Methods of single-molecule energy landscape reconstruction with optical traps

Optical traps facilitate measurement of force and position as single molecules of DNA, RNA, or protein are unfolded and refolded. The effective energy landscape of a biomolecule can be reconstructed from the force and position data, providing insight into its structure and regulatory functions. We have developed new experimental and analytical methods to reconstruct energy landscapes by taking advantage of the harmonic constraint of an optical trap. We demonstrate the effectiveness of these methods using a model DNA hairpin and then apply these methods to study problems of practical biophysical interest. CCR5 mRNA has been demonstrated to stimulate -1 programmed ribosomal frameshifting and we measure its structural properties. We measure the binding energy of a GA/AG tandem mismatch, one of many mismatches with unusual properties. We use our single-molecule methods to reproduce bulk measurements of the nearest-neighbor DNA base-pair free energy parameters and we consider possible refinements to the model. We also study an alternative method of measuring energy landscapes, Dynamic Force Spectroscopy (DFS), and conduct experiments on DNA quadruplexes to demonstrate the effectiveness of DFS with optical traps. Finally, we develop theory to elucidate the role of noise in optical trap measurements of energy landscapes

Noise associated with nonconservative forces in optical traps

It is known that for a particle held in an optical trap the interaction of thermal fluctuations with a nonconservative scattering force can cause a persistent nonequilibrium probability flux in the particle position. We investigate position fluctuations associated with this nonequilibrium flux analytically and through simulation. We introduce a model which reproduces the nonequilibrium effects, and in which the magnitude of additional position fluctuations can be calculated in closed form. The ratio of additional nonconservative fluctuations to direct thermal fluctuations scales inversely with the square root of trap power, and is small for typical experimental parameters. In a simulated biophysical experiment the nonconservative scattering force does not significantly increase the observed fluctuations in the length of a double-stranded DNA tether

The Physical Conditions in Starbursts Derived from Bayesian Fitting of Mid-IR SEDS: 30 Doradus as a Template

To understand and interpret the observed Spectral Energy Distributions (SEDs) of starbursts, theoretical or semi-empirical SED models are necessary. Yet, while they are well-founded in theory, independent verification and calibration of these models, including the exploration of possible degeneracies between their parameters, are rarely made. As a consequence, a robust fitting method that leads to unique and reproducible results has been lacking. Here we introduce a novel approach based on Bayesian analysis to fit the Spitzer-IRS spectra of starbursts using the SED models proposed by Groves et al. (2008). We demonstrate its capabilities and verify the agreement between the derived best fit parameters and actual physical conditions by modelling the nearby, well-studied, giant HII region 30 Dor in the LMC. The derived physical parameters, such as cluster mass, cluster age, ISM pressure and covering fraction of photodissociation regions, are representative of the 30 Dor region. The inclusion of the emission lines in the modelling is crucial to break degeneracies. We investigate the limitations and uncertainties by modelling sub-regions, which are dominated by single components, within 30 Dor. A remarkable result for 30 Doradus in particular is a considerable contribution to its mid-infrared spectrum from hot ({\simeq} 300K) dust. The demonstrated success of our approach will allow us to derive the physical conditions in more distant, spatially unresolved starbursts.Comment: 17 pages, 10 figures. Accepted por publication in the Astrophysical Journa

Mechanical unfolding of long human telomeric RNA (TERRA)

[EN] We report the first single molecule investigation of TERRA molecules. By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplex blocks, which unfold at lower forces than their DNA counterparts.This work was supported by grants from the Spanish Ministry of Science and Innovation (grants RYC2007-01765 to JRA-G, BFU2011-30295-C02-01 to AV, and CTQ2010-21567-C02-02 to CG). MG was supported by the FPI fellowship BES-2009-027909. RB and EH-G were supported by Comunidad de Madrid, grant CAM-S2009MAT-1507. AV acknowledges an institutional grant from the Fundacion Ramon Areces to the CBMSO. JRA-G wants to thank Prof. J. L. Carrascosa and Prof. J. M. Valpuesta (CNB-CSIC) for their continuous support and encouragement in this research. We also acknowledge the excellent technical assistance of Beatriz de Pablos (CBMSO).Garavís, M.; Bocanegra, R.; Herrero-Galán, E.; González, C.; Villasante, A.; Arias-Gonzalez, JR. (2013). Mechanical unfolding of long human telomeric RNA (TERRA). Chemical Communications. 49(57):6397-6399. https://doi.org/10.1039/c3cc42981dS639763994957De Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes & Development, 19(18), 2100-2110. doi:10.1101/gad.1346005Blackburn, E. H. (1991). Structure and function of telomeres. Nature, 350(6319), 569-573. doi:10.1038/350569a0Biffi, G., Tannahill, D., McCafferty, J., & Balasubramanian, S. (2013). Quantitative visualization of DNA G-quadruplