TAMRA was enhanced selectively by Na(I) cation that was explained by the dequenching of TAMRA emission originated from the peculiarity of the basket-type structure of Na(I)-quadruplex. Anisotropy of FAM-21 (but not 21-TAMRA) appeared to be sensitive to the G-quadruplex formation, showing significant increase with an increase in cation concentration and indicating some restrictions in rotational depolarization of FAM. FRET experiments revealed that all tested cations caused quenching of FAM fluorescence in PSO, but only Na + and K + ions produced sensitized emission of TAMRA acceptor. Higher FRET efficiency observed in the presence of sodium ion was attributed to the specific spectral factor and steric interactions in the basket-type Na(I)-quadruplex.

  2. Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

    PubMed

    Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

    2016-12-01

    In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors.

  3. A TR-FRET-based functional assay for screening activators of CARM1.

    PubMed

    Zeng, Hao; Wu, Jiacai; Bedford, Mark T; Sbardella, Gianluca; Hoffmann, F Michael; Bi, Kun; Xu, Wei

    2013-05-10

    Epigenetics is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that twofold overexpression of CARM1 in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, thus leading to the hypothesis that activating CARM1 by chemical activators might be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved (TR) FRET assay that uses poly(A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen TR-FRET assay uses MCF7 cells expressing GFP-PABP1 fusion protein through BacMam gene delivery system, methyl-PABP1 specific antibody, and terbium-labeled secondary antibody. This assay has been validated as reflecting the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions. PMID:23585185

  4. Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells.

    PubMed Central

    Tramier, Marc; Gautier, Isabelle; Piolot, Tristan; Ravalet, Sylvie; Kemnitz, Klaus; Coppey, Jacques; Durieux, Christiane; Mignotte, Vincent; Coppey-Moisan, Maïté

    2002-01-01

    By using a novel time- and space-correlated single-photon counting detector, we show that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to herpes simplex virus thymidine kinase (TK) monomers can be used to reveal homodimerization of TK in the nucleus and cytoplasm of live cells. However, the quantification of energy transfer was limited by the intrinsic biexponential fluorescence decay of the donor CFP (lifetimes of 1.3 +/- 0.2 ns and 3.8 +/- 0.4 ns) and by the possibility of homodimer formation between two TK-CFP. In contrast, the heterodimerization of the transcriptional factor NF-E2 in the nucleus of live cells was quantified from the analysis of the fluorescence decays of GFP in terms of 1) FRET efficiency between GFP and DsRed chromophores fused to p45 and MafG, respectively, the two subunits of NF-E2 (which corresponds to an interchromophoric distance of 39 +/- 1 A); and 2) fractions of GFP-p45 bound to DsRed-MafG (constant in the nucleus, varying in the range of 20% to 70% from cell to cell). The picosecond resolution of the fluorescence kinetics allowed us to discriminate between very short lifetimes of immature green species of DsRed-MafG and that of GFP-p45 involved in FRET with DsRed-MafG. PMID:12496124

  5. Single-Molecule Pull-Down FRET to Dissect the Mechanisms of Biomolecular Machines.

    PubMed

    Kahlscheuer, Matthew L; Widom, Julia; Walter, Nils G

    2015-01-01

    Spliceosomes are multimegadalton RNA-protein complexes responsible for the faithful removal of noncoding segments (introns) from pre-messenger RNAs (pre-mRNAs), a process critical for the maturation of eukaryotic mRNAs for subsequent translation by the ribosome. Both the spliceosome and ribosome, as well as many other RNA and DNA processing machineries, contain central RNA components that endow biomolecular complexes with precise, sequence-specific nucleic acid recognition, and versatile structural dynamics. Single-molecule fluorescence (or Förster) resonance energy transfer (smFRET) microscopy is a powerful tool for the study of local and global conformational changes of both simple and complex biomolecular systems involving RNA. The integration of biochemical tools such as immunoprecipitation with advanced methods in smFRET microscopy and data analysis has opened up entirely new avenues toward studying the mechanisms of biomolecular machines isolated directly from complex biological specimens, such as cell extracts. Here, we detail the general steps for using prism-based total internal reflection fluorescence microscopy in exemplary single-molecule pull-down FRET studies of the yeast spliceosome and discuss the broad application potential of this technique.

  6. Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis.

    PubMed

    Tomov, Toma E; Tsukanov, Roman; Masoud, Rula; Liber, Miran; Plavner, Noa; Nir, Eyal

    2012-03-01

    Among the advantages of the single-molecule approach when used to study biomolecular structural dynamics and interaction is its ability to distinguish between and independently observe minor subpopulations. In a single-molecule Förster resonance energy transfer (FRET) and alternating laser excitation diffusion experiment, the various populations are apparent in the resultant histograms. However, because histograms are calculated based on the per-burst mean FRET and stoichiometry ratio and not on the internal photon distribution, much of the acquired information is lost, thereby reducing the capabilities of the method. Here we suggest what to our knowledge is a novel statistical analysis tool that significantly enhances these capabilities, and we use it to identify and isolate static and dynamic subpopulations. Based on a kernel density estimator and a proper photon distribution analysis, for each individual burst, we calculate scores that reflect properties of interest. Specifically, we determine the FRET efficiency and brightness ratio distributions and use them to reveal 1), the underlying structure of a two-state DNA-hairpin and a DNA hairpin that is bound to DNA origami; 2), a minor doubly labeled dsDNA subpopulation concealed in a larger singly labeled dsDNA; and 3), functioning DNA origami motors concealed within a larger subpopulation of defective motors. Altogether, these findings demonstrate the usefulness of the proposed approach. The method was developed and tested using simulations, its rationality is described, and a computer algorithm is provided.

  7. Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

    PubMed

    Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

    2016-12-01

    In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. PMID:27612755

  8. Single-Molecule Pull-Down FRET to Dissect the Mechanisms of Biomolecular Machines.

    PubMed

    Kahlscheuer, Matthew L; Widom, Julia; Walter, Nils G

    2015-01-01

    Spliceosomes are multimegadalton RNA-protein complexes responsible for the faithful removal of noncoding segments (introns) from pre-messenger RNAs (pre-mRNAs), a process critical for the maturation of eukaryotic mRNAs for subsequent translation by the ribosome. Both the spliceosome and ribosome, as well as many other RNA and DNA processing machineries, contain central RNA components that endow biomolecular complexes with precise, sequence-specific nucleic acid recognition, and versatile structural dynamics. Single-molecule fluorescence (or Förster) resonance energy transfer (smFRET) microscopy is a powerful tool for the study of local and global conformational changes of both simple and complex biomolecular systems involving RNA. The integration of biochemical tools such as immunoprecipitation with advanced methods in smFRET microscopy and data analysis has opened up entirely new avenues toward studying the mechanisms of biomolecular machines isolated directly from complex biological specimens, such as cell extracts. Here, we detail the general steps for using prism-based total internal reflection fluorescence microscopy in exemplary single-molecule pull-down FRET studies of the yeast spliceosome and discuss the broad application potential of this technique. PMID:26068753

  9. A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy.

    PubMed

    Day, Richard N; Tao, Wen; Dunn, Kenneth W

    2016-11-01

    Genetically encoded fluorescent protein (FP)-based biosensor probes are useful tools for monitoring cellular events in living cells and tissues. Because these probes were developed for one-photon excitation approaches, their broad two-photon excitation (2PE) and poorly understood photobleaching characteristics have made their implementation in studies using two-photon laser-scanning microscopy (TPLSM) challenging. Here we describe a protocol that simplifies the use of Förster resonance energy transfer (FRET)-based biosensors in TPLSM. First, the TPLSM system is evaluated and optimized using FRET standards expressed in living cells, which enables the determination of spectral bleed-through (SBT) and the confirmation of FRET measurements from the known standards. Next, we describe how to apply the approach experimentally using a modified version of the A kinase activity reporter (AKAR) protein kinase A (PKA) biosensor as an example-first in cells in culture and then in hepatocytes in the liver of living mice. The microscopic imaging can be accomplished in a day in laboratories that routinely use TPLSM.

  10. Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening

    SciTech Connect

    Johansson, Daniel X.; Brismar, Hjalmar . E-mail: mats.persson@ki.se

    2007-01-12

    The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5 {+-} 1.5% (mean {+-} SEM) and 8.0 {+-} 0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8 {+-} 2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1 {+-} 1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals.

  11. Time-dependent FRET with single enzymes: domain motions and catalysis in H(+)-ATP synthases.

    PubMed

    Bienert, Roland; Zimmermann, Boris; Rombach-Riegraf, Verena; Gräber, Peter

    2011-02-25

    H(+)-ATP synthases are molecular machines which couple transmembrane proton transport with ATP synthesis from ADP and inorganic phosphate by a rotational mechanism. Single-pair fluorescence resonance energy transfer (spFRET) in single molecules is a powerful tool to analyse conformational changes. It is used to investigate subunit movements in H(+)-ATP synthases from E. coli (EF(0)F(1)) and from spinach chloroplasts (CF(0)F(1)) during catalysis. The enzymes are incorporated into liposome membranes, and this allows the generation of a transmembrane pH difference, which is necessary for ATP synthesis. After labelling of appropriate sites on different subunits with fluorescence donor and acceptor, the kinetics of spFRET are measured. Analysis of the E(FRET) traces reveals rotational movement of the ε and γ subunits in 120° steps with opposite directions during ATP synthesis and ATP hydrolysis. The stepped movement is characterized by a 120° step faster than 1 ms followed by a rest period with an average dwell time of 15 ms, which is in accordance with the turnover time of the enzyme. In addition to the three conformational states during catalysis, also an inactive conformation is found, which is observed after catalysis.

  12. Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors.

    PubMed

    Kamioka, Yuji; Sumiyama, Kenta; Mizuno, Rei; Sakai, Yoshiharu; Hirata, Eishu; Kiyokawa, Etsuko; Matsuda, Michiyuki

    2012-01-01

    Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

  13. FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets.

    PubMed

    Kuznetsov, Andrey; Bindokas, Vytautas P; Marks, Jeremy D; Philipson, Louis H

    2005-07-01

    Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential (V(m)) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from V(m) -70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in V(m). When V(m) was measured simultaneously with intracellular Ca2+, all active cells showed tight coupling of V(m) to islet cell Ca2+ changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans. PMID:15758044

  14. Real-time detecting gelatinases activity in living cells by FRET imaging

    NASA Astrophysics Data System (ADS)

    Yang, Jie; Zhang, Zhihong; Liu, Bifeng; Luo, Qingming

    2006-01-01

    Degradation of the extracellular matrix by Matrix metalloproteinases (MMPs) not only enhances tumor invasion, but also affects tumor cell behaviour and leads to cancer progression. To monitor gelatinases (contain MMP2 and MMP9) activity in living cells, we constructed a vector that encoded a gelatinases recognition site (GRS) between citrine (mutation of EYFP Q69M) in N terminal and ECFP in C terminal. Because Gelatinases are secretory proteins and act outside of cell, an expressing vector displayed the fusion protein on cellular surface was used for this FRET gene probe. On expression of YFP-GRS-ECFP in MCF-7 cells that expressed no gelatinases, we were able to observe the efficient transfer of energy from excited ECFP to YFP within the YFP-GRS-ECFP molecule. However, the fusion protein YFP-GRS-ECFP was expressed in MDA-MB 453s cell line with high secretory gelatinases, so YFP-GRS-ECFP was cleaved by gelatinases, no such transfer of energy was detected and fluorescence signal disappeared in YFP channel since YFP protein was cut down. Moreover, Doxycycline, a MMP inhibitor, could make FRET signal increase and fluorescence signal appeared in YFP channel. Thus, the FRET probe YFP-GRS-ECFP can sensitively and reliably monitor gelatinases activation in living cells and can be used for screening MMP inhibitors.

  15. Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

    PubMed Central

    Tsytlonok, Maksym; Ibrahim, Shehu M.; Rowling, Pamela J.E.; Xu, Wenshu; Ruedas-Rama, Maria J.; Orte, Angel; Klenerman, David; Itzhaki, Laura S.

    2015-01-01

    Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated. PMID:25565106

  16. FRET based biosensor for detection of active NF-kB

    NASA Astrophysics Data System (ADS)

    Baldini, Francesco; Citti, Lorenzo; Domenici, Claudio; Giannetti, Ambra; Tedeschi, Lorena; Vo-Dinh, Tuan; Wabuyele, Musundi B.

    2005-05-01

    The Nuclear Factor kB is a transcription factor, ubiquitously expressed, involved in the regulation of a large number of genes and in a variety of human disease including inflammation, asthma, atherosclerosis, AIDS, septic shock, arthritis and cancer. The critical need for a simple and direct method to evaluate the quantity of active NF-kB in a biological sample can be addressed using a suitable and reusable biosensor. For this purpose, a novel method, using fluorescence resonance energy transfer (FRET), to detect the active form of NF-kB binding a specific DNA sequence has been developed. A single-stranded DNA (ssDNA) with auto-complementary sequence has been properly designed and synthesized. In order to evaluate FRET due to the DNA/protein binding interaction taking place between double-stranded DNA (dsDNA) immobilized in a capillary wall and NF-kB proteins, a highly sensitive FRET-based biosensor system developed in our laboratory was used. Preliminary results show that our system was capable of detecting the active form of NF-kB protein with a detection efficiency of about 90% and that the system has a good regenerability.

  17. A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy.

    PubMed

    Day, Richard N; Tao, Wen; Dunn, Kenneth W

    2016-11-01

    Genetically encoded fluorescent protein (FP)-based biosensor probes are useful tools for monitoring cellular events in living cells and tissues. Because these probes were developed for one-photon excitation approaches, their broad two-photon excitation (2PE) and poorly understood photobleaching characteristics have made their implementation in studies using two-photon laser-scanning microscopy (TPLSM) challenging. Here we describe a protocol that simplifies the use of Förster resonance energy transfer (FRET)-based biosensors in TPLSM. First, the TPLSM system is evaluated and optimized using FRET standards expressed in living cells, which enables the determination of spectral bleed-through (SBT) and the confirmation of FRET measurements from the known standards. Next, we describe how to apply the approach experimentally using a modified version of the A kinase activity reporter (AKAR) protein kinase A (PKA) biosensor as an example-first in cells in culture and then in hepatocytes in the liver of living mice. The microscopic imaging can be accomplished in a day in laboratories that routinely use TPLSM. PMID:27685098

  18. Real-time single cell analysis of molecular mechanism of apoptosis and proliferation using FRET technique

    NASA Astrophysics Data System (ADS)

    Chen, Tongsheng; Xing, Da; Gao, Xuejuan; Wang, Fang

    2006-09-01

    Bcl-2 family proteins (such as Bid and Bak/Bax) and 14-3-3 proteins play a key role in the mitochondria-mediated cell apoptosis induced by cell death factors such as TNF-α and lower power laser irradiation (LPLI). In this report, fluorescence resonance energy transfer (FRET) has been used to study the molecular mechanism of apoptosis in living cells on a fluorescence scanning confocal microscope. Based on the genetic code technique and the green fluorescent proteins (GFPs), single-cell dynamic analysis of caspase3 activation, caspase8 activation, and PKCs activation are performed during apoptosis induced by laser irradiation in real-time. To investigate the cellular effect and mechanism of laser irradiation, human lung adenocarcinoma cells (ASTC-a-1) transfected with plasmid SCAT3 (pSCAT3)/ CKAR FRET reporter, were irradiated and monitored noninvasively with both FRET imaging. Our results show that high fluence lower power laser irradiation (HFLPLI) can induce an increase of caspase3 activation and a decrease of PKCs activation, and that LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs.

  19. FRET Fluctuation Spectroscopy of Diffusing Biopolymers: Contributions of Conformational Dynamics and Translational Diffusion

    PubMed Central

    Gurunathan, Kaushik; Levitus, Marcia

    2009-01-01

    The use of Fluorescence Correlation Spectroscopy (FCS) to study conformational dynamics in diffusing biopolymers requires that the contributions to the signal due to translational diffusion are separated from those due to conformational dynamics. A simple approach that has been proposed to achieve this goal involves the analysis of fluctuations in Fluorescence Resonance Energy Transfer (FRET) efficiency. In this work, we investigate the applicability of this methodology by combining Monte Carlo simulations and experiments. Results show that diffusion does not contribute to the measured fluctuations in FRET efficiency in conditions where the relaxation time of the kinetic process is much shorter than the mean transit time of the molecules in the optical observation volume. However, in contrast to what has been suggested in previous work, the contributions of diffusion are otherwise significant. Neglecting the contributions of diffusion can potentially lead to an erroneous interpretation of the kinetic mechanisms. As an example, we demonstrate that the analysis of FRET fluctuations in terms of a purely kinetic model would generally lead to the conclusion that the system presents complex kinetic behavior even for an idealized two-state system PMID:20030305

  20. CO2 Capture with Enzyme Synthetic Analogue

    SciTech Connect

    Cordatos, Harry

    2010-11-08

    Overview of an ongoing, 2 year research project partially funded by APRA-E to create a novel, synthetic analogue of carbonic anhydrase and incorporate it into a membrane for removal of CO2 from flue gas in coal power plants. Mechanism background, preliminary feasibility study results, molecular modeling of analogue-CO2 interaction, and program timeline are provided.

  1. Continuous analogues of matrix factorizations

    PubMed Central

    Townsend, Alex; Trefethen, Lloyd N.

    2015-01-01

    Analogues of singular value decomposition (SVD), QR, LU and Cholesky factorizations are presented for problems in which the usual discrete matrix is replaced by a ‘quasimatrix’, continuous in one dimension, or a ‘cmatrix’, continuous in both dimensions. Two challenges arise: the generalization of the notions of triangular structure and row and column pivoting to continuous variables (required in all cases except the SVD, and far from obvious), and the convergence of the infinite series that define the cmatrix factorizations. Our generalizations of triangularity and pivoting are based on a new notion of a ‘triangular quasimatrix’. Concerning convergence of the series, we prove theorems asserting convergence provided the functions involved are sufficiently smooth. PMID:25568618

  2. Fully analogue photonic reservoir computer.

    PubMed

    Duport, François; Smerieri, Anteo; Akrout, Akram; Haelterman, Marc; Massar, Serge

    2016-03-03

    Introduced a decade ago, reservoir computing is an efficient approach for signal processing. State of the art capabilities have already been demonstrated with both computer simulations and physical implementations. If photonic reservoir computing appears to be promising a solution for ultrafast nontrivial computing, all the implementations presented up to now require digital pre or post processing, which prevents them from exploiting their full potential, in particular in terms of processing speed. We address here the possibility to get rid simultaneously of both digital pre and post processing. The standalone fully analogue reservoir computer resulting from our endeavour is compared to previous experiments and only exhibits rather limited degradation of performances. Our experiment constitutes a proof of concept for standalone physical reservoir computers.

  3. Fully analogue photonic reservoir computer

    PubMed Central

    Duport, François; Smerieri, Anteo; Akrout, Akram; Haelterman, Marc; Massar, Serge

    2016-01-01

    Introduced a decade ago, reservoir computing is an efficient approach for signal processing. State of the art capabilities have already been demonstrated with both computer simulations and physical implementations. If photonic reservoir computing appears to be promising a solution for ultrafast nontrivial computing, all the implementations presented up to now require digital pre or post processing, which prevents them from exploiting their full potential, in particular in terms of processing speed. We address here the possibility to get rid simultaneously of both digital pre and post processing. The standalone fully analogue reservoir computer resulting from our endeavour is compared to previous experiments and only exhibits rather limited degradation of performances. Our experiment constitutes a proof of concept for standalone physical reservoir computers. PMID:26935166

  4. FRET Sensor for Erythrosine Dye Based on Organic Nanoparticles: Application to Analysis of Food Stuff.

    PubMed

    Mahajan, Prasad G; Bhopate, Dhanaji P; Kolekar, Govind B; Patil, Shivajirao R

    2016-07-01

    An aqueous suspension of fluorescent nanoparticles (PHNNPs) of naphthol based fluorescent organic compound 1-[(Z)-(2-phenylhydrazinylidene) methyl] naphthalene -2-ol (PHN) were prepared using reprecipitation method shows bathochromically shifted aggregation induced enhanced emission (AIEE) in the spectral region where erythrosine (ETS) food dye absorbs strongly. The average size of 72.6 nm of aqueous suspension of PHNNPs obtained by Dynamic light scattering results shows a narrow particle size distribution. The negative zeta potential of nano probe (-22.6 mV) responsible to adsorb oppositely charged analyte on its surface and further permit to bind nano probe and analyte within the close distance proximity required for efficient fluorescence resonance energy transfer (FRET) to take place from donor (PHNNPs) to acceptor (ETS). Systematic FRET experiments performed by measuring fluorescence quenching of PHNNPs with successive addition of ETS solution exploited the use of the PHNNPs as a novel nano probe for the detection of ETS in aqueous solution with extremely lower limit of detection equal to 3.6 nM (3.1 ng/mL). The estimation of photo kinetic and thermodynamic parameters such as quenching rate constant, enthalpy change (∆H), Gibbs free energy change (∆G) and entropy change (∆S) was obtained by the quenching results obtained at different constant temperatures which were found to fit the well-known Stern-Volmer relation. The mechanism of binding and fluorescence quenching of PHNNPs by ETS food dye is proposed on the basis of results obtained in photophysical studies, thermodynamic parameter, energy transfer efficiency, critical energy transfer distance (R0) and distance of approach between donor-acceptor molecules (r). The proposed FRET method based on fluorescence quenching of PHNNPs was successfully applied to develop an analytical method for estimation of ETS from food stuffs without interference of other complex ingredients. Graphical Abstract A

  5. FRET Sensor for Erythrosine Dye Based on Organic Nanoparticles: Application to Analysis of Food Stuff.

    PubMed

    Mahajan, Prasad G; Bhopate, Dhanaji P; Kolekar, Govind B; Patil, Shivajirao R

    2016-07-01

    An aqueous suspension of fluorescent nanoparticles (PHNNPs) of naphthol based fluorescent organic compound 1-[(Z)-(2-phenylhydrazinylidene) methyl] naphthalene -2-ol (PHN) were prepared using reprecipitation method shows bathochromically shifted aggregation induced enhanced emission (AIEE) in the spectral region where erythrosine (ETS) food dye absorbs strongly. The average size of 72.6 nm of aqueous suspension of PHNNPs obtained by Dynamic light scattering results shows a narrow particle size distribution. The negative zeta potential of nano probe (-22.6 mV) responsible to adsorb oppositely charged analyte on its surface and further permit to bind nano probe and analyte within the close distance proximity required for efficient fluorescence resonance energy transfer (FRET) to take place from donor (PHNNPs) to acceptor (ETS). Systematic FRET experiments performed by measuring fluorescence quenching of PHNNPs with successive addition of ETS solution exploited the use of the PHNNPs as a novel nano probe for the detection of ETS in aqueous solution with extremely lower limit of detection equal to 3.6 nM (3.1 ng/mL). The estimation of photo kinetic and thermodynamic parameters such as quenching rate constant, enthalpy change (∆H), Gibbs free energy change (∆G) and entropy change (∆S) was obtained by the quenching results obtained at different constant temperatures which were found to fit the well-known Stern-Volmer relation. The mechanism of binding and fluorescence quenching of PHNNPs by ETS food dye is proposed on the basis of results obtained in photophysical studies, thermodynamic parameter, energy transfer efficiency, critical energy transfer distance (R0) and distance of approach between donor-acceptor molecules (r). The proposed FRET method based on fluorescence quenching of PHNNPs was successfully applied to develop an analytical method for estimation of ETS from food stuffs without interference of other complex ingredients. Graphical Abstract A

  6. FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D

    NASA Technical Reports Server (NTRS)

    Bruno, John G.

    2013-01-01

    Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is

  7. Monitoring of dual bio-molecular events using FRET biosensors based on mTagBFP/sfGFP and mVenus/mKOκ fluorescent protein pairs.

    PubMed

    Su, Ting; Pan, Shaotao; Luo, Qingming; Zhang, Zhihong

    2013-08-15

    Fluorescent protein (FP)-based Förster resonance energy transfer (FRET) biosensors are powerful tools for dynamically measuring cellular molecular events because they offer high spatial and temporal resolution in living cells. Despite the broad use of FP-based FRET biosensors in cell biology, imaging of multiple molecular events (multi-parameter molecular imaging) in single cells using current FRET pairs remains difficult because it usually requires a control group for additional data calibration. Hence, spectrally compatible FRET pairs that do not require complex image calibration are the key to widespread applications of FP-based FRET biosensors in multi-parameter molecular imaging. Here, we report a new combination of spectrally distinguishable FRET pairs for dual-parameter molecular imaging: mTagBFP/sfGFP (blue and green FP, B/G) and mVenus/mKOκ (yellow and orange FP, Y/O). We demonstrate that additional image correction is not necessary for these dual FRET pairs. Using these dual FRET pairs, we achieve simultaneous imaging of Src and Ca(2+) signaling in single living cells stimulated with epithelial growth factor (EGF). By converting traditional FRET biosensors into B/G and Y/O-based biosensors, additional applications are available to elucidate the dynamic relationships of multiple molecular events within a single living cell.

  8. Quantitative FRET (Förster Resonance Energy Transfer) analysis for SENP1 protease kinetics determination.

    PubMed

    Liu, Yan; Liao, Jiayu

    2013-02-21

    Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle (1,3,6,7). The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate (8-13). However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs. Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) (9,10,14,15). The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor

  9. ETP-0474: Evaluation of Electroless Nickel Coatings to Achieve Interference Fit in the RSRM Without Fretting

    NASA Technical Reports Server (NTRS)

    Schaffnit, William O.

    1997-01-01

    Part of the redesign of the SRMs for the Space Shuttle involved the substitution of three new capture cylinders for three of the previously used cylinders. These new cylinders mate with the old standard case segments in each of the three field joints. The new capture cylinders contain an integral capture latch on the tang end which mates with a case clevis during stackup at KSC. The capture cylinders also contain a groove in the capture latch to provide for a third 0-ring in the joint and are designed to achieve a metal-to- metal interference fit between the capture latch and the mating clevis. An unexpected fretting problem has occurred on the tang capture feature and the inner clevis leg interference fit surfaces on flight hardware since STS-26. Varying degrees of fretting damage have been found on the case segments from different flight motors. Fretting is a wear phenomena that occurs when two tightly fitting metal surfaces are subject to cyclic relative motion of extremely small amplitudes (generally less than 0.010-inch) in the absence of adequate lubrication. It is adhesive ("cold" - welding) in nature and vibration is its essential causative factor. This problem has manifested itself on the flight motors as a series of pits and axial gouges on the inside diameter (ID) surfaces of the inner clevis legs and the outside diameter (OD) surfaces of the tang capture features. The problem occurs in varying degrees of severity in all of the field joints. It is not believed that fretting is a flight safety issue. However, it could become a reusability issue if left unattended. Fretting has been encountered in other industries for many years and measures that will prevent or reduce it have been devised. These include: elimination or reduction of vibration (amplitudes and/or frequencies), elimination of slip, improved lubrication between parts, increased surface separation, increased interference, inducing residual compressive stresses in the surfaces of the mating

  10. Structural Heterogeneity and Quantitative FRET Efficiency Distributions of Polyprolines through a Hybrid Atomistic Simulation and Monte Carlo Approach

    PubMed Central

    Hoefling, Martin; Lima, Nicola; Haenni, Dominik; Seidel, Claus A. M.; Schuler, Benjamin; Grubmüller, Helmut

    2011-01-01

    Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single

  11. Plant Volatile Analogues Strengthen Attractiveness to Insect

    PubMed Central

    Sun, Yufeng; Yu, Hao; Zhou, Jing-Jiang; Pickett, John A.; Wu, Kongming

    2014-01-01

    Green leaf bug Apolygus lucorum (Meyer-Dür) is one of the major pests in agriculture. Management of A. lucorum was largely achieved by using pesticides. However, the increasing population of A. lucorum since growing Bt cotton widely and the increased awareness of ecoenvironment and agricultural product safety makes their population-control very challenging. Therefore this study was conducted to explore a novel ecological approach, synthetic plant volatile analogues, to manage the pest. Here, plant volatile analogues were first designed and synthesized by combining the bioactive components of β-ionone and benzaldehyde. The stabilities of β-ionone, benzaldehyde and analogue 3 g were tested. The electroantennogram (EAG) responses of A. lucorum adult antennae to the analogues were recorded. And the behavior assay and filed experiment were also conducted. In this study, thirteen analogues were acquired. The analogue 3 g was demonstrated to be more stable than β-ionone and benzaldehyde in the environment. Many of the analogues elicited EAG responses, and the EAG response values to 3 g remained unchanged during seven-day period. 3 g was also demonstrated to be attractive to A. lucorum adults in the laboratory behavior experiment and in the field. Its attractiveness persisted longer than β-ionone and benzaldehyde. This indicated that 3 g can strengthen attractiveness to insect and has potential as an attractant. Our results suggest that synthetic plant volatile analogues can strengthen attractiveness to insect. This is the first published study about synthetic plant volatile analogues that have the potential to be used in pest control. Our results will support a new ecological approach to pest control and it will be helpful to ecoenvironment and agricultural product safety. PMID:24911460

  12. Synthesis and biological evaluation of febrifugine analogues.

    PubMed

    Mai, Huong Doan Thi; Thanh, Giang Vo; Tran, Van Hieu; Vu, Van Nam; Vu, Van Loi; Le, Cong Vinh; Nguyen, Thuy Linh; Phi, Thi Dao; Truong, Bich Ngan; Chau, Van Minh; Pham, Van Cuong

    2014-12-01

    A series of febrifugine analogues were designed and synthesized. Antimalarial activity evaluation of the synthetic compounds indicated that these derivatives had a strong inhibition against both chloroquine-sensitive and -resistant Plasmodium falciparum parasites. Many of them were found to be more active than febrifugine hydrochloride. The tested analogues had also a significant cytotoxicity against four cancer cell lines (KB, MCF7, LU1 and HepG2). Among the synthetic analogues, two compounds 17b and 17h displayed a moderate cytotoxicity while they exhibited a remarkable antimalarial activity. PMID:25632466

  13. A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET).

    PubMed

    Lerner, Eitan; Ploetz, Evelyn; Hohlbein, Johannes; Cordes, Thorben; Weiss, Shimon

    2016-07-01

    Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein-DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

  14. Characterization of the fretting corrosion behavior, surface and debris from head-taper interface of two different modular hip prostheses.

