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

    PubMed

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

    2016-06-20

    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

  3. Gas-phase lifetimes of nucleobase analogues by picosecond pumpionization and streak techniques.

    PubMed

    Blaser, Susan; Frey, Hans-Martin; Heid, Cornelia G; Leutwyler, Samuel

    2014-01-01

    The picosecond (ps) timescale is relevant for the investigation of many molecular dynamical processes such as fluorescence, nonradiative relaxation, intramolecular vibrational relaxation, molecular rotation and intermolecular energy transfer, to name a few. While investigations of ultrafast (femtosecond) processes of biological molecules, e.g. nucleobases and their analogues in the gas phase are available, there are few investigations on the ps time scale. We have constructed a ps pump-ionization setup and a ps streak camera fluorescence apparatus for the determination of lifetimes of supersonic jet-cooled and isolated molecules and clusters. The ps pump-ionization setup was used to determine the lifetimes of the nucleobase analogue 2-aminopurine (2AP) and of two 2AP˙(H2O)n water cluster isomers with n=1 and 2. Their lifetimes lie between 150 ps and 3 ns and are strongly cluster-size dependent. The ps streak camera setup was used to determine accurate fluorescence lifetimes of the uracil analogue 2-pyridone (2PY), its self-dimer (2PY)2, two isomers of its trimer (2PY)3 and its tetramer (2PY)4, which lie in the 7-12 ns range. PMID:24983611

  4. Reactions of β-Propiolactone with Nucleobase Analogues, Nucleosides, and Peptides

    PubMed Central

    Uittenbogaard, Joost P.; Zomer, Bert; Hoogerhout, Peter; Metz, Bernard

    2011-01-01

    β-Propiolactone is often applied for inactivation of viruses and preparation of viral vaccines. However, the exact nature of the reactions of β-propiolactone with viral components is largely unknown. The purpose of the current study was to elucidate the chemical modifications occurring on nucleotides and amino acid residues caused by β-propiolactone. Therefore, a set of nucleobase analogues was treated with β-propiolactone, and reaction products were identified and quantified. NMR revealed at least one modification in either deoxyguanosine, deoxyadenosine, or cytidine after treatment with β-propiolactone. However, no reaction products were found from thymidine and uracil. The most reactive sides of the nucleobase analogues and nucleosides were identified by NMR. Furthermore, a series of synthetic peptides was used to determine the conversion of reactive amino acid residues by liquid chromatography-mass spectrometry. β-Propiolactone was shown to react with nine different amino acid residues. The most reactive residues are cysteine, methionine, and histidine and, to a lesser degree, aspartic acid, glutamic acid, tyrosine, lysine, serine, and threonine. Remarkably, cystine residues (disulfide groups) do not react with β-propiolactone. In addition, no reaction was observed for β-propiolactone with asparagine, glutamine, and tryptophan residues. β-Propiolactone modifies proteins to a larger extent than expected from current literature. In conclusion, the study determined the reactivity of β-propiolactone with nucleobase analogues, nucleosides, and amino acid residues and elucidated the chemical structures of the reaction products. The study provides detailed knowledge on the chemistry of β-propiolactone inactivation of viruses. PMID:21868382

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Inhibition of Siderophore Biosynthesis in Mycobacterium tuberculosis with Nucleoside Bisubstrate Analogues: Structure–Activity Relationships of the Nucleobase Domain of 5′-O-[N-(Salicyl)sulfamoyl]adenosine

    PubMed Central

    Neres, João; Labello, Nicholas P.; Somu, Ravindranadh V.; Boshoff, Helena I.; Wilson, Daniel J.; Vannada, Jagadeshwar; Chen, Liqiang; Barry, Clifton E.; Bennett, Eric M.; Aldrich, Courtney C.

    2009-01-01

    5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) is a prototype for a new class of antitubercular agents that inhibit the aryl acid adenylating enzyme (AAAE) known as MbtA involved in biosynthesis of the mycobactins. Herein, we report the structure-based design, synthesis, biochemical, and biological evaluation of a comprehensive and systematic series of analogues, exploring the structure–activity relationship of the purine nucleobase domain of Sal-AMS. Significantly, 2-phenyl-Sal-AMS derivative 26 exhibited exceptionally potent antitubercular activity with an MIC99 under iron-deficient conditions of 0.049 µM while the N-6-cyclopropyl-Sal-AMS 16 led to improved potency and to a 64-enhancement in activity under iron-deficient conditions relative to iron-replete conditions, a phenotype concordant with the designed mechanism of action. The most potent MbtA inhibitors disclosed here display in vitro antitubercular activity superior to most current first line TB drugs, and these compounds are also expected to be useful against a wide range of pathogens that require aryl-capped siderphores for virulence. PMID:18690677

  11. Microhydration of Deprotonated Nucleobases

    NASA Astrophysics Data System (ADS)

    Wincel, Henryk

    2016-05-01

    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.

  12. 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.

  13. 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.

  14. 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

  15. Synthesis and fluorescence characteristics of ATP-based FRET probes.

    PubMed

    Hardt, Norman; Hacker, Stephan M; Marx, Andreas

    2013-12-28

    Adenosine triphosphate (ATP) analogues labelled with two dyes suitable for undergoing Förster Resonance Energy Transfer (FRET) have the potential to be valuable tools to continuously study the enzymatic activity of ATP consuming enzymes. Here, we present a synthesis strategy that allows obtaining these ATP analogues in a straight-forward manner. Earlier studies indicate that modifying ATP at the O2'- and the γ-position is a very promising starting point for the design of these probes. We synthesized probes modified with five different combinations of dyes attached to these positions and investigated their fluorescence characteristics in the non-cleaved state as well as after enzymatic hydrolysis. All presented probes largely change their fluorescence characteristics upon cleavage. They include ratiometric FRET probes as well as dark quenched analogues. For typical in vitro applications a combination of the sulfonated polymethine dyes Sulfo-Cy3 and Sulfo-Cy5 seems to be most promising due to their excellent solubility in aqueous buffer and a large change of fluorescence characteristics upon cleavage. For this combination of dyes we also synthesized analogues modified at the γ- and the C2- or the O3'-position, respectively, as these attachment sites are also well accepted by certain ATP consuming enzymes. These analogues show comparably large changes in fluorescence characteristics. Overall, we present new ATP-based FRET probes that have the potential to enable monitoring the enzymatic activity of ATP consuming enzymes. PMID:24173528

  16. 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.

  17. 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, alumimum bronze with a polyester additive was most effective at reducing wear and minimizing fretting damage to the mating uncoated surface, by means of a self-lubricating 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.

  18. 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.

  19. FRET and mechanobiology

    PubMed Central

    2009-01-01

    Since the development of green fluorescent protein (GFP) and other fluorescent proteins (FPs) with distinct colors, genetically-encoded probes and biosensors have been widely applied to visualize the molecular localization and activities in live cells. In particular, biosensors based on fluorescence resonance energy transfer (FRET) have significantly advanced our understanding of the dynamic molecular hierarchy at subcellular levels. These biosensors have also been extensively applied in recent years to study how cells perceive the mechanical environment and transmit it into intracellular molecular signals (i.e. mechanotransduction). In this review, we will first provide a brief introduction of the recent development of FPs. Different FRET biosensors based on FPs will then be described. The last part of the review will be dedicated to the introduction of examples applying FRET biosensors to visualize mechanotransduction in live cells. In summary, the integration of FRET technology and the different cutting-edge mechanical stimulation systems can provide powerful tools to allow the elucidation of the mechanisms regulating mechanobiology at cellular and molecular levels in normal and pathophysiological conditions. PMID:20016756

  20. 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

  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. Mechanics of fretting fatigue

    NASA Astrophysics Data System (ADS)

    Hills, D. A.

    1994-06-01

    Several aspects of the mechanics of cracks originating at sites of fretting are considered. It is argued that the problem may be distilled into three separate parts: the contact problem itself in full or partial slip, the initiation of a crack from a surface suffering severe distress, and the propagation of a crack under combined contact and bulk loading. The first of these may be solved by either a classical or numerical means, while the last merely requires the careful use of fracture mechanics. However, it is the second element which remains elusive to quantify, and the influence of the intrinsic length scales in the problem, including contact length, surface roughness and amplitude of relative tangential displacement on initiation conditions, is discussed and explored.

  3. 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

  4. 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

  5. Communication: Photoactivation of nucleobase bound platinum(II) metal complexes: probing the influence of the nucleobase.

    PubMed

    Sen, Ananya; Dessent, Caroline E H

    2014-12-28

    We present UV laser action spectra (220-300 nm) of isolated nucleobase-bound Pt(II)(CN)4(2-) complexes, i.e., Pt(CN)4(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 λmax ∼ 4.7 eV absorption band (nucleobase localized chromophore) and a subsequent increase in the absorption intensity towards higher spectral-energy (Pt(CN)4(2-) localized chromophore). However, strikingly different band widths are observed across the series of complexes, decreasing in the order Pt(CN)4(2-)⋅Thymine > Pt(CN)4(2-)⋅Uracil > Pt(CN)4(2-)⋅Adenine > Pt(CN)4(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. PMID:25554122

  6. 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.

  7. FRET enhanced fluorescent nanodiamonds.

    PubMed

    Fudala, Rafal; Raut, Sangram; Maliwal, Badri P; Zerda, T W; Gryczynski, Ignacy; Simanek, Eric; Borejdo, Julian; Rich, Ryan; Akopova, Irina; Gryczynski, Zygmunt

    2014-01-01

    Fluorescent nanodiamonds (FNDs) are one of the new and very promising biocompatible nanomaterials that can be used both as a fluorescence imaging agent and a highly versatile platform for controlled functionalization to target and deliver a wide spectrum of therapeutic agents. Among the remarkable fluorescence properties are excellent photostability, emission between 600-700nm, quantum yield of 1 and moderately long fluorescence lifetimes. However the low absorption cross section of fluorescent (N-V)(-) centers limits FNDs' brightness. In this work we show that an approach based on the Forster resonance energy transfer (FRET) may significantly enhance the fluorescence signal observed from a single ND. We demonstrate that organic dyes (fluorophores) attached to the FND surface can efficiently transfer the excitation energy to (N-V)(-) centers. Multiple dyes positioned in close proximity to the ND facile surface may serve as harvesting antennas transferring excitation energy to the fluorescent centers. We propose that, with the help of some of the functional groups present on the FND surface, we can either directly link flurophores or use scalable dendrimer chemistry to position many organic dyes at a calibrated distance. Also, the remaining multiple functional groups will be still available for particle targeting and drug delivery. This opens a new way for designing a new type of theranostics particles of ultrahigh brightness, high photostability, specific targeting, and high capacity for drug delivery. PMID:22394126

  8. 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

  9. Correlation of nucleoside and nucleobase transporter gene expression with antimetabolite drug cytotoxicity.

    PubMed

    Lu, Xin; Gong, Shimei; Monks, Anne; Zaharevitz, Daniel; Moscow, Jeffrey A

    2002-01-01

    Antimetabolite drugs that inhibit nucleic acid metabolism are widely used in cancer chemotherapy. Nucleoside and nucleobase transporters are important for the cellular uptake of nucleic acids and their corresponding anticancer analogue drugs. Thus, these transporters may play a role both in antimetabolite drug sensitivity, by mediating the uptake of nucleoside analogues, and in antimetabolite drug resistance, by mediating the uptake of endogenous nucleosides that may rescue cells from toxicity. Therefore, we examined the relation of the expression of nucleoside and nucleobase transporters to antimetabolite cytotoxicity. We measured the RNA levels of all eight known nucleoside and nucleobase transporters in 50 cell lines included in the National Cancer Institute's Anticancer Drug Screen panel. RNA levels of concentrative nucleoside transporters (CNTs), equilibrative nucleoside transporters (ENTs) and nucleobase transporters (NCBTs) were determined by quantitative RT-PCR using real-time fluorescence acquisition. This method was validated by measuring the expression of the MDR1 gene, and correlating our results with independently determined measurements of MDR1 RNA levels and protein function in these cell lines. We then correlated the pattern of RNA levels to the pattern of cytotoxicity of anticancer drugs in the NCI drug screen database using the COMPARE analysis. Several hypothesized relations between transporter gene expression and cytotoxicity, based upon known interactions between certain nucleoside analogues and transporter proteins, were not observed, suggesting that expression of individual transporters may not be a significant determinant of the cytotoxicity of these drugs. The most closely correlated drug cytotoxicity patterns to transporter gene expression patterns (where increased expression corresponds to increase sensitivity) included those between CNT1 and O6-methylguanine and between ENT2 and hydroxyurea. We also observed that p53 status influenced

  10. 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.