    PubMed

    Dos Santos, Claudio T; Barbosa, Cassio; Monteiro, Maurício J; Abud, Ibrahim C; Caminha, Ieda M V; Roesler, Carlos R M

    2016-09-01

    Modular hip prostheses are flexible to match anatomical variations and to optimize mechanical and tribological properties of each part by using different materials. However, micromotions associated with the modular components can lead to fretting corrosion and, consequently, to release of debris which can cause adverse local tissue reactions in human body. In the present study, the surface damage and residues released during in vitro fretting corrosion tests were characterized by stereomicroscope, SEM and EDS. Two models of modular hip prosthesis were studied: Model SS/Ti Cementless whose stem was made of ASTM F136 Ti-6Al-4V alloy and whose metallic head was made of ASTM F138 austenitic stainless steel, and Model SS/SS Cemented with both components made of ASTM F138 stainless steel. The fretting corrosion tests were evaluated according to the criteria of ASTM F1875 standard. Micromotions during the test caused mechanical wear and material loss in the head-taper interface, resulting in fretting-corrosion. Model SS/SS showed higher grade of corrosion. Different morphologies of debris predominated in each model studied. Small and agglomerated particles were observed in the Model SS/Ti and irregular particles in the Model SS/SS. After 10 million cycles, the Model SS/Ti was more resistant to fretting corrosion than the Model SS/SS. PMID:27179766

  15. Development of confocal immunofluorescence FRET microscopy to Investigate eNOS and GSNOR localization and interaction in pulmonary endothelial cells

    NASA Astrophysics Data System (ADS)

    Rehman, Shagufta; Brown-Steinke, Kathleen; Palmer, Lisa; Periasamy, Ammasi

    2015-03-01

    Confocal FRET microscopy is a widely used technique for studying protein-protein interactions in live or fixed cells. Endothelial nitric oxide synthase (eNOS) and S-nitrosoglutathione reductase (GSNOR) are enzymes involved in regulating the bioavailability of S-nitrosothiols (SNOs) in the pulmonary endothelium and have roles in the development of pulmonary arterial hypertension. Labeling of endogenous proteins to better understand a disease process can be challenging. We have used immunofluorescence to detect endogenous eNOS and GSNOR in primary pulmonary endothelial cells to co-localize these proteins as well as to study their interaction by FRET. The challenge has been in selecting the right immunofluorescence labeling condition, right antibody, the right blocking reagent, the right FRET pair and eliminating cross-reactivity of secondary antibodies. We have used Alexa488 and Alexa568 as a FRET pair. After a series of optimizations, the data from Confocal Laser Scanning Microscopy (CLSM) demonstrate co-localization of eNOS and GSNOR in the perinuclear region of the pulmonary endothelial cell primarily within the cis-Golgi with lower levels of co-localization seen within the trans-Golgi. FRET studies demonstrate, for the first time, interaction between eNOS and GSNOR in both murine and bovine pulmonary endothelial cells. Further characterization of eNOSGSNOR interaction and the subcellular location of this interaction will provide mechanistic insight into the importance of S-nitrosothiol signaling in pulmonary biology, physiology and pathology.

  16. Characterization of the fretting corrosion behavior, surface and debris from head-taper interface of two different modular hip prostheses.

    PubMed

    Dos Santos, Claudio T; Barbosa, Cassio; Monteiro, Maurício J; Abud, Ibrahim C; Caminha, Ieda M V; Roesler, Carlos R M

    2016-09-01

    Modular hip prostheses are flexible to match anatomical variations and to optimize mechanical and tribological properties of each part by using different materials. However, micromotions associated with the modular components can lead to fretting corrosion and, consequently, to release of debris which can cause adverse local tissue reactions in human body. In the present study, the surface damage and residues released during in vitro fretting corrosion tests were characterized by stereomicroscope, SEM and EDS. Two models of modular hip prosthesis were studied: Model SS/Ti Cementless whose stem was made of ASTM F136 Ti-6Al-4V alloy and whose metallic head was made of ASTM F138 austenitic stainless steel, and Model SS/SS Cemented with both components made of ASTM F138 stainless steel. The fretting corrosion tests were evaluated according to the criteria of ASTM F1875 standard. Micromotions during the test caused mechanical wear and material loss in the head-taper interface, resulting in fretting-corrosion. Model SS/SS showed higher grade of corrosion. Different morphologies of debris predominated in each model studied. Small and agglomerated particles were observed in the Model SS/Ti and irregular particles in the Model SS/SS. After 10 million cycles, the Model SS/Ti was more resistant to fretting corrosion than the Model SS/SS.

  17. Fretting behavior of NiTi shape memory alloy against long bone in the imitated human physiological solution

    NASA Astrophysics Data System (ADS)

    Yin, Y.; Xu, Y. T.; Xia, T. D.; Da, G. Z.

    2007-07-01

    The environment of orthopaedic implants sometimes induces vibrations at the contact of the modular prostheses components. In this paper the fretting behavior of NiTi SMAs against human bones in the imitated human physiological solution was studied at various displacement amplitudes and Ph value. Surface micrograph after fretting was observed by MEF3 microscope. Appearance of fretting scar was measured by 2206 roughness tester. The result shows that the friction coefficient between the bone and NiTi SMAs pairs declined due to the lubrication effect of Hank's solution, and which increased when Ph value of fluid was not 7.2 due to the corrosion. So the friction coefficient at acid and alkali Hank's solution is higher than those at the neutral solution and ambient air condition. Generally speaking, the friction coefficient between the bone and NiTi SMAs tend to be stable with the increasing amplitude at all test conditions. It is because that the surface was oxidized to restrain the forming of wear debris and the further development of fretting scars. Although the length and width of the wear scars in simulation body fluid are smaller than that at ambient air condition, the surface of NiTi SMAs damaged is characterized by deep scratches with debris particles within the contact area. Fretting regime of NiTi/bones pairs exhibits the mixed regime at ambient air condition and the slip regime in the Hank's solution.

  18. Proposal of a New Method for Measuring Förster Resonance Energy Transfer (FRET) Rapidly, Quantitatively and Non-Destructively

    PubMed Central

    Helm, Paul Johannes

    2012-01-01

    The process of radiationless energy transfer from a chromophore in an excited electronic state (the “donor”) to another chromophore (an “acceptor”), in which the energy released by the donor effects an electronic transition, is known as “Förster Resonance Energy Transfer” (FRET). The rate of energy transfer is dependent on the sixth power of the distance between donor and acceptor. Determining FRET efficiencies is tantamount to measuring distances between molecules. A new method is proposed for determining FRET efficiencies rapidly, quantitatively, and non-destructively on ensembles containing donor acceptor pairs: at wavelengths suitable for mutually exclusive excitations of donors and acceptors, two laser beams are intensity-modulated in rectangular patterns at duty cycle ½ and frequencies f1 and f2 by electro-optic modulators. In an ensemble exposed to these laser beams, the donor excitation is modulated at f1, and the acceptor excitation, and therefore the degree of saturation of the excited electronic state of the acceptors, is modulated at f2. Since the ensemble contains donor acceptor pairs engaged in FRET, the released donor fluorescence is modulated not only at f1 but also at the beat frequency Δf: = |f1 − f2|. The depth of the latter modulation, detectable via a lock-in amplifier, quantitatively indicates the FRET efficiency. PMID:23202903

  19. A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement–Förster-Type Resonance Energy Transfer (PIFE-FRET)

    PubMed Central

    2016-01-01

    Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein–DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems. PMID:27184889

  20. Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

    PubMed

    Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

    2015-01-01

    Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002-0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer. PMID:26290434

  1. Nucleobases and prebiotic molecules in organic residues produced from the ultraviolet photo-irradiation of pyrimidine in NH(3) and H(2)O+NH(3) ices.