  11. 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.

  12. Latest advances in biomaterials: from deoxyribonucleic acid to nucleobases

    NASA Astrophysics Data System (ADS)

    Ouchen, Fahima; Gomez, Eliot; Joyce, Donna; Williams, Adrienne; Kim, Steve; Heckman, Emily; Johnson, Lewis; Yaney, Perry; Venkat, Narayanan; Steckl, Andrew; Kajzar, François; Rau, Ileana; Pawlicka, Agnieszka; Prasad, Paras; Grote, James

    2014-03-01

    This paper is a review of the recent research in bio-based materials for photonics and electronics applications. Materials that we have been working with include: deoxyribonucleic acid (DNA)-based biopolymers and nucleobases. We will highlight work on increasing the ionic conductivity of DNA-based membranes, enhancing the direct (DC) current and photoconductivity of DNA-based biopolymers, crosslinking of DNA-based biopolymers and promising applications for DNA nucleobases. Key

  13. 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

  14. 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

  15. Excitation of nucleobases from a computational perspective I: reaction paths.

    PubMed

    Giussani, Angelo; Segarra-Martí, Javier; Roca-Sanjuán, Daniel; Merchán, Manuela

    2015-01-01

    The main intrinsic photochemical events in nucleobases can be described on theoretical grounds within the realm of non-adiabatic computational photochemistry. From a static standpoint, the photochemical reaction path approach (PRPA), through the computation of the respective minimum energy path (MEP), can be regarded as the most suitable strategy in order to explore the electronically excited isolated nucleobases. Unfortunately, the PRPA does not appear widely in the studies reported in the last decade. The main ultrafast decay observed experimentally for the gas-phase excited nucleobases is related to the computed barrierless MEPs from the bright excited state connecting the initial Franck-Condon region and a conical intersection involving the ground state. At the highest level of theory currently available (CASPT2//CASPT2), the lowest excited (1)(ππ*) hypersurface for cytosine has a shallow minimum along the MEP deactivation pathway. In any case, the internal conversion processes in all the natural nucleobases are attained by means of interstate crossings, a self-protection mechanism that prevents the occurrence of photoinduced damage of nucleobases by ultraviolet radiation. Many alternative and secondary paths have been proposed in the literature, which ultimately provide a rich and constructive interplay between experimentally and theoretically oriented research. PMID:24264958

  16. 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.

  17. 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.

  18. DNA-mediated excitonic upconversion FRET switching

    DOE PAGESBeta

    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

  19. Recognition of DNA sequencing through binding of nucleobases to graphene

    NASA Astrophysics Data System (ADS)

    Zaffino, Valentina

    Graphene is one of the most promising materials in nanotechnology. Its large surface to volume ratio, high conductivity and electron mobility at room temperature are outstanding properties for use in DNA sensors. For this study, we used Density Functional Theory (DFT), ?with and without the inclusion of van der Waals (vdW) interactions, ?to investigate the adsorption of nucleobases (cytosine, guanine, adenine, thymine, and uracil) on pristine graphene and graphene with defects (Divacancy and Stone-Wales). We investigated the performance of two types of vdW-DF functional (optB86b-vdW and rPW86-vdW), as well as the PBE functional, and their description of the adsorption geometry and electronic structure of the nucleobase-graphene systems.The inclusion of defects results in an increase in binding energy, closer adsorption of the molecule to graphene and greater buckling in both the graphene structure and nucleobase.

  20. 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

  1. 1'-Homonucleosides and their structural analogues: A review.

    PubMed

    Wróblewski, Andrzej E; Głowacka, Iwona E; Piotrowska, Dorota G

    2016-08-01

    Nucleoside analogues belong to an important class of antiviral and anticancer drugs. Insertion of a methylene fragment between the anomeric carbon and pyrimidine or purine bases transforms nucleosides into 1'-homonucleosides. When compared with nucleosides this modification lengthens the separation between HO-C5' of pentofuranoside fragments and nitrogen (N1 or N9) atoms of nucleobases, lowers the steric and electronic interactions between nucleobases and sugar rings, introduces greater flexibility around a CH2-Base bond and thus allows for more rotational freedom, and since the anomeric effect no longer operates any sugar or pseudosugar moiety exists in its unique conformation and experiences specific conformational mobility and hydrolysis of the C1'-CH2Base bond by cellular enzymes is no longer feasible. This review covers 1'-homonucleosides with a tetrahydrofuran ring and its nitrogen and sulfur analogues as well as those containing a cyclopentane moiety as a sugar replacer. Achievements in syntheses of sugar or pseudosugar scaffolds are of primary interest since pathways to install nucleobases are well recognized. Whenever possible, the biological activity, mostly antiviral and antitumor but sometimes as inhibitors of specific enzymes, will be presented and discussed to help identify structural features responsible for the particular mode of action and thus possible therapeutic significance. PMID:27128178

  2. Photoreponsive Hybrid Nanoparticles with Inherent FRET Activity.

    PubMed

    Achilleos, Demetra S; Hatton, T Alan; Vamvakaki, Maria

    2016-06-14

    The photoactivated inherent fluorescence resonance energy transfer (FRET) properties of a hard-and-soft hybrid nanosystem comprising poly(1'-(2-methacryloxyethyl)-3',3'-dimethyl-6-nitrospiro-(2H-1-benzopyran-2,2'-indoline))-co-poly[2-(dimethylamino)ethyl methacrylate] (PSPMA-co-PDMAEMA) random copolymer brushes on silica nanoparticles are described. This unique FRET process is switched on by the simultaneous generation of isomer X and merocyanine (MC), which are bipolar in nature and comprise donor-acceptor dyads, from a single spiropyran (SP) chromophore upon UV irradiation. These X-MC species exhibit sufficient lifetimes to allow the read-out of the FRET process. The phenomenon is gradually switched off because of the thermal relaxation of the bipolar chromophores. This inherent property of the nanoemitters is employed in the development of biosensors of high specificity by monitoring variations in the FRET efficiency and lifetime of the hybrids in the presence of biological substances. More specifically, bovine serum albumin (BSA) augments the formation of MC species and retards the MC photobleaching process, leading to the enhancement of the FRET efficiency and lifetime, respectively. On the other hand, amino acid l-histidine further retards the MC thermal relaxation and prolongs the FRET process. We envisage that this platform opens new perspectives in the development of novel, optical nanosensors for applications in various fields including healthcare products and environmental monitoring. PMID:27222922

  3. 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

  4. Photochemical etiology of promising ancestors of the RNA nucleobases.

    PubMed

    Brister, M M; Pollum, M; Crespo-Hernández, C E

    2016-07-27

    RNA is a product of chemical and biological evolution and the identification of its heterocyclic ancestors is essential for understanding the molecular origins of life. Among a diverse array of selection pressures thought to have shaped the composition of the nucleobases on prebiotic Earth, protection against intense ultraviolet radiation must have been essential. In this contribution, a detailed spectroscopic and photophysical investigation of barbituric acid and 2,4,6-triaminopyrimidine, two promising candidates for the prebiotic ancestors of RNA nucleobases, is presented in aqueous solution. It is shown that although these pyrimidine derivatives absorb ultraviolet radiation strongly, both compounds possess efficient electronic relaxation mechanisms for dissipating most of the absorbed ultraviolet energy to their aqueous environment as heat within hundreds of femtoseconds, thus safeguarding their chemical integrity. In fact, these two heterocyclic compounds rival the photostability observed in the canonical nucleobases in aqueous solution, thus supporting the recent proposal that both barbituric acid and 2,4,6-triaminopyrimidine are promising ancestors of the RNA nucleobases. PMID:26898746

  5. 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

  6. Formation of a stable triplex incorporating a CG interrupting site by a new WNA derivative containing 3-aminopyrazole as a nucleobase.

    PubMed

    Uchida, Yuko; Taniguchi, Yosuke; Aoki, Eriko; Togo, Mieko; Sasaki, Shigeki

    2008-01-01

    Triplex-forming oligonucleotides (TFOs) bind within the major groove of duplex DNA in a sequence-specific manner, and have attracted much interest as genomic tools. However, as the triplex DNA is formed by the interaction between the TFOs and homopurine/homopyrimidine sequences of the target duplex DNA, the stable triplex formation is prevented by one pyrimidine base in the homopurine strand. Previously, we developed the nucleoside analogues (WNA: W-shaped nucleoside analogues) that furnish an aromatic ring as a stacking part and a nucleobase as a recognition part onto the bicyclic skeleton. Selective recognition of a TA and a CG interrupting site has been achieved by WNA-beta T and WNA-beta C, respectively. In the subsequent study, it was found that the triplex formation by the WNA analogues depend on its neighbouring bases within the TFO. In this paper, we describe the synthesis and the evaluation of the triplex forming ability of WNA-beta 3AP, having 3-aminopyrazole (3AP) as a nucleobase. It is remarkable that the TFO containing the WNA-beta 3AP recognizes the CG interrupting site with high selectivity in the TFO sequence of 3'-GZG-5', in which the previous WNA-beta C did not show the stabilizing effect. PMID:18776291

  7. 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.

  8. 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.

  9. Intracellular protein interaction mapping with FRET hybrids

    PubMed Central

    You, Xia; Nguyen, Annalee W.; Jabaiah, Abeer; Sheff, Mark A.; Thorn, Kurt S.; Daugherty, Patrick S.

    2006-01-01

    A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types. PMID:17130455

  10. 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. PMID:26210398

  11. 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

  12. 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.

  13. Glucose-Nucleobase Pseudo Base Pairs: Biomolecular Interactions within DNA.

    PubMed

    Vengut-Climent, Empar; Gómez-Pinto, Irene; Lucas, Ricardo; Peñalver, Pablo; Aviñó, Anna; Fonseca Guerra, Célia; Bickelhaupt, F Matthias; Eritja, Ramón; González, Carlos; Morales, Juan C

    2016-07-18

    Noncovalent forces rule the interactions between biomolecules. Inspired by a biomolecular interaction found in aminoglycoside-RNA recognition, glucose-nucleobase pairs have been examined. Deoxyoligonucleotides with a 6-deoxyglucose insertion are able to hybridize with their complementary strand, thus exhibiting a preference for purine nucleobases. Although the resulting double helices are less stable than natural ones, they present only minor local distortions. 6-Deoxyglucose stays fully integrated in the double helix and its OH groups form two hydrogen bonds with the opposing guanine. This 6-deoxyglucose-guanine pair closely resembles a purine-pyrimidine geometry. Quantum chemical calculations indicate that glucose-purine pairs are as stable as a natural T-A pair. PMID:27328804

  14. 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.

  15. Supramolecular gels made from nucleobase, nucleoside and nucleotide analogs.

    PubMed

    Peters, Gretchen Marie; Davis, Jeffery T

    2016-06-01

    Supramolecular or molecular gels are attractive for various applications, including diagnostics, tissue scaffolding and targeted drug release. Gelators derived from natural products are of particular interest for biomedical purposes, as they are generally biocompatible and stimuli-responsive. The building blocks of nucleic acids (i.e. nucleobases, nucleosides, and nucleotides) are desirable candidates for supramolecular gelation as they readily engage in reversible, noncovalent interactions. In this review, we describe a number of organo- and hydrogels formed through the assembly of nucleosides, nucleotides, and their derivatives. While natural nucleosides and nucleotides generally require derivatization to induce gelation, guanosine and its corresponding nucleotides are well known gelators. This unique gelating ability is due to propensity of the guanine nucleobase to self-associate into stable higher-order assemblies, such as G-ribbons, G4-quartets, and G-quadruplexes. PMID:27146863

  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. 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.

  18. Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells.

    PubMed

    Zhang, Jiang; Zhang, Lili; Chai, Liuying; Yang, Fangfang; Du, Mengyan; Chen, Tongsheng

    2016-09-01

    3-cube-based Förster resonance energy transfer (FRET) microscopy, a sensitized acceptor FRET quantification method, has been widely used to visualize dynamic protein-protein interaction in living cells. Determining the FRET sensitized-quenching transition factor (G factor) of a particular donor-acceptor pair and optical system is crucial for 3-cube FRET quantification. We here improved the acceptor photobleaching-based G factor determination method (termed as mPb-G) and the two-plasmid-based G factor determination method (termed as mTP-G) for rapid and reliable measurement of the G factor. mTP-G method determines G factor by simultaneously detecting three images of cells exclusively expressing each of two tandem constructs with multiple donors and multiple acceptors. This method circumvents switchover of the cells exclusively expressing each of the two constructs. mPb-G method images G factor by detecting three images of cells expressing a donor-acceptor tandem FRET construct before and after partially photobleaching acceptor. We performed the two methods on our dual-channel wide-field FRET microscope to obtain reliable G factor, and also measured the FRET efficiency and acceptor-to-donor concentration ratio of tandem constructs with different acceptor-donor stoichiometries in living HepG2 cells. mTP-G and mPb-G methods provide two simple and reliable tools for determining the G factor, in turn, quantitatively measuring FRET signal and monitoring dynamic biochemical processes in living cells. PMID:27239984

  19. 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.

  20. 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

  1. 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

  2. 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. PMID:26530400

  3. 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

  4. Efficient self-assembly in water of long noncovalent polymers by nucleobase analogues.

    PubMed

    Cafferty, Brian J; Gállego, Isaac; Chen, Michael C; Farley, Katherine I; Eritja, Ramon; Hud, Nicholas V

    2013-02-20

    Molecular self-assembly is widely appreciated to result from a delicate balance between several noncovalent interactions and solvation effects. However, current design approaches for achieving self-assembly in water with small, synthetic molecules do not consider all aspects of the hydrophobic effect, in particular the requirement of surface areas greater than 1 nm(2) for an appreciable free energy of hydration. With the concept of a minimum hydrophobic surface area in mind, we designed a system that achieves highly cooperative self-assembly in water. Two weakly interacting low-molecular-weight monomers (cyanuric acid and a modified triaminopyrimidine) are shown to form extremely long supramolecular polymer assemblies that retain water solubility. The complete absence of intermediate assemblies means that the observed equilibrium is between free monomers and supramolecular assemblies. These observations are in excellent agreement with literature values for the free energy of nucleic acid base interactions as well as the calculated free energy penalty for the exposure of hydrophobic structures in water. The results of our study have implications for the design of new self-assembling structures and hydrogel-forming molecules and may provide insights into the origin of the first RNA-like polymers. PMID:23394182

  5. 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. PMID:24976360

  6. 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.

  7. Fluorescent 2-Aminopyridine Nucleobases for Triplex-Forming Peptide Nucleic Acids.

    PubMed

    Cheruiyot, Samwel K; Rozners, Eriks

    2016-08-17

    Development of new fluorescent peptide nucleic acids (PNAs) is important for fundamental research and practical applications. The goal of this study was the design of fluorogenic nucleobases for incorporation in triplex-forming PNAs. The underlying design principle was the use of a protonation event that accompanied binding of a 2-aminopyridine (M) nucleobase to a G-C base pair as an on switch for a fluorescence signal. Two fluorogenic nucleobases, 3-(1-phenylethynyl)-M and phenylpyrrolo-M, were designed, synthesized and studied. The new M derivatives provided modest enhancement of fluorescence upon protonation but showed reduced RNA binding affinity and quenching of fluorescence signal upon triple-helix formation with cognate double-stranded RNA. Our study illustrates the principal challenges of design and provides guidelines for future improvement of fluorogenic PNA nucleobases. The 3-(1-phenylethynyl)-M may be used as a fluorescent nucleobase to study PNA-RNA triple-helix formation. PMID:27223320

  8. Probing nucleobase photo protection with soft x-rays

    NASA Astrophysics Data System (ADS)

    Gühr, Markus

    2013-05-01

    We [1] present a new method for ultrafast spectroscopy of molecular photoexcited dynamics. The technique uses a pair of femtosecond pulses: a photoexcitation pulse initiating excited state dynamics followed by a soft x-ray (SXR) probe pulse that core ionizes certain atoms inside the molecule. We observe the Auger decay of the core hole as a function of delay between the photoexcitation and SXR pulses. The core hole decay is particularly sensitive to the local valence electrons near the core and shows new types of propensity rules, compared to dipole selection rules in SXR absorption or emission spectroscopy. We apply the delayed ultrafast x-ray Auger probing (DUXAP) method to the specific problem of nucleobase photoprotection to demonstrate its potential. The ultraviolet photoexcited ππ * states of nucleobases are prone to chemical reactions with neighboring bases. To avoid this, the single molecules funnel the ππ * population to lower lying electronic states on an ultrafast timescale under violation of the Born-Oppenheimer approximation. The new type of propensity rule, which is confirmed by Auger decay simulations, allows us to have increased sensitivity on the direct relaxation from the ππ * state to the vibrationally hot electronic ground state. For the nucleobase thymine, we measure a decay of the ππ * state and a subsequent filling of the vibrationally hot ground state in 300 fs. This work was supported by the AMOS program within the Chemical Sciences, Geosciences, and Biosciences Division of the Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy.Portions of this research were carried out at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. LCLS is an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. Other portions of this research were carried out at the Advanced Light Source, which is supported by the Director, Office of

  9. 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

  10. 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

  11. 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.

  12. The effect of lubricant additives on fretting wear

    NASA Astrophysics Data System (ADS)

    Qiu, Y.; Roylance, B. J.

    1992-10-01

    The effect of lubricant additives on fretting wear has been investigated using a ball-on-plate machine. The test results confirm that the antiwear additives, e.g. phospho-sulphurized terpene, sulphurized esters and sulphurized paraffins, are effective in reducing friction and wear. Examination of worn surfaces by optical and electron microscope inspection indicated the presence of thin films which had been deposited under fretting action when using oils containing these additives. Unlubricated fretting wear occurred in the scuffing region. In contrast, the lubricated fretting wear with the lubricating oils containing the antiwear additives took place in the mixed lubrication region. In lubricated fretting wear, the size of the wear particles was smaller than with dry fretting wear.

  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. 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.

  15. Coupled thermoelastic analysis of fretting contacts

    NASA Astrophysics Data System (ADS)

    Ganapathy, Harish

    Fretting fatigue is the contact phenomenon occurring when two bodies in contact experience oscillatory loads. The surface tribology and contact stress evolution in a fretting contact has been studied using coupled thermoelastic analysis. Both, an aluminum and titanium alloy have been studied. Full-field real-time in-situ temperature maps of the contact region and its vicinity have been obtained using a multi-element infrared camera. The distinguishing features of the contact including the sliding regime, partial slip contact, bulk stress effects, boundary conditions effects etc. have been successfully captured using temperature measurement of the order of millikelvin. The coupled thermoclastic response of aluminum and titanium alloy has been successfully characterized, including the mean stress effect. A full coupled thermoelastic finite element model with Coulomb friction, frictional heating and gap conductance, has been used to predict the experimental temperatures. Changes in loads and changes in the coefficient of friction produce changes in different areas of the temperature field. The coupled thermoelastic effect may be used as a powerful tool to guide the march towards the complete understanding of the phenomenon of fretting. The method has been successfully used to guide the finite element analysis of a lap joint specimen.

  16. 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.

  17. Distinction of nucleobases – a tip-enhanced Raman approach

    PubMed Central

    Treffer, Regina; Lin, Xiumei; Bailo, Elena; Deckert-Gaudig, Tanja

    2011-01-01

    Summary The development of novel DNA sequencing methods is one of the ongoing challenges in various fields of research seeking to address the demand for sequence information. However, many of these techniques rely on some kind of labeling or amplification steps. Here we investigate the intrinsic properties of tip-enhanced Raman scattering (TERS) towards the development of a novel, label-free, direct sequencing method. It is known that TERS allows the acquisition of spectral information with high lateral resolution and single-molecule sensitivity. In the presented experiments, single stranded adenine and uracil homopolymers were immobilized on different kinds of substrates (mica and gold nanoplates) and TERS experiments were conducted, which demonstrated the reproducibility of the technique. To elucidate the signal contributions from the specific nucleobases, TERS spectra were collected on single stranded calf thymus DNA with arbitrary sequence. The results show that, while the Raman signals with respect to the four nucleobases differ remarkably, specific markers can be determined for each respective base. The combination of sensitivity and reproducibility shows that the crucial demands for a sequencing procedure are met. PMID:22003468

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  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. Influence of Fretting Wear on Lifetime of Tin Plated Connectors

    NASA Astrophysics Data System (ADS)

    Ikeda, Hirosaka; Ito, Tetsuya; Sawada, Shigeru; Hattori, Yasuhiro; Saitoh, Yasushi; Tamai, Terutaka; Iida, Kazuo

    Due to the recent increase in electronic devices mounted on automobiles, a large number of connectors, especially low-cost tin plated connectors are being used. As a result, their contact reliability has become problematic. Furthermore, for the connectors which are subjected to fretting wear caused by heat cycle and vibrations, the contact resistance increases because of wear of tin and deposition of oxides, which generates problems of poor contact. This study is intended to analyze the change in contact resistance of tin plated connectors from the start of fretting wear to the end of their lifetime from the viewpoint of practical reliability, and to observe the trace and the characteristics of fretting wear microscopically. This study found that wear and oxidation of tin plated connectors start immediately with fretting wear, and thus accumulation of abrasion powder on fretting areas causes connectors to reach to the end of their useful lifetime quickly. Especially, it was demonstrated that amplitude of fretting has a considerable influence on a connector's lifetime. It is made clear that air-tightness, so-called “gas-tight” of tin in a fretting area influences fretting wear considerably.

  6. Remote field eddy current inspection of support plate fretting wear

    SciTech Connect

    Shatat, A.; Atherton, D.L.

    1997-03-01

    This article demonstrates how the remote field eddy current technique might be extended to measure support plate fretting wear in heat exchanger tubes. A finite element analysis was used to examine the plate`s effect on the eddy current signal. Experimental data lend support to a suggested multifrequency method for sizing fretting grooves.

  7. 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

  8. 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

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

    PubMed

    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. 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

  12. X-Ray Photoelectron Spectroscopy (XPS) of Bacteriorhodopsin Analogues Synthesized from Fluorophenyl Retinals

    NASA Astrophysics Data System (ADS)

    Takahashi, Masae; Takahashi, Takashi; Tokunaga, Fumio; Murano, Kentaro; Tsujimoto, Kazuo; Sagawa, Takasi

    1984-04-01

    The two external point-charge (TEPC) model for bacteriorhodopsin (bR) has been examined by X-ray photoelectron spectroscopy (XPS) and CNDO/S molecular orbital calculations. A main concern was given to a point charge near the β-ionone ring. XPS measurements were carried out on fluorophenyl retinal (F-ret) and their derivatives (Schiff base, protonated Schiff base and bacteriorhodopsin analogues (F-bR)), paying close attention to the chemical shift of the F 1s core level. No meaningful differences were observed among these species although numerical calculations on an assumption of the TEPC model have predicted the chemical shift of about 3 eV between F-ret and F-bR. This fact has arisen a serious question to the validity of the TEPC model. The same conclusion has been reached by the present study of absorption maxima of F-ret and their derivatives.