    PubMed

    Nuevo, Michel; Milam, Stefanie N; Sandford, Scott A

    2012-04-01

    Although not yet identified in the interstellar medium (ISM), N-heterocycles including nucleobases-the information subunits of DNA and RNA-are present in carbonaceous chondrites, which indicates that molecules of biological interest can be formed in non-terrestrial environments via abiotic pathways. Recent laboratory experiments and ab initio calculations have already shown that the irradiation of pyrimidine in pure H(2)O ices leads to the formation of a suite of oxidized pyrimidine derivatives, including the nucleobase uracil. In the present work, NH(3):pyrimidine and H(2)O:NH(3):pyrimidine ice mixtures with different relative proportions were irradiated with UV photons under astrophysically relevant conditions. Liquid- and gas-chromatography analysis of the resulting organic residues has led to the detection of the nucleobases uracil and cytosine, as well as other species of prebiotic interest such as urea and small amino acids. The presence of these molecules in organic residues formed under abiotic conditions supports scenarios in which extraterrestrial organics that formed in space and were subsequently delivered to telluric planets via comets and meteorites could have contributed to the inventory of molecules that triggered the first biological reactions on their surfaces. PMID:22519971

  2. A new route for the prebiotic synthesis of nucleobases and hydantoins in water/ice solutions involving the photochemistry of acetylene.

    PubMed

    Menor-Salván, César; Marín-Yaseli, Margarita R

    2013-05-10

    The origin of nucleobases and other heterocycles is a classic question in the chemistry of the origins of life. The construction of laboratory models for the abiotic synthesis of nitrogen heterocycles in plausible natural conditions also aids the understanding and prediction of chemical species in the Solar System. Here, we report a new explanation for the origin of hydantoins, purines, and pyrimidines in eutectic water/ice/urea solutions driven by ultraviolet irradiation (in the 185-254 nm range, UVC) of acetylene under anoxic conditions. An analysis of the products indicates the synthesis of hydantoin and 5-hydroxyhydantoin, the purines uric acid, xanthine, and guanine, and the pyrimidines uracil and cytosine. The synthesis occurred together with the photo-oxidation of bases in a complex process for which possible pathways are proposed. In conclusion, an acetylene-containing atmosphere could contribute to the origin of nucleobases in the presence of a urea/water system by an HCN-independent mechanism. The presence of ice has a dual role as a favorable medium for the synthesis of nucleobases and protection against degradation and as a source of free radicals for the synthesis of highly oxidized heterocycles. A mechanism for the origin of hydantoins and uracil from urea in plausible conditions for prebiotic chemistry is also proposed.

  3. Comparative characterization of nucleotides, nucleosides and nucleobases in Abelmoschus manihot roots, stems, leaves and flowers during different growth periods by UPLC-TQ-MS/MS.

    PubMed

    Du, Le-yue; Qian, Da-wei; Jiang, Shu; Shang, Er-xin; Guo, Jian-ming; Liu, Pei; Su, Shu-lan; Duan, Jin-ao; Zhao, Min

    2015-12-01

    Nucleotides, nucleosides and nucleobases have been proven as important bioactive compounds related to many physiological processes. Abelmoschus manihot (L.) Medicus from the family of Malvaceae is an annual herbal plant of folk medicine widely distributed in Oceania and Asia. However, up to now, no detailed information could be available for the types and contents of nucleotides, nucleosides and nucleobases contained in A. manihot roots, stems, leaves as well as the flowers. In the present study, an UPLC-TQ-MS/MS method was established for detection of the twelve nucleotides, nucleosides and nucleobases. The validated method was successfully applied to identify the 12 analytes in different parts of A. manihot harvested at ten growth periods. 2'-deoxyinosine was not detected in all of the A. manihot samples. The data demonstrated that the distribution and concentration of the 12 compounds in A. manihot four parts were arranged in a decreasing order as leaf>flower>stem>root. Based on the results, the leaves and flowers of A. manihot could be developed as health products possessed nutraceutical and bioactive properties in the future. This method might also be utilized for the quality control of the A. manihot leaves and other herbal medicines being rich in nucleotides, nucleosides and nulecobases.

  4. A new route for the prebiotic synthesis of nucleobases and hydantoins in water/ice solutions involving the photochemistry of acetylene.

    PubMed

    Menor-Salván, César; Marín-Yaseli, Margarita R

    2013-05-10

    The origin of nucleobases and other heterocycles is a classic question in the chemistry of the origins of life. The construction of laboratory models for the abiotic synthesis of nitrogen heterocycles in plausible natural conditions also aids the understanding and prediction of chemical species in the Solar System. Here, we report a new explanation for the origin of hydantoins, purines, and pyrimidines in eutectic water/ice/urea solutions driven by ultraviolet irradiation (in the 185-254 nm range, UVC) of acetylene under anoxic conditions. An analysis of the products indicates the synthesis of hydantoin and 5-hydroxyhydantoin, the purines uric acid, xanthine, and guanine, and the pyrimidines uracil and cytosine. The synthesis occurred together with the photo-oxidation of bases in a complex process for which possible pathways are proposed. In conclusion, an acetylene-containing atmosphere could contribute to the origin of nucleobases in the presence of a urea/water system by an HCN-independent mechanism. The presence of ice has a dual role as a favorable medium for the synthesis of nucleobases and protection against degradation and as a source of free radicals for the synthesis of highly oxidized heterocycles. A mechanism for the origin of hydantoins and uracil from urea in plausible conditions for prebiotic chemistry is also proposed. PMID:23536286

  5. Comparative characterization of nucleotides, nucleosides and nucleobases in Abelmoschus manihot roots, stems, leaves and flowers during different growth periods by UPLC-TQ-MS/MS.

    PubMed

    Du, Le-yue; Qian, Da-wei; Jiang, Shu; Shang, Er-xin; Guo, Jian-ming; Liu, Pei; Su, Shu-lan; Duan, Jin-ao; Zhao, Min

    2015-12-01

    Nucleotides, nucleosides and nucleobases have been proven as important bioactive compounds related to many physiological processes. Abelmoschus manihot (L.) Medicus from the family of Malvaceae is an annual herbal plant of folk medicine widely distributed in Oceania and Asia. However, up to now, no detailed information could be available for the types and contents of nucleotides, nucleosides and nucleobases contained in A. manihot roots, stems, leaves as well as the flowers. In the present study, an UPLC-TQ-MS/MS method was established for detection of the twelve nucleotides, nucleosides and nucleobases. The validated method was successfully applied to identify the 12 analytes in different parts of A. manihot harvested at ten growth periods. 2'-deoxyinosine was not detected in all of the A. manihot samples. The data demonstrated that the distribution and concentration of the 12 compounds in A. manihot four parts were arranged in a decreasing order as leaf>flower>stem>root. Based on the results, the leaves and flowers of A. manihot could be developed as health products possessed nutraceutical and bioactive properties in the future. This method might also be utilized for the quality control of the A. manihot leaves and other herbal medicines being rich in nucleotides, nucleosides and nulecobases. PMID:26551204

  6. Nucleobases and Prebiotic Molecules in Organic Residues Produced from the Ultraviolet Photo-Irradiation of Pyrimidine in NH3 and H2O+NH3 Ices

    NASA Technical Reports Server (NTRS)

    Nuevo, Michel; Milam, Stefanie N.; Sandford, Scott

    2012-01-01

    Although not yet identified in the interstellar medium (ISM), N-heterocycles including nucleobases the information subunits of DNA and RNA are present in carbonaceous chondrites, which indicates that molecules of biological interest can be formed in non-terrestrial environments via abiotic pathways. Recent laboratory experiments and ab-initio calculations have already shown that the irradiation of pyrimidine in pure H2O ices leads to the formation of a suite of oxidized pyrimidine derivatives, including the nucleobase uracil. In the present work, NH3:pyrimidine and H2O:NH3:pyrimidine ice mixtures with different relative proportions were irradiated with UV photons under astrophysically relevant conditions. Liquid- and gas-chromatography analysis of the resulting organic residues has led to the detection of the nucleobases uracil and cytosine, as well as other species of prebiotic interest such as urea and small amino acids. The presence of these molecules in organic residues formed under abiotic conditions supports scenarios in which extraterrestrial organics that formed in space and were subsequently delivered to telluric planets via comets and meteorites could have contributed to the inventory of molecules that triggered the first biological reactions on their surfaces.