  13. Photo-Irradiation of Pyrimidine in Interstellar Ice Analogs: Searching for Nucleobases

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

    Nucleobases have been detected in meteorites and possibly form in space. The functionalization of PAHs from UV photons in mixed ices has proven effective in the lab. Here we investigate how irradiation affects pyrimidine in interstellar ice analogs.

  14. Nucleobases and Other Prebiotic Species from the Ultraviolet Irradiation of Pyrimidine in Astrophysical Ices

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

    We discuss the results of UV irradiation of ices containing pyrimidine and show that such processing efficiently forms the nucleobases uracil and cytosine, but not thymine, a pattern similar to what is seen in carbonaceous meteorites.

  15. Formation of Nucleobases from the UV Photo-Irradiation of Pyrimidine in Astrophysical Ice Analogs

    NASA Astrophysics Data System (ADS)

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

    2010-04-01

    This work shows how pyrimidic nucleobases (uracil, cytosine, etc.) can be formed under abiotic conditions from the UV irradiation of pyrimidine in astrophysical ices. The formation mechanisms and the photo-stability of such compounds are discussed.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  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. IODINE MONOCHLORIDE FACILITATED DEGLYCOSYLATION, ANOMERIZATION, AND ISOMERIZATION OF 3-SUBSTITUTED THYMIDINE ANALOGUES

    PubMed Central

    Khalil, Ahmed; Ishita, Keisuke; Ali, Tehane; Tiwari, Rohit; Riachy, Ramy; Toppino, Antonio; Hasabelnaby, Sherifa; Sayfullin, Naum; Oliver, Allen G.; Gallucci, Judith; Huang, Zhenguo; Tjarks, Werner

    2014-01-01

    The reaction of thymidine, 3-mono-, and 3,3′,5′-trialkylsubstitued thymidine analogues with iodine monochloride (ICl) was investigated. Treatment with ICl resulted in rapid deglycosylation, anomerization, and isomerization of thymidine and 3-substituted thymidine analogues under various reaction conditions leading to the formation of the nucleobases as the major products accompanied by minor formation of α-furanosidic-, α-pyranosidic- and β-pyranosidic nucleosides. On the other hand, 3,3′,5′-trisubstitued thymidine analogues were only deglycosylated and anomerized. These results are similar to those observed for the acidic hydrolysis of the glycoside bond in nucleosides, but were presumably caused by the Lewis acid character of an iodine electrophile. PMID:25372994

  6. Friction and fretting wear characteristics of different diamond-like carbon coatings against alumina in water-lubricated fretting conditions.

    PubMed

    Watabe, Tsukasa; Amanov, Auezhan; Tsuboi, Ryo; Sasaki, Shinya

    2013-12-01

    Diamond-like carbon (DLC) coatings typically show low friction and high wear resistance. In this study, the friction and fretting wear characteristics of PVD, CVD and CVD-Si DLC coatings were investigated against an alumina (Al2O3) ball under water-lubricated fretting conditions. The objective of this study is to investigate and compare the friction and fretting wear characteristics of those DLC coatings at various fretting frequencies. The test results showed that the PVD DLC coating led to a lower friction coefficient and a higher resistance to fretting wear compared to those of the CVD and CVD-Si DLC coatings. However, the CVD DLC coating showed that the fretting wear resistance decreases with increasing frequency, while no significant difference in fretting wear resistances of the PVD and CVD-Si DLC coatings was observed. Quantitative surface analyses of the specimens were performed using an energy dispersive spectroscopy (EDS), a laser scanning microscope (LSM), a scanning electron microscope (SEM), an atomic force microscope (AFM) and the Raman spectroscopy. PMID:24266210

  7. 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

  8. 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.

  9. 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. PMID:27220279

  10. 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.

  11. Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy.

    PubMed Central

    Parker, M D; Hyde, R J; Yao, S Y; McRobert, L; Cass, C E; Young, J D; McConkey, G A; Baldwin, S A

    2000-01-01

    Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (K(m) 0.32+/-0.05 mM) than the pyrimidine nucleoside uridine (K(m) 3.5+/-1.1 mM). It also efficiently transports nucleobases such as adenine (K(m) 0.32+/-0.10 mM) and hypoxanthine (K(m) 0.41+/-0.1 mM), and anti-viral 3'-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR [6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, or nitrobenzylthioinosine] and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs. PMID:10861212

  12. 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.

  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. Nucleobases in Space: Laboratory Studies of Polycyclic Aromatic Nitrogen Heterocycles

    NASA Technical Reports Server (NTRS)

    Elsila, Jamie; Mattioda, Andy; Bernstein, Max; Sandford, Scott; Hudgins, Doug

    2005-01-01

    Polycyclic Aromatic Nitrogen Heterocycles (PANHs) are heterocyclic aromatics Le., PAHs with carbon atoms replaced by a nitrogen atom. These molecules have been detected in meteorite extracts, and in general these nitrogen heterocycles are of astrobiological interest since this class of molecules include nucleobases, basic components of our nucleic acids. These compounds are predicted to be present in the interstellar medium and in Titan tholin, but have received relatively little attention. We will present spectra and reactions of PANHs, frozen in solid H2O at 12 K, conditions germane to astronomical observations. In contrast to simple PAHs, that do not interact strongly with solid H2O, the nitrogen atoms in PANHs are potentially capable of hydrogen bonding with H20 changing their spectra, complicating their remote detection on the surfaces of icy bodies. Moreover, we have studied the photo-chemistry of these interesting compounds under astrophysical conditions and will use our lab studies to assess a potential interstellar heritage of these compounds in carbonaceous chondrites.

  15. 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

  16. Metal Ion-Mediated Nucleobase Recognition by the ZTP Riboswitch.

    PubMed

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

    2015-07-23

    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. As an important intermediate in purine biosynthesis, ZMP also serves as a proxy for the concentration of N10-formyl-tetrahydrofolate, 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 base, ribose sugar, and phosphate moieties of the ligand. 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 chemically similar compounds such as ATP/AMP. PMID:26144884

  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. 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

  19. 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

  20. Pulse-shaping based two-photon FRET stoichiometry

    PubMed Central

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

    2015-01-01

    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. PMID:25836193

  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. 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.

  3. 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).

  4. Intravital FRET: Probing Cellular and Tissue Function in Vivo.

    PubMed

    Radbruch, Helena; Bremer, Daniel; Mothes, Ronja; Günther, Robert; Rinnenthal, Jan Leo; Pohlan, Julian; Ulbricht, Carolin; Hauser, Anja E; Niesner, Raluca

    2015-01-01

    The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo-ratiometrically and time-resolved by fluorescence lifetime imaging-and show their concrete application in the context of neuroinflammation in adult mice. PMID:26006244

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. Identification of nucleobases using variable currents through graphene nanopores: A first principles study

    NASA Astrophysics Data System (ADS)

    Haraldsen, J. T.; McFarland, H.; Ahmed, T.; Zhu, J.-X.; Balatsky, A. V.

    2015-03-01

    Nanopore-based technology has the potential to be an efficient method for DNA/RNA base sequencing, as well as an identifier of other biomolecules. However, the thickness of the nanopore substrate is critical for the identification of individual nucleobases due to resulting noise and resolution problems. Recently, graphene has been suggested as a possible nanopore substrate due to its single atomic thickness and robust strength. In this study, we examine a possible device mechanism for the voltage dependence of nucleobases passing through a graphene nanopore. We utilize density functional theory with a generalized gradient approach on a graphene ribbon with a nucleobase in order to calculate the transmission spectra for each base. Transmission spectra for each base allows for the calculation of the ballistic current and differential current as a function of voltage. We show that applying various bias voltages across a graphene ribbon for the general, energy-minimized position of the translocated nucleobase, it is possible to distinguish individual bases using the resulting current. Overall, our goal is to improve nanopore device design by helping to further DNA/RNA nucleobase identification and sequencing.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. Evolving a polymerase for hydrophobic base analogues.

    PubMed

    Loakes, David; Gallego, José; Pinheiro, Vitor B; Kool, Eric T; Holliger, Philipp

    2009-10-21

    Hydrophobic base analogues (HBAs) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. While extensive synthetic efforts have yielded examples of HBAs with favorable substrate properties, their discovery has remained challenging. Here we describe a complementary strategy for improving HBA substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (CSR) with the archetypal HBA 5-nitroindole (d5NI) and its derivative 5-nitroindole-3-carboxamide (d5NIC) as selection substrates. Starting from a repertoire of chimeric polymerases generated by molecular breeding of DNA polymerase genes from the genus Thermus, we isolated a polymerase (5D4) with a generically enhanced ability to utilize HBAs. The selected polymerase. 5D4 was able to form and extend d5NI and d5NIC (d5NI(C)) self-pairs as well as d5NI(C) heteropairs with all four bases with efficiencies approaching, or exceeding, those of the cognate Watson-Crick pairs, despite significant distortions caused by the intercalation of the d5NI(C) heterocycles into the opposing strand base stack, as shown by nuclear magnetic resonance spectroscopy (NMR). Unlike Taq polymerase, 5D4 was also able to extend HBA pairs such as Pyrene: varphi (abasic site), d5NI: varphi, and isocarbostyril (ICS): 7-azaindole (7AI), allowed bypass of a chemically diverse spectrum of HBAs, and enabled PCR amplification with primers comprising multiple d5NI(C)-substitutions, while maintaining high levels of catalytic activity and fidelity. The selected polymerase 5D4 promises to expand the range of nucleobase analogues amenable to replication and should find numerous applications, including the synthesis and replication of nucleic acid polymers with expanded chemical and functional diversity. PMID:19778048

  15. 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

  16. 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.

  17. An application of the van der Waals density functional: Hydrogen bonding and stacking interactions between nucleobases.

    PubMed

    Cooper, Valentino R; Thonhauser, T; Langreth, David C

    2008-05-28

    We apply the van der Waals density functional (vdW-DF) to study hydrogen bonding and stacking interactions between nucleobases. The excellent agreement of our results with high level quantum chemical calculations highlights the value of the vdW-DF for first-principles investigations of biologically important molecules. Our results suggest that, in the case of hydrogen-bonded nucleobase pairs, dispersion interactions reduce the cost of propeller twists while having a negligible effect on buckling. Furthermore, the efficient scaling of DFT methods allowed for the easy optimization of separation distance between nucleobase stacks, indicating enhancements in the interaction energy of up to 3 kcalmol over previous fixed distance calculations. We anticipate that these results are significant for extending the vdW-DF method to model larger vdW complexes and biological molecules. PMID:18513005

  18. A graphene field-effect transistor as a molecule-specific probe of DNA nucleobases.

    PubMed

    Dontschuk, Nikolai; Stacey, Alastair; Tadich, Anton; Rietwyk, Kevin J; Schenk, Alex; Edmonds, Mark T; Shimoni, Olga; Pakes, Chris I; Prawer, Steven; Cervenka, Jiri

    2015-01-01

    Fast and reliable DNA sequencing is a long-standing target in biomedical research. Recent advances in graphene-based electrical sensors have demonstrated their unprecedented sensitivity to adsorbed molecules, which holds great promise for label-free DNA sequencing technology. To date, the proposed sequencing approaches rely on the ability of graphene electric devices to probe molecular-specific interactions with a graphene surface. Here we experimentally demonstrate the use of graphene field-effect transistors (GFETs) as probes of the presence of a layer of individual DNA nucleobases adsorbed on the graphene surface. We show that GFETs are able to measure distinct coverage-dependent conductance signatures upon adsorption of the four different DNA nucleobases; a result that can be attributed to the formation of an interface dipole field. Comparison between experimental GFET results and synchrotron-based material analysis allowed prediction of the ultimate device sensitivity, and assessment of the feasibility of single nucleobase sensing with graphene. PMID:25800494

  19. Efficient enzyme-free copying of all four nucleobases templated by immobilized RNA

    NASA Astrophysics Data System (ADS)

    Deck, Christopher; Jauker, Mario; Richert, Clemens

    2011-08-01

    The transition from inanimate materials to the earliest forms of life must have involved multiplication of a catalytically active polymer that is able to replicate. The semiconservative replication that is characteristic of genetic information transfer requires strands that contain more than one type of nucleobase. Short strands of RNA can act as catalysts, but attempts to induce efficient self-copying of mixed sequences (containing four different nucleobases) have been unsuccessful with ribonucleotides. Here we show that inhibition by spent monomers, formed by the hydrolysis of the activated nucleotides, is the cause for incomplete extension of growing daughter strands on RNA templates. Immobilization of strands and periodic displacement of the solution containing the activated monomers overcome this inhibition. Any of the four nucleobases (A/C/G/U) is successfully copied in the absence of enzymes. We conclude therefore that in a prebiotic world, oligoribonucleotides may have formed and undergone self-copying on surfaces.