  7. Antimicrobial activity of resveratrol analogues.

    PubMed

    Chalal, Malik; Klinguer, Agnès; Echairi, Abdelwahad; Meunier, Philippe; Vervandier-Fasseur, Dominique; Adrian, Marielle

    2014-01-01

    Stilbenes, especially resveratrol and its derivatives, have become famous for their positive effects on a wide range of medical disorders, as indicated by a huge number of published studies. A less investigated area of research is their antimicrobial properties. A series of 13 trans-resveratrol analogues was synthesized via Wittig or Heck reactions, and their antimicrobial activity assessed on two different grapevine pathogens responsible for severe diseases in the vineyard. The entire series, together with resveratrol, was first evaluated on the zoospore mobility and sporulation level of Plasmopara viticola (the oomycete responsible for downy mildew). Stilbenes displayed a spectrum of activity ranging from low to high. Six of them, including the most active ones, were subsequently tested on the development of Botrytis cinerea (fungus responsible for grey mold). The results obtained allowed us to identify the most active stilbenes against both grapevine pathogens, to compare the antimicrobial activity of the evaluated series of stilbenes, and to discuss the relationship between their chemical structure (number and position of methoxy and hydroxy groups) and antimicrobial activity. PMID:24918540

  8. Space analogue studies in Antarctica.

    PubMed

    Lugg, D; Shepanek, M

    1999-01-01

    Medical research has been carried out on the Australian National Antarctic Research Expeditions (ANARE) for 50 years. As an extension of this program collaborative Australian/United States research on immunology, microbiology, psychology and remote medicine has produced important data and insight on how humans adapt to the stress of extreme isolation, confinement and the harsh environment of Antarctica. An outstanding analogue for the isolation and confinement of space missions (especially planetary outposts), ANARE has been used as an international research platform by Australia and the United States since 1993. Collaborative research has demonstrated a lowered responsiveness of the immune system under the isolation and confinement of Antarctic winter-over; a reduction of almost 50% in T cell proliferation to mitogen phytohaemogglutinin, as well as changes in latent herpesvirus states and the expansion of the polyclonal latent Epstein-Barr virus infected B cell populations. Although no clinically significant disease has been found to result from these immune changes, research is currently assessing the effects of psychological factors on the immune system. This and associated research performed to date and its relevance to both organisations is discussed, and comment made on possible extensions to the program in both medical and other fields.

  9. Antimicrobial activity of resveratrol analogues.

    PubMed

    Chalal, Malik; Klinguer, Agnès; Echairi, Abdelwahad; Meunier, Philippe; Vervandier-Fasseur, Dominique; Adrian, Marielle

    2014-06-10

    Stilbenes, especially resveratrol and its derivatives, have become famous for their positive effects on a wide range of medical disorders, as indicated by a huge number of published studies. A less investigated area of research is their antimicrobial properties. A series of 13 trans-resveratrol analogues was synthesized via Wittig or Heck reactions, and their antimicrobial activity assessed on two different grapevine pathogens responsible for severe diseases in the vineyard. The entire series, together with resveratrol, was first evaluated on the zoospore mobility and sporulation level of Plasmopara viticola (the oomycete responsible for downy mildew). Stilbenes displayed a spectrum of activity ranging from low to high. Six of them, including the most active ones, were subsequently tested on the development of Botrytis cinerea (fungus responsible for grey mold). The results obtained allowed us to identify the most active stilbenes against both grapevine pathogens, to compare the antimicrobial activity of the evaluated series of stilbenes, and to discuss the relationship between their chemical structure (number and position of methoxy and hydroxy groups) and antimicrobial activity.

  10. Space analogue studies in Antarctica

    NASA Technical Reports Server (NTRS)

    Lugg, D.; Shepanek, M.

    1999-01-01

    Medical research has been carried out on the Australian National Antarctic Research Expeditions (ANARE) for 50 years. As an extension of this program collaborative Australian/United States research on immunology, microbiology, psychology and remote medicine has produced important data and insight on how humans adapt to the stress of extreme isolation, confinement and the harsh environment of Antarctica. An outstanding analogue for the isolation and confinement of space missions (especially planetary outposts), ANARE has been used as an international research platform by Australia and the United States since 1993. Collaborative research has demonstrated a lowered responsiveness of the immune system under the isolation and confinement of Antarctic winter-over; a reduction of almost 50% in T cell proliferation to mitogen phytohaemogglutinin, as well as changes in latent herpesvirus states and the expansion of the polyclonal latent Epstein-Barr virus infected B cell populations. Although no clinically significant disease has been found to result from these immune changes, research is currently assessing the effects of psychological factors on the immune system. This and associated research performed to date and its relevance to both organisations is discussed, and comment made on possible extensions to the program in both medical and other fields.

  11. Space analogue studies in Antarctica

    NASA Astrophysics Data System (ADS)

    Lugg, D.; Shepanek, M.

    1999-09-01

    Medical research has been carried out on the Australian National Antarctic Research Expeditions (ANARE) for 50 years. As an extension of this program collaborative Australian/United States research on immunology, microbiology, psychology and remote medicine has produced important data and insight on how humans adapt to the stress of extreme isolation, confinement and the harsh environment of Antarctica. An outstanding analogue for the isolation and confinement of space missions (especially planetary outposts), ANARE has been used as an international research platform by Australia and the United States since 1993. Collaborative research has demonstrated a lowered responsiveness of the immune system under the isolation and confinement of Antarctic winter-over; a reduction of almost 50% in T cell proliferation to mltogen phytohaemogglutinin, as well as changes in latent herpesvirus states and the expansion of the polyclonal latent Epstein-Barr virus infected B cell populations. Although no clinically significant disease has been found to result from these immune changes, research is currently assessing the effects of psychological factors on the immune system. This and associated research performed to date and its relevance to both organisations is discussed, and comment made on possible extensions to the program in both medical and other fields.

  12. Condensed matter analogues of cosmology

    NASA Astrophysics Data System (ADS)

    Kibble, Tom; Srivastava, Ajit

    2013-10-01

    It is always exciting when developments in one branch of physics turn out to have relevance in a quite different branch. It would be hard to find two branches farther apart in terms of energy scales than early-universe cosmology and low-temperature condensed matter physics. Nevertheless ideas about the formation of topological defects during rapid phase transitions that originated in the context of the very early universe have proved remarkably fruitful when applied to a variety of condensed matter systems. The mathematical frameworks for describing these systems can be very similar. This interconnection has led to a deeper understanding of the phenomena in condensed matter systems utilizing ideas from cosmology. At the same time, one can view these condensed matter analogues as providing, at least in a limited sense, experimental access to the phenomena of the early universe for which no direct probe is possible. As this special issue well illustrates, this remains a dynamic and exciting field. The basic idea is that when a system goes through a rapid symmetry-breaking phase transition from a symmetric phase into one with spontaneously broken symmetry, the order parameter may make different choices in different regions, creating domains that when they meet can trap defects. The scale of those domains, and hence the density of defects, is constrained by the rate at which the system goes through the transition and the speed with which order parameter information propagates. This is what has come to be known as the Kibble-Zurek mechanism. The resultant scaling laws have now been tested in a considerable variety of different systems. The earliest experiments illustrating the analogy between cosmology and condensed matter were in liquid crystals, in particular on the isotropic-to-nematic transition, primarily because it is very easy to induce the phase transition (typically at room temperature) and to image precisely what is going on. This field remains one of the

  13. Fretting-corrosion in Hip Implant Modular Junctions: New Experimental Set-up and Initial Outcome

    PubMed Central

    Royhman, D.; Patel, M.; Runa, M.J.; Jacobs, J.J.; Hallab, N.J.; Wimmer, M.A.; Mathew, M.T.