  20. Understanding Stacking Interactions between an Aromatic Ring and Nucleobases in Aqueous Solution: Experimental and Theoretical Study.

    PubMed

    Kataev, Evgeny A; Shumilova, Tatiana A; Fiedler, Benjamin; Anacker, Tony; Friedrich, Joachim

    2016-08-01

    Stacking interactions between aromatic compounds and nucleobases are crucial in recognition of nucleotides and nucleic acids, but a comprehensive understanding of the strength and selectivity of these interactions in aqueous solution has been elusive. To this end, model complexes have been designed and analyzed by experiment and theory. For the first time, stacking free energies between five nucleobases and anthracene were determined experimentally from thermodynamic double mutant cycles. Three different experimental methods were proposed and evaluated. The dye prefers to bind nucleobases in the order (kcal/mol): G (1.3) > T (0.9) > U (0.8) > C (0.5) > A (0.3). The respective trend of interaction free energies extracted from DFT calculations correlates to that obtained experimentally. Analysis of the data suggests that stacking interactions dominate over hydrophobic effects in an aqueous solution and can be predicted with DFT calculations. PMID:27314892

  1. A graphene field-effect transistor as a molecule-specific probe of DNA nucleobases

    NASA Astrophysics Data System (ADS)

    Dontschuk, Nikolai; Stacey, Alastair; Tadich, Anton; Rietwyk, Kevin J.; Schenk, Alex; Edmonds, Mark T.; Shimoni, Olga; Pakes, Chris I.; Prawer, Steven; Cervenka, Jiri

    2015-03-01

    Fast and reliable DNA sequencing is a long-standing target in biomedical research. Recent advances in graphene-based electrical sensors have demonstrated their unprecedented sensitivity to adsorbed molecules, which holds great promise for label-free DNA sequencing technology. To date, the proposed sequencing approaches rely on the ability of graphene electric devices to probe molecular-specific interactions with a graphene surface. Here we experimentally demonstrate the use of graphene field-effect transistors (GFETs) as probes of the presence of a layer of individual DNA nucleobases adsorbed on the graphene surface. We show that GFETs are able to measure distinct coverage-dependent conductance signatures upon adsorption of the four different DNA nucleobases; a result that can be attributed to the formation of an interface dipole field. Comparison between experimental GFET results and synchrotron-based material analysis allowed prediction of the ultimate device sensitivity, and assessment of the feasibility of single nucleobase sensing with graphene.

  2. 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.

  3. 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.

  4. Synthesis of novel conjugates of a saccharide, amino acids, nucleobase and the evaluation of their cell compatibility.

    PubMed

    Yuan, Dan; Du, Xuewen; Shi, Junfeng; Zhou, Ning; Baoum, Abdulgader Ahmed; Xu, Bing

    2014-01-01

    This article reports the synthesis of a novel type of conjugate of three fundamental biological build blocks (i.e., saccharide, amino acids, and nucleobase) and their cell compatibility. The facile synthesis starts with the synthesis of nucleobase and saccharide derivatives, then uses solid-phase peptide synthesis (SPPS) to build the peptide segment (Phe-Arg-Gly-Asp or naphthAla-Phe-Arg-Gly-Asp with fully protected groups), and later, an amidation reaction in liquid phase connects these three parts together. The overall yield of these multiple step synthesis is about 34%. Besides exhibiting excellent solubility, these conjugates of saccharide-amino acids-nucleobase (SAN), like the previously reported conjugates of nucleobase-amino acids-saccharide (NAS) and nucleobase-saccharide-amino acids (NSA), are mammalian cell compatible. PMID:25383110

  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. 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

  7. An examination of faying surface fretting in single lap splices

    NASA Astrophysics Data System (ADS)

    Brown, Adam

    While fretting damage in mechanically fastened joints is widely acknowledged as a common source of crack nucleation, little work is available in the open literature on the role that fretting damage plays in the fatigue life of a riveted joint. To expand on the limited knowledge available, a study was undertaken on fretting fatigue in thin-sheet riveted fuselage lap joints. In joints constructed out of 1 mm thick 2024-T3 aluminum sheet the rivet forming load was found to have a significant effect on the location of fretting damage and crack nucleation. This effect was observed for splices riveted with machine countersunk and with universal rivets. The shift in the location of peak fretting damage and crack nucleation with changing rivet forming loads was investigated through numerical and experimental methods. A predictive model based on the critical plane Smith-Watson-Topper strain life equation was applied to the complex geometry of the single lap splice and was shown to be effective in predicting the fretting fatigue life as well as the location of fretting-induced crack nucleation. Basing this model on an explicit finite element simulation allowed for the inclusion of compressive residual stresses generated during rivet forming. Key to the proper functionality of the predictive model was to have a validated finite element model from which results for the stress and strain field in the loaded component could be obtained. In addition to the predictive model, a series of splice coupon and simplified geometry fretting fatigue tests were performed. The tests showed that, at higher rivet forming loads, crack nucleation is on the faying surface away from the hole edge and that the type of surface condition is important to the fretting fatigue life of the splice. The discovery of this variation with surface treatment at high rivet forming loads is important as more research is showing the benefit of using load-controlled rivet forming and higher rivet forming loads in

  8. 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).

  9. Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells

    PubMed Central

    Day, Richard N.; Davidson, Michael W.

    2012-01-01

    Summary The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts, and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs. PMID:22396229

  10. DNA Stains as Surrogate Nucleobases in Fluorogenic Hybridization Probes.

    PubMed

    Hövelmann, Felix; Seitz, Oliver

    2016-04-19

    The increasing importance assigned to RNA dynamics in cells and tissues calls for probe molecules that enable fluorescence microscopy imaging in live cells. To achieve this goal, fluorescence dyes are conjugated with oligonucleotides so as to provide strong emission upon hybridization with the target molecule. The impressive 10(3)-fold fluorescence intensification observed when DNA stains such as thiazole orange (TO) interact with double-stranded DNA is intriguing and prompted the exploration of oligonucleotide conjugates. However, nonspecific interactions of DNA stains with polynucleotides tend to increase background, which would affect the contrast achievable in live-cell imaging. This Account describes the development of DNA-stain-labeled hybridization probes that provide high signal-to-background. We focus on our contributions in context with related advances from other laboratories. The emphasis will be on the requirements of RNA imaging in live cells. To reduce background, intercalator dyes such as TO were appended to peptide nucleic acid (PNA), which is less avidly recognized by DNA stains than DNA/RNA. Constraining the TO dye as a nucleobase surrogate in "forced intercalation (FIT) probes" improved the target specificity, presumably by helping to prevent unspecific interactions. The enforcement of TO intercalation between predetermined base pairs upon formation of the probe-target duplex provided for high brightness and enabled match/mismatch selectivity beyond stringency of hybridization. We show examples that highlight the use of PNA FIT probes in the imaging of mRNA, miRNA, and lncRNA in living cells. The "FIT approach" was recently extended to DNA probes. Signal brightness can become limiting when low-abundance targets ought to be visualized over cellular autofluorescence. We discuss strategies that further the brightness of signaling by FIT probes. Multilabeling with identical dyes does not solve the brightness issue. To avoid self-quenching, we

  11. Interactions of the "piano-stool" [ruthenium(II) (eta6-arene)(en)CL]+ complexes with water and nucleobases; ab initio and DFT study.

    PubMed

    Futera, Zdenek; Klenko, Julia; Sponer, Judit E; Sponer, Jirí; Burda, Jaroslav V

    2009-09-01

    Piano stool ruthenium complexes of the composition [Ru(II)(eta6-arene)(en)Cl](+/2+) (en = ethylenediamine) represent an emerging class of cisplatin-analogue anticancer drug candidates. In this study, we use computational quantum chemistry to characterize the structure, stability and reactivity of these compounds. All these structures were optimized at DFT(B3LYP)/6-31G(d) level and their single point properties were determined by the MP2/6-31++G(2df,2pd) method. Thermodynamic parameters and rate constants were determined for the aquation process, as a replacement of the initial chloro ligand by water and subsequent exchange reaction of aqua ligand by nucleobases. The computations were carried out at several levels of DFT and ab initio theories (B3LYP, MP2 and CCSD) utilizing a range of bases sets (from 6-31G(d) to aug-cc-pVQZ). Excellent agreement with experimental results for aquation process was obtained at the CCSD level and reasonable match was achieved also with the B3LYP/6-31++G(2df,2pd) method. This level was used also for nucleobase-water exchange reaction where a smaller rate constant for guanine exchange was found in comparison with adenine. Although adenine follows a simple replacement mechanism, guanine complex passes by a two-step mechanism. At first, Ru-O6(G) adduct is formed, which is transformed through a chelate TS2 to the Ru-N7(G) final complex. In case of guanine, the exchange reaction is more favorable thermodynamically (releasing in total by about 8 kcal/mol) but according to our results, the rate constant for guanine substitution is slightly smaller than the analogous constant in adenine case when reaction course from local minimum is considered. PMID:19090568

  12. Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes.

    PubMed

    Chowdhury, Sandipan Roy; Chauhan, Pradeep S; Dedkova, Larisa M; Bai, Xiaoguang; Chen, Shengxi; Talukder, Poulami; Hecht, Sidney M

    2016-05-01

    Described herein are the synthesis and photophysical characterization of a library of aryl-substituted oxazole- and thiazole-based dipeptidomimetic analogues, and their incorporation into position 66 of green fluorescent protein (GFP) in lieu of the natural fluorophore. These fluorescent analogues resemble the fluorophore formed naturally by GFP. As anticipated, the photophysical properties of the analogues varied as a function of the substituents at the para position of the phenyl ring. The fluorescence emission wavelength maxima of compounds in the library varied from ∼365 nm (near-UV region) to ∼490 nm (visible region). The compounds also exhibited a large range of quantum yields (0.01-0.92). The analogues were used to activate a suppressor tRNACUA and were incorporated into position 66 of GFP using an in vitro protein biosynthesizing system that employed engineered ribosomes selected for their ability to incorporate dipeptides. Four analogues with interesting photophysical properties and reasonable suppression yields were chosen, and the fluorescent proteins (FPs) containing these fluorophores were prepared on a larger scale for more detailed study. When the FPs were compared with the respective aminoacyl-tRNAs and the actual dipeptide analogues, the FPs exhibited significantly enhanced fluorescence intensities at the same concentrations. Part of this was shown to be due to the presence of the fluorophores as an intrinsic element of the protein backbone. There were also characteristic shifts in the emission maxima, indicating the environmental sensitivity of these probes. Acridon-2-ylalanine and oxazole 1a were incorporated into positions 39 and 66 of GFP, respectively, and were shown to form an efficient Förster resonance energy transfer (FRET) pair, demonstrating that the analogues can be used as FRET probes. PMID:27050631

  13. 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

  14. Borromean three-body FRET in frozen Rydberg gases

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

    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.

  15. Riboswitch Structure and Dynamics by smFRET Microscopy

    PubMed Central

    Suddala, Krishna C.; Walter, Nils G.

    2016-01-01

    Riboswitches are structured non-coding RNA elements that control the expression of their embedding messenger RNAs by sensing the intracellular concentration of diverse metabolites. As the name suggests, riboswitches are dynamic in nature so that studying their inherent conformational dynamics and ligand-mediated folding is important for understanding their mechanism of action. Single molecule fluorescence energy transfer (smFRET) microscopy is a powerful and versatile technique for studying the folding pathways and intra- and intermolecular dynamics of biological macromolecules, especially RNA. The ability of smFRET to monitor intramolecular distances and their temporal evolution make it a particularly insightful tool for probing the structure and dynamics of riboswitches. Here, we detail the general steps for using prism-based total internal reflection fluorescence (TIRF) microscopy for smFRET studies of the structure, dynamics and ligand binding mechanisms of riboswitches. PMID:25432756

  16. Synthesis of carbocyclic nucleoside analogs with five-membered heterocyclic nucleobases

    PubMed Central

    Cho, Jong hyun; Coats, Steven J.; Schinazi, Raymond F.

    2015-01-01

    New carbocyclic nucleoside analogs with five-membered heterocyclic nucleobases were synthesized and evaluated as potential anti-HIV and anti-HCV agents. Among the synthesized carbocyclic nucleoside analogs, the pyrazole amide 15f exhibited modest selective anti-HIV-1 activity (EC50 = 24 µM). PMID:26028788

  17. 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.

  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. Chemoenzymatic synthesis and utilization of a SAM analog with an isomorphic nucleobase.

    PubMed

    Vranken, C; Fin, A; Tufar, P; Hofkens, J; Burkart, M D; Tor, Y

    2016-07-14

    SalL, an enzyme that catalyzes the synthesis of SAM from l-methionine and 5'-chloro-5'-deoxyoadenosine, is shown to accept 5'-chloro-5'-deoxythienoadenosine as a substrate and facilitate the synthesis of a synthetic SAM analog with an unnatural nucleobase. This synthetic cofactor is demonstrated to replace SAM in the DNA methylation reaction with M.TaqI. PMID:27270873

  20. Nucleobase-functionalized ABC triblock copolymers: self-assembly of supramolecular architectures.

    PubMed

    Zhang, Keren; Fahs, Gregory B; Aiba, Motohiro; Moore, Robert B; Long, Timothy E

    2014-08-21

    RAFT polymerization afforded acrylic ABC triblock copolymers with self-complementary nucleobase-functionalized external blocks and a low-Tg soft central block. ABC triblock copolymers self-assembled into well-defined lamellar microphase-separated morphologies for potential applications as thermoplastic elastomers. Complementary hydrogen bonding within the hard phase facilitated self-assembly and enhanced mechanical performance. PMID:24984613

  1. 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

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

    PubMed

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

    2016-07-01

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

  3. Identification and Functional Characterization of the First Nucleobase Transporter in Mammals

    PubMed Central

    Yamamoto, Syunsuke; Inoue, Katsuhisa; Murata, Tomoaki; Kamigaso, Syunsuke; Yasujima, Tomoya; Maeda, Jun-ya; Yoshida, Yukihiro; Ohta, Kin-ya; Yuasa, Hiroaki

    2010-01-01

    Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodium-dependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 μm. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use. PMID:20042597

  4. 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.

  5. Solution structures of purine base analogues 9-deazaguanine and 9-deazahypoxanthine.

    PubMed

    Karnawat, Vishakha; Puranik, Mrinalini

    2016-03-01

    Deaza analogues of nucleobases are potential drugs against infectious diseases caused by parasites. A caveat is that apart from binding their target parasite enzymes, they also bind and inhibit enzymes of the host. In order to design derivatives of deaza analogues which specifically bind target enzymes, knowledge of their molecular structure, protonation state, and predominant tautomers at physiological conditions is essential. We have employed resonance Raman spectroscopy at an excitation wavelength of 260 nm, to decipher solution structure of 9-deazaguanine (9DAG) and 9-deazahypoxanthine (9DAH). These are analogues of guanine and hypoxanthine, respectively, and have been exploited to study static complexes of nucleobase binding enzymes. Such enzymes are known to perturb pKa of their ligands, and thus, we also determined solution structures of these analogues at two, acidic and alkaline, pH. Structure of each possible protonation state and tautomer was computed using density functional theoretical calculations. Species at various pHs were identified based on isotopic shifts in experimental wavenumbers and by comparing these shifts with corresponding computed isotopic shifts. Our results show that at physiological pH, N1 of pyrimidine ring in 9DAG and 9DAH bears a proton. At lower pH, N3 is place of protonation, and at higher pH, deprotonation occurs at N1 position. The proton at N7 of purine ring remains intact even at pH 12.5. We have further compared these results with naturally occurring nucleotides. Our results identify key vibrational modes which can report on hydrogen bonding interactions, protonation and deprotonation in purine rings upon binding to the active site of enzymes. PMID:25894214

  6. 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.

  7. Chromophoric Nucleoside Analogues: Synthesis and Characterization of 6-Aminouracil-Based Nucleodyes.

    PubMed

    Freeman, Noam S; Moore, Curtis E; Wilhelmsson, L Marcus; Tor, Yitzhak

    2016-06-01

    Nucleodyes, visibly colored chromophoric nucleoside analogues, are reported. Design criteria are outlined and the syntheses of cytidine and uridine azo dye analogues derived from 6-aminouracil are described. Structural analysis shows that the nucleodyes are sound structural analogues of their native nucleoside counterparts, and photophysical studies demonstrate that the nucleodyes are sensitive to microenvironmental changes. Quantum chemical calculations are presented as a valuable complementary tool for the design of strongly absorbing nucleodyes, which overlap with the emission of known fluorophores. Förster critical distance (R0) calculations determine that the nucleodyes make good FRET pairs with both 2-aminopurine (2AP) and pyrrolocytosine (PyC). Additionally, unique tautomerization features exhibited by 5-(4-nitrophenylazo)-6-oxocytidine (8) are visualized by an extraordinary crystal structure. PMID:27128151

  8. A FRET-based probe with a chemically deactivatable quencher.

    PubMed

    Leriche, Geoffray; Budin, Ghyslain; Darwich, Zeinab; Weltin, Denis; Mély, Yves; Klymchenko, Andrey S; Wagner, Alain

    2012-03-28

    A new concept of a chemically deactivatable quencher is proposed for a FRET-based probe that turns-on its fluorescence by either an enzymatic cleavage or a chemical reagent (sodium dithionite). This concept allowed us to quantify the caspase-3 cleavage activity in solution and to reveal unreacted probes in cell experiments. PMID:22327268

  9. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET.

    PubMed

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

    2016-03-29

    Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs). 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, validating our binding assay against the gold standard isothermal calorimetry (ITC), and using 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

  10. 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...

  11. 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

  12. 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

  13. 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.

  14. Accurate single-molecule FRET studies using multiparameter fluorescence detection.

    PubMed

    Sisamakis, Evangelos; Valeri, Alessandro; Kalinin, Stanislav; Rothwell, Paul J; Seidel, Claus A M

    2010-01-01

    In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing important insight into how biological molecules function. So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. In particular, Förster resonance energy transfer (FRET) studies of single molecules have shown that biomolecules may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of smFRET data are challenging since they can be complicated due to many artifacts arising from the complex photophysical behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amounts of contaminants. We demonstrate that the simultaneous acquisition of a maximum of fluorescence parameters by multiparameter fluorescence detection (MFD) allows for a robust assessment of all possible artifacts arising from smFRET and offers unsurpassed capabilities regarding the identification and analysis of individual species present in a population of molecules. After a short introduction, the data analysis procedure is described in detail together with some experimental considerations. The merits of MFD are highlighted further with the presentation of some applications to proteins and nucleic acids, including accurate structure determination based on FRET. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility, and position of fluorophores have to be taken into account when determining FRET-related distances with high accuracy. Furthermore, the broad time resolution (picoseconds to hours) of MFD allows for kinetic studies that resolve interconversion events between various subpopulations as a biomolecule of interest explores its

  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

    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. 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

  18. 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

  19. Perrin and Förster unified: Dual-laser triple-polarization FRET (3polFRET) for interactions at the Förster-distance and beyond.

    PubMed

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

    2016-04-01

    Dual laser flow cytometric energy transfer (FCET)--elaborated by Trón et al. in 1984--is an efficient and rapid way of measuring FRET on large cell populations. FRET efficiency and the donor and acceptor concentrations are determined from one donor and two acceptor signals. In this communication this method is extended towards the domain of receptor dynamics by the detection of polarized components of the three intensities. By enabling a complete description of the proximity and dynamics of FRET-systems, the new measuring scheme allows a more refined description of both the structure and dynamics of cell surface receptor clusters at the nano-scale and beyond. Associated donor fraction, limiting anisotropy and rotational correlation time of the donor, acceptor anisotropy and cell-by-cell estimation of the orientation factor for FRET (κ2) are available in the steady state on a single FRET sample in a very rapid and statistically efficient way offered by flow cytometry. For a more sensitive detection of conformational changes the "polarized FRET indices"--quantities composed from FRET efficiency and anisotropies--are proposed. The method is illustrated by measurements on a FRET system with changing FRET-fraction and on a two donor-one acceptor-system, when the existence of receptor trimers are proven by the detection of "hetero-FRET induced homo-FRET relief", i.e. the diminishing of homo-FRET between the two donors in the presence of a donor quencher. The method also offers higher sensitivity for assessing conformational changes at the nano-scale, due to its capability for the simultaneous detection of changes of proximity and relative orientations of the FRET donor and acceptor. Although the method has been introduced in the context of FRET, it is more general: It can be used for monitoring triple-anisotropy correlations also in those cases when FRET actually does not occur, e.g. for interactions occuring beyond the Förster-distance R0. Interpretation of κ2 has

  20. 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. PMID:27003481

  1. Formation of Nucleobases from the UV Irradiation of Pyrimidine in Interstellar Ice Analogs

    NASA Technical Reports Server (NTRS)

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

    2010-01-01

    Previous laboratory simulations showed that complex molecules, including prebiotic compounds/can be formed under interstellar conditions from the vacuum UV irradiation of interstellar ice analogs containing H2O, CO, NH3 etc. Although some complex prebiotic species have not been confirmed In the interstellar medium, they are known to be present in meteorites. Nucleobases, the building blocks of DNA and RNA, have also been detected in meteorites. Here, we present a study of the formation of pyrimidine-based compounds from the UV irradiation of pyrimidine in H2O- and/or NH3-ices at 20-30 K, Our results show that various derivatives, induding the nucleobases uracil and cytosine, are formed under these conditions.