    2015-01-01

    Modern hip prostheses feature a modular implant design with at least one tapered junction. This design can lead to several complications due to the introduction of additional interfaces, which are subjected to various loading conditions and micromotion. The main objective of current study is to develop a fretting corrosion apparatus, which is able characterize the mechanical and electrochemical behaviour of various existing metal alloy couples during fretting motion. This study describes the design and the main considerations during the development of a novel fretting corrosion apparatus, as well as determination of the machine compliance and the initial testing results. Machine compliance considerations and frictional interactions of the couples are discussed in detail. For the preliminary tests, metal alloy pins, made of Ti6Al4V and wrought high-carbon CoCrMo were mechanically polished to a surface roughness of less than 20nm. 2 pins (Diameter = 11mm) of either Ti6Al4V or CoCrMo were loaded onto a Ti6Al4V alloy rod at a normal force of 200N. The interface types included: Ti6Al4V-Ti6Al4V-Ti6Al4V, Ti6Al4V-Ti6Al4V-CoCrMo, and CoCrMo-Ti6Al4V-CoCrMo. The Ti6Al4V rod articulated against the metal alloy pins in a sinusoidal fretting motion with a displacement amplitude of ±50μm. Bovine calf serum (30g/L of protein content) was selected as a lubricant and tested at 2 different pH levels (pH 3.0 and 7.6). In all cases, current and friction energy were monitored during the fretting process. The results indicated distinct, material-specific current evolutions and friction energies. No significant differences were observed in electrochemical or mechanical behaviour in response to pH change. In general, Ti6Al4V-Ti6Al4V-Ti6Al4V couples displayed the earliest passivation and superior electrochemical behaviour compared to Ti6Al4V-Ti6Al4V-CoCrMo and CoCrMo-Ti6Al4V-CoCrMo under fretting conditions. In addition, fluctuations in current were observed in specific regions at all

  14. Sulfur analogues of psychotomimetic agents. Monothio analogues of mescaline and isomescaline.

    PubMed

    Jacob, P; Shulgin, A T

    1981-11-01

    Two monothio analogues of mescaline and three monothio analogues of 2,3,4-trimethoxyphenethylamine (isomescaline) have been synthesized and characterized. Only the two mescaline analogues (3-and 4-thiomescaline) were found to be psychotomimetics in man, being 6 and 12 times more potent than mescaline, respectively. All five compounds can serve as substrates for bovine plasma monoamine oxidase in vitro, but no positive correlation is apparent between the extent of enzymatic degradation and human psychotomimetic potency.

  15. A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins

    PubMed Central

    Booth, E.A.; Thorner, J.

    2016-01-01

    Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein–protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level “ruler” because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤ 10 nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins. PMID:27473902

  16. A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins.

    PubMed

    Booth, E A; Thorner, J

    2016-01-01

    Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein-protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level "ruler" because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤10nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins. PMID:27473902

  17. Fretting Wear Properties of TiCN-Ni Cermets: Influence of Load and Secondary Carbide Addition

    NASA Astrophysics Data System (ADS)

    Manoj Kumar, B. V.; Basu, Bikramjit

    2008-03-01

    The increasing demand for TiCN-based cermets in tribological applications necessitates a thorough understanding of the influence of experimental as well as material parameters on the friction and wear properties. In optimizing microstructure and properties, secondary carbides are added to baseline TiCN-Ni cermet. The present work aims at evaluating the fretting wear behavior of Ti(CN)-Ni cermets containing various secondary carbides, such as WC, NbC, TaC, and HfC, against steel at different loading (2, 6, and 10 N) conditions. The evolution of tangential frictional force for the investigated cermets was analyzed in terms of fretting logs and fretting loops. The topographical characterization of worn surfaces was performed, using laser surface profilometry and a scanning electron microscope (SEM) equipped with energy-dispersive spectroscopy (EDS) capability. The steady-state coefficient of friction (COF) was minimum (0.33) for a TiCN-20Ni cermet/steel tribocouple, while a maximum COF (0.47) was recorded for TiCN-20Ni-10HfC cermet/steel at a 2-N load. The wear rate of the cermets varied in the range of 1.7 × 10-6 to 3.5 × 10-6 mm3/Nm. The TiCN-20Ni-10HfC cermet exhibited poor wear resistance among investigated cermets. The dominant wear mechanisms were found as abrasion and tribolayer formation. The dominance of abrasion is explained in terms of cumulative energy dissipation.

  18. Utilizing homogenous FRET to extend molecular photonic wires beyond 30 nm (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Diaz, Sebastian A.; Buckhout-White, Susan; Ancona, Mario G.; Melinger, Joseph S.; Medintz, Igor L.

    2015-10-01

    Molecular photonic wires (MPWs) present interesting applications in energy harvesting, artificial photosynthesis, and nano-circuitry. MPWs allow the directed movement of energy at the nanoscopic level. Extending the length of the energy transfer with a minimal loss in efficiency would overcome an important hurdle in allowing MPWs to reach their potential. We investigated Homogenous Förster Resonance Energy Transfer (HomoFRET) as a means to achieve this goal. We designed a simple, self-assembled DNA nanostructure with specifically placed dyes (Alexa488-Cy3-Cy3.5-Alexa647-Cy5.5) at a distance of 3.4 nm, a separation at which energy transfer should theoretically be very high. The input of the wire was at 466 nm with an output up to 697 nm. Different structures were studied where the Cy3.5 section of the MPW was extended from one to six repeats. We found that though the efficiency cost is not null, HomoFRET can be extended up to six repeat dyes with only a 22% efficiency loss when compared to a single step system. The advantage is that these six repeats created a MPW which was 17 nm longer, almost 2.5 times the initial length. To confirm the existence of HomoFRET between the Cy3.5 repeats fluorescence lifetime and fluorescence lifetime anisotropy was measured. Under these conditions we are able to demonstrate the energy transfer over a distance of 30.4 nm, with an end-to-end efficiency of 2.0%, by utilizing a system with only five unique dyes.

  19. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

    PubMed

    Raina, Shweta A; Tsai, Jeffrey; Samsó, Montserrat; Fessenden, James D

    2012-01-01

    Fluorescent protein (FP) insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM) maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET) measurements were used to localize green fluorescent protein (GFP) insertions within the ryanodine receptor type 1 (RyR1), a large intracellular Ca(2+) release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK)-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

  20. The structure activity relationship of discodermolide analogues.

    PubMed

    Shaw, Simon J

    2008-03-01

    The marine polyketide discodermolide is a member of a class of natural products that stabilize microtubules. Many analogues have been synthesized suggesting that few changes can be made to the internal carbon backbone. Both ends of the molecule, however, can be modified. The majority of analogues have been generated via modification of the lactone region. This suggests that significant simplifications can be made in this region provided that the lactone moiety is maintained.