  2. First-Principles Photoemission Spectroscopy of DNA and RNA Nucleobases from Koopmans-Compliant Functionals.

    PubMed

    Nguyen, Ngoc Linh; Borghi, Giovanni; Ferretti, Andrea; Marzari, Nicola

    2016-08-01

    The need to interpret ultraviolet photoemission data strongly motivates the refinement of first-principles techniques that are able to accurately predict spectral properties. In this work, we employ Koopmans-compliant functionals, constructed to enforce piecewise linearity in approximate density functionals, to calculate the structural and electronic properties of DNA and RNA nucleobases. Our results show that not only ionization potentials and electron affinities are accurately predicted with mean absolute errors of <0.1 eV, but also that calculated photoemission spectra are in excellent agreement with experimental ultraviolet photoemission spectra. In particular, the role and contribution of different tautomers to the photoemission spectra are highlighted and discussed in detail. The structural properties of nucleobases are also investigated, showing an improved description with respect to local and semilocal density-functional theory. Methodologically, our results further consolidate the role of Koopmans-compliant functionals in providing, through orbital-density-dependent potentials, accurate electronic and spectral properties. PMID:27267665

  3. 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. PMID:25118044

  4. Time-resolved homo-FRET studies of biotin-streptavidin complexes.

    PubMed

    Andreoni, Alessandra; Nardo, Luca; Rigler, Rudolf

    2016-09-01

    Förster resonance energy transfer is a mechanism of fluorescence quenching that is notably useful for characterizing properties of biomolecules and/or their interactions. Here we study water-solutions of Biotin-Streptavidin complexes, in which Biotin is labeled with a rigidly-bound fluorophore that can interact by Förster resonance energy transfer with the fluorophores labeling the other, up to three, Biotins of the same complex. The fluorophore, Atto550, is a Rhodamine analogue. We detect the time-resolved fluorescence decay of the fluorophores with an apparatus endowed with single-photon sensitivity and temporal resolution of ~30ps. The decay profiles we observe for samples containing constant Biotin-Atto550 conjugates and varying Streptavidin concentrations are multi-exponential. Each decay component can be associated with the rate of quenching exerted on each donor by each of the acceptors that label the other Biotin molecules, depending on the binding site they occupy. The main features that lead to this result are that (i) the transition dipole moments of the up-to-four Atto550 fluorophores that label the complexes are fixed as to both relative positions and mutual orientations; (ii) the fluorophores are identical and the role of donor in each Biotin-Streptavidin complex is randomly attributed to the one that has absorbed the excitation light (homo-FRET). Obviously the high-temporal resolution of the excitation-detection apparatus is necessary to discriminate among the fluorescence decay components. PMID:27494295

  5. Site Covalent Modification of Methionyl Peptides for the Production of FRET Complexes.

    PubMed

    Jadhav, Pramodkumar D; Shen, Jianheng; Sammynaiken, Ramaswami; Reaney, Martin J T

    2015-11-16

    Flax cyclic peptides (orbitides, linusorbs (LOs)) [1-8-NαC],[1-MetO2]-linusorb B1 ([MetO2]-LO1) and [1-9-NαC],[1-MetO2]-linusorb B2 ([MetO2]-LO2) are biologically active. These LOs lack active nuclei commonly used in peptide modification. We have developed reactions to activate methionine methyl sulphide to produce stable derivatives. In these reactions, LOs are converted to sulfonium intermediates and subsequently to derivatives containing active nuclei while preserving their fundamental structures. The reaction conditions preserved cyclic peptide fundamental structure and organic solvent solubility. [Met]-LO1 and [Met]-LO2 analogues containing activated groups (-CN, -COOEt, and -NH2 ) in the form of methionine, methionine (S)-oxide, and methionine (S,S)-dioxide amino acids were synthesized and characterized by LCMS and 1D and 2D NMR spectroscopy. Coumarin orbitide complexes produced in this manner bind Eu(3+) yielding FRET compounds that absorb energy through coumarin antennae and emit photons at lanthanide wavelengths. PMID:26434760

  6. 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.

  7. Multifunctional, Biocompatible Supramolecular Hydrogelators Consist Only of Nucleobase, Amino Acid, and Glycoside

    PubMed Central

    Li, Xinming; Kuang, Yi; Shi, Junfeng; Gao, Yuan; Lin, Hsin-Chieh; Xu, Bing

    2011-01-01

    The integration of nucleobase, amino acid, and glycoside into a single molecule results in a novel class of supramolecular hydrogelators, which not only exhibit biocompatibility and biostability, but also facilitate the entry of nucleic acids into cytosol and nuclei of cells. This work illustrates a simple way to generate an unprecedented molecular architecture from the basic biological building blocks for the development of sophisticated soft nanomaterials, including supramolecular hydrogels. PMID:21928792

  8. 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

  9. 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

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

    PubMed

    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

    2016-06-01

    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

  11. 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

  12. 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

  13. 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.

  14. 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.

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

    PubMed

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

    2016-05-01

    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

  16. 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

  17. Hydrogen bond formation between the naturally modified nucleobase and phosphate backbone

    PubMed Central

    Sheng, Jia; Zhang, Wen; Hassan, Abdalla E. A.; Gan, Jianhua; Soares, Alexei S.; Geng, Song; Ren, Yi; Huang, Zhen

    2012-01-01

    Natural RNAs, especially tRNAs, are extensively modified to tailor structure and function diversities. Uracil is the most modified nucleobase among all natural nucleobases. Interestingly, >76% of uracil modifications are located on its 5-position. We have investigated the natural 5-methoxy (5-O-CH3) modification of uracil in the context of A-form oligonucleotide duplex. Our X-ray crystal structure indicates first a H-bond formation between the uracil 5-O-CH3 and its 5′-phosphate. This novel H-bond is not observed when the oxygen of 5-O-CH3 is replaced with a larger atom (selenium or sulfur). The 5-O-CH3 modification does not cause significant structure and stability alterations. Moreover, our computational study is consistent with the experimental observation. The investigation on the uracil 5-position demonstrates the importance of this RNA modification at the atomic level. Our finding suggests a general interaction between the nucleobase and backbone and reveals a plausible function of the tRNA 5-O-CH3 modification, which might potentially rigidify the local conformation and facilitates translation. PMID:22641848

  18. DNA STRETCHING AND OPTIMISATION OF NUCLEOBASE RECOGNITION FOR ENZYMATIC NANOPORE SEQUENCING

    PubMed Central

    Stoddart, David; Franceschini, Lorenzo; Heron, Andrew; Bayley, Hagan; Maglia, Giovanni

    2015-01-01

    In nanopore sequencing, where single DNA strands are electrophoretically translocated through a nanopore and the resulting ionic signal is used to identify the four DNA bases, an enzyme has been used to ratchet the nucleic acid stepwise through the pore at a controlled speed. In this work, we investigated the ability of αHL nanopores to distinguish the four DNA bases under conditions that are compatible with the activity of DNA-handling enzymes. Our findings suggest that in immobilised strands, the applied potential exerts a force on DNA (~ 10 pN at +160 mV) that increases the distance between nucleobases by about 2.2 Å/V. The four nucleobases can be resolved over wide ranges of applied potentials (from +60 mV to +220 mV in 1 m KCl) and ionic strengths (from 200 mM KCl to 1 M KCl at +160 mV) and nucleobase recognition can be improved when the ionic strength on the side of the DNA-handling enzyme is low, while the ionic strength on the opposite side is high. PMID:25648138

  19. Single Cell FRET Analysis for the Identification of Optimal FRET-Pairs in Bacillus subtilis Using a Prototype MEM-FLIM System

    PubMed Central

    Detert Oude Weme, Ruud G. J.; Kovács, Ákos T.; de Jong, Sander J. G.; Veening, Jan-Willem; Siebring, Jeroen; Kuipers, Oscar P.

    2015-01-01

    Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET). Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM). For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV)-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells. PMID:25886351

  20. 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

  1. 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.

  2. 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.

  3. 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

  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. 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

  6. 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

  7. 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

  8. Quantum dots and fluorescent protein FRET-based biosensors.

    PubMed

    Boeneman, Kelly; Delehanty, James B; Susumu, Kimihiro; Stewart, Michael H; Deschamps, Jeffrey R; Medintz, Igor L

    2012-01-01

    There has been considerable recent interest in the creation of nanoparticle-biomolecule hybrid materials for uses such as in vitro and in vivo biosensing, biological imaging, and drug -delivery. Nanoparticles have a high surface to volume ratio, making them capable of being decorated with -various biomolecules on their surface which retain their biological activity. Techniques to bind these biomolecules to nanoparticle surfaces are also advancing rapidly. Here we demonstrate hybrid materials assembled around CdSe/ZnS core/shell semiconductor quantum dots (QDs). These intrinsically fluorescent materials are conjugated to the fluorescent proteins YFP, mCherry and the light harvesting complex b-phycoerythrin (b-PE). QDs have fluorescent properties that make them ideal as donor fluorophores for Förster resonance energy transfer (FRET) while the fluorescent proteins are able to act as FRET acceptors displaying many advantages over organic dyes. We examine FRET interactions between QDs and all three fluorescent proteins. Furthermore, we show QD-mCherry hybrid materials can be utilized for in vitro biosensing of caspase-3 enzymatic activity. We further show that QDs and fluorescent proteins can be conjugated together intracellularly with strong potential for live-cell imaging and biosensing applications. PMID:22101713

  9. 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

  10. 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.

  11. Functionalized Tricyclic Cytosine Analogues Provide Nucleoside Fluorophores with Improved Photophysical Properties and a Range of Solvent Sensitivities

    PubMed Central

    Rodgers, Brittney J.; Elsharif, Nada A.; Vashisht, Nisha; Mingus, Macy M.; Mulvahill, Mark A.; Stengel, Gudrun; Kuchta, Robert D.

    2014-01-01

    Tricyclic cytosines (tC and tCO frameworks) have emerged as a unique class of fluorescent nucleobase analogues that minimally perturb the structure of B-form DNA and that are not quenched in duplex nucleic acids. Systematic derivatization of these frameworks is a likely approach to improve on and diversify photophysical properties, but has not so far been examined. Synthetic methods were refined to improve on tolerance for electron donating and electron withdrawing groups, resulting in a series of eight new, fluorescent cytidine analogues. Photophysical studies show that substitution of the framework results in a pattern of effects largely consistent across tC and tCO and provides nucleoside fluorophores that are brighter than either parent. Moreover, a range of solvent sensitivities is observed, offering promise that this family of probes can be extended to new applications that require reporting on the local environment. PMID:24311229

  12. 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

  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. 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.

  15. The mechanics and tribology of fretting fatigue with application to riveted lap joints

    NASA Astrophysics Data System (ADS)

    Szolwinski, Matthew Paul

    Fretting is the synergistic combination of wear, corrosion, and fatigue damage mechanisms driven by the partial slip of contacting surfaces. The surface microslip and near-surface contact stresses associated with fretting can lead to severe reduction in service lifetimes of contacting components as diversified as bearings, turbine blades and mechanically-fastened joints, both structural and biological. This tribologically induced degradation has come under close scrutiny by those responsible for maintaining aging fleets of both commercial and military aircraft. Thus a critical need exists for predicting fretting crack nucleation in riveted aluminum. aircraft joints. Fulfilling this need requires characterizing both the near-surface mechanics and intimately-related tribology of fretting. To this end, a well characterized experimental setup has been developed to generate carefully controlled and monitored fretting contacts to investigate the nature of the near-surface conditions. Included in this investigation were in-situ observations of the fretting contact stress field via a non-invasive thermal imaging technique and a characterization of the evolution of friction under partial slip conditions. With specific qualitative and quantitative understanding of these near-surface conditions, a series of fretting fatigue experiments have been conducted to validate a mechanics-based model for predicting fretting fatigue crack nucleation. Finally, efforts have been directed toward extending this understanding of fretting crack nucleation to riveted aircraft structure through modeling of the riveting process and a related experimental program designed to link riveting process parameters and fretting damage in single-lap joint structures. This work focuses specifically on determination of the residual stresses induced during rivet installation and the morphological characterization of fretting fatigue damage in the riveted test specimens manufactured under controlled

  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. Supramolecular hydrogels formed by the conjugates of nucleobases, Arg-Gly-Asp (RGD) peptides, and glucosamine

    PubMed Central

    Li, Xinming; Du, Xuewen; Gao, Yuan; Shi, Junfeng; Kuang, Yi

    2012-01-01

    Here we report the generation of a novel class of supramolecular hydrogelators based on the integration of nucleobase, Arg-Gly-Asp (RGD) peptides, and glucosamine in a single molecule. These novel small molecule hydrogelators self-assemble in water to form stable supramolecular nanofibers/hydrogels and exhibit useful biostability. This approach provides a new opportunity for systematic exploration of the self-assembly of small biomolecules by varying any individual segment to generate a large array of supramolecular hydrogels for biological functions and for biomedical applications. PMID:22844343

  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. Fretting induced plasticity in blade/disk contacts

    NASA Astrophysics Data System (ADS)

    Gao, Guofeng

    Finite element analyses of fretting contacts were developed for different pad geometries and material combinations. A better understanding of the surface tractions and life predictions for crack nucleation and propagation is achieved by including the effects of plasticity and the representative crack in the finite element modeling. Pure elastic finite element results were validated with the existing elastic solution suite based on Singular Integral Equations (SIEs). The elastic-perfectly-plastic analysis of a rigid cylindrical pad on an isotropic half-space was compared with the results in the literature. Different shapes of rigid indenters were examined and the results were compared with those from the SIEs. Fretting contacts with similar and dissimilar isotropic materials were studied. The effects of plasticity on fretting life were highlighted by comparing the results from pure elastic analyses and elastic-plastic analyses. Finite element modelings involving SCN materials with different rotations of material principle axes were also investigated. The crack nucleation life was calculated with the Modified Manson-McKnight model. This model, modified by stressed area approach, usually generates significantly conservative estimates when pure elastic stress results are used. When plasticity is taken into account, the life prediction accuracy for crack nucleation was proven to be improved tremendously. A representative crack was incorporated into the FEM model at the trailing edge of contact to study its effects on surface tractions and crack propagation life. The originally smooth pressure distribution has a singularity at the edge of contact and the contact size expands due to the crack growth. The crack propagation life was evaluated by using the modified crack closure method and the Paris law.

  20. Photophysical properties of pyrrolocytosine, a cytosine fluorescent base analogue.

    PubMed

    Nguyen, Quynh L; Spata, Vincent A; Matsika, Spiridoula

    2016-07-27

    The photophysical behavior of pyrrolocytosine (PC), a fluorescent base analogue of cytosine, has been investigated using theoretical approaches. The similarities between the PC and cytosine structures allow PC to maintain the pseudo-Watson-Crick base-pairing arrangement with guanine. Cytosine, similar to the other natural nucleobases, is practically non-fluorescent, because of ultrafast radiationless decay occurring through conical intersections. PC displays a much higher fluorescence quantum yield than cytosine, making it an effective fluorescent marker to study the structure, function, and dynamics of DNA/RNA complexes. Similar to 2-aminopurine, a constitutional isomer of adenine that base-pairs with thymine, PC's fluorescence is quenched when it is incorporated into a dinucleotide or a trinucleotide. In this work we examine the photophysical properties of isolated PC, microhydrated PC, as well as, complexes where PC is either base-stacked or hydrogen-bonded with guanine. Our results indicate that hydration affects the radiationless decay pathways in PC by destabilizing conical intersections. The calculations of dimers and trimers show that the radiative decay is affected by π stacking, while the presence of charge transfer states between PC and guanine may contribute to radiationless decay. PMID:27251599

  1. 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…

  2. 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.

  3. 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.

  4. 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.

  5. Polyfluorophore Excimers and Exciplexes as FRET Donors in DNA

    PubMed Central

    Teo, Yin Nah; Kool, Eric T.

    2009-01-01

    We describe studies aimed at testing whether oligomeric exciplex- and excimer fluorophores conjugated to DNA have the potential to act as donors for energy transfer by the Förster mechanism. Oligodeoxyfluorosides (ODFs) are composed of stacked, electronically interacting fluorophores replacing the bases on a DNA scaffold. The monomer chromophores in the twenty tetramer-length ODFs studied here include pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and a nonfluorescent spacer (S); these are conjugated in varied combinations at the 3’ end of a 14mer DNA probe sequence. In the absence of an acceptor chromophore, many of the ODF-DNAs show broad, unstructured long-wavelength emission peaks characteristic of excimer and exciplex excited states, similar to what has been observed for unconjugated ODFs. Although such delocalized excited states have been widely studied, we know of no prior report of their use in FRET. We tested the ability of the twenty ODFs to donate energy to Cy5 and TAMRA dyes conjugated to a complementary strand of DNA, with these acceptors oriented either at the near or far end of the ODF-conjugated probes. Results showed that a number of the ODF fluorophores exhibited relatively efficient energy transfer characteristic of the Förster mechanism, as judged by drops in donor emission quantum yield and fluorescence lifetime, accompanied by increases in intensity of acceptor emission bands. Excimer/exciplex bands in the donors were selectively quenched while shorter-wavelength monomer emission stayed relatively constant, consistent with the notion that the delocalized excited states, rather than individual fluorophores, are the donors. Interestingly, only specific sequences of ODFs were able to act as donors, while others did not, even though their emission wavelengths were similar. The new FRET donors possess large Stokes shifts, which can be beneficial for multiple applications. In addition, all ODFs can be excited at a single

  6. Tumor specific lung cancer diagnostics with multiplexed FRET immunoassays

    NASA Astrophysics Data System (ADS)

    Geißler, D.; Hill, D.; Löhmannsröben, H.-G.; Thomas, E.; Lavigne, A.; Darbouret, B.; Bois, E.; Charbonnière, L. J.; Ziessel, R. F.; Hildebrandt, N.

    2010-02-01

    An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

  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. A Study on Fretting Fatigue Life in Elevated Temperature for Incoloy 800

    NASA Astrophysics Data System (ADS)

    Kwon, Jae Do; Woo, Seung Wan; Chung, Il Sup; Yoon, Dong Hwan; Park, Dae Kyu

    Incoloy 800, which is used within steam generator tubes, is a heat resistant material since it is an iron-nickel-chromium alloy. However, construction of a systematic database is needed to receive integrity data defecting insurance of specific data about room and elevated temperature fretting fatigue behavior for Incoloy 800. Accordingly, this study investigates the specific change in fatigue limitations under the condition of the fretting fatigue as compared to that under the condition of the plain fatigue by performing plain and fretting fatigue tests on Incoloy 800 at 320°C, real operating temperature and at room-temperature, respectively. The change in the frictional force is measured during the fretting fatigue testing against the repeated cycle, and the mechanism of fretting fatigue is investigated through the observation of the fatigue-fracture surface.

  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

    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.

  11. Aggregation-Induced-Emissive Molecule Incorporated into Polymeric Nanoparticulate as FRET Donor for Observing Doxorubicin Delivery.

    PubMed

    Han, Xiongqi; Liu, De-E; Wang, Tieyan; Lu, Hongguang; Ma, Jianbiao; Chen, Qixian; Gao, Hui

    2015-10-28

    Tetraphenylethene (TPE) derivatives characterized with distinct aggregation-induced-emission, attempted to aggregate with doxorubicin (Dox) to formulate the interior compartment of polymeric nanoparticulate, served as fluorescence resonance energy transfer (FRET) donor to promote emission of acceptor Dox. Accordingly, this FRET formulation allowed identification of Dox in complexed form by detecting FRET. Important insight into the Dox releasing can be subsequently explored by extracting complexed Dox (FRET) from the overall Dox via direct single-photon excitation of Dox. Of note, functional catiomers were used to complex with FRET partners for a template formulation, which was verified to induce pH-responsive release in the targeted subcellular compartment. Hence, this well-defined multifunctional system entitles in situ observation of the drug releasing profile and insight on drug delivery journey from the tip of injection vein to the subcellular organelle of the targeted cells. PMID:26448180

  12. 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

    2016-01-01

    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

  13. 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

  14. Structural Analogues of Selfotel.

    PubMed

    Dziuganowska, Zofia A; Ślepokura, Katarzyna; Volle, Jean-Noël; Virieux, David; Pirat, Jean-Luc; Kafarski, Paweł

    2016-06-17

    A small library of phosphonopiperidylcarboxylic acids, analogues of NMDA antagonist selfotel (CGS 19755), was synthesized. First, the series of aromatic esters was obtained via a palladium-catalyzed cross-coupling reaction (Hirao coupling) of dialkyl phosphites with bromopyridinecarboxylates, followed by their hydrolysis. Then, hydrogenation of the resulting phosphonopyridylcarboxylic acids over PtO2 yielded the desired phosphonopiperidylcarboxylic acids. NMR studies indicated that the hydrogenation reaction proceeds predominantly by cis addition. Several compounds were obtained as monocrystal structures. Preliminary biological studies performed on cultures of neurons suggest that the obtained compounds possess promising activity toward NMDA receptors. PMID:27187758

  15. 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.

  16. 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.

  17. 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.

  18. 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. PMID:26758444

  19. 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.

  20. 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

  1. 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. PMID:26607270

  2. Ultraviolet Irradiation of Pyrimidine in Interstellar Ice Analogs: Formation and Stability of Nucleobases

    NASA Astrophysics Data System (ADS)

    Milam, Stefanie; Nuevo, Michel; Sandford, Scott; Elsila, Jamie; Dworkin, Jason

    The detection of amino acids in organic residues formed by the UV photolysis of 10 K ices representative of interstellar and cometary environments (H2 O, CO, CO2 , CH3 OH, NH3 , etc.) show that molecules of prebiotic interest could potentially form in space. The detection of amino acids in meteorites supports a scenario where the organic molecules required for life are of extraterrestrial origin. Nucleobases, the informational units of RNA and DNA, have also been detected in meteorites and constitute another family of prebiotic compounds that can possibly form in interstellar environments. These molecules are functionalized heterocyclic aromatic species. There are two classes of nucleobases: pyrimidines (e.g. thymine, uracil, and cytosine) and purines (e.g. adenine and guanine). The functionalization of PAHs from UV photolysis in mixed molecular ices has been proven effective in the laboratory. This work aims at studying how UV irradiation affects pyrimidine in interstellar ice analogs. In particular, we show how H2 O/ pyrimidine mixtures lead to the production of oxidized compounds and study their photostability.

  3. First-principles photoemission spectroscopy in DNA and RNA nucleobases from Koopmans-compliant functionals

    NASA Astrophysics Data System (ADS)

    Nguyen, Ngoc Linh; Borghi, Giovanni; Ferretti, Andrea; Marzari, Nicola

    The determination of spectral properties of the DNA and RNA nucleobases from first principles can provide theoretical interpretation for experimental data, but requires complex electronic-structure formulations that fall outside the domain of applicability of common approaches such as density-functional theory. In this work, we show that Koopmans-compliant functionals, constructed to enforce piecewise linearity in energy functionals with respect to fractional occupation-i.e., with respect to charged excitations-can predict not only frontier ionization potentials and electron affinities of the nucleobases with accuracy comparable or superior with that of many-body perturbation theory and high-accuracy quantum chemistry methods, but also the molecular photoemission spectra are shown to be in excellent agreement with experimental ultraviolet photoemsision spectroscopy data. The results highlight the role of Koopmans-compliant functionals as accurate and inexpensive quasiparticle approximations to the spectral potential, which transform DFT into a novel dynamical formalism where electronic properties, and not only total energies, can be correctly accounted for.

  4. 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.

  5. 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.

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

    PubMed

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

    2015-10-01

    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

  7. Studying DNA Looping by Single-Molecule FRET

    PubMed Central

    Le, Tung T.; Kim, Harold D.

    2014-01-01

    Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA. PMID:24998459

  8. 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

  9. Functional Studies of DNA-Protein Interactions Using FRET Techniques.

    PubMed

    Blouin, Simon; Craggs, Timothy D; Lafontaine, Daniel A; Penedo, J Carlos

    2015-01-01

    Protein-DNA interactions underpin life and play key roles in all cellular processes and functions including DNA transcription, packaging, replication, and repair. Identifying and examining the nature of these interactions is therefore a crucial prerequisite to understand the molecular basis of how these fundamental processes take place. The application of fluorescence techniques and in particular fluorescence resonance energy transfer (FRET) to provide structural and kinetic information has experienced a stunning growth during the past decade. This has been mostly promoted by new advances in the preparation of dye-labeled nucleic acids and proteins and in optical sensitivity, where its implementation at the level of individual molecules has opened a new biophysical frontier. Nowadays, the application of FRET-based techniques to the analysis of protein-DNA interactions spans from the classical steady-state and time-resolved methods averaging over large ensembles to the analysis of distances, conformational changes, and enzymatic reactions in individual protein-DNA complexes. This chapter introduces the practical aspects of applying these methods for the study of protein-DNA interactions. PMID:26404147

  10. 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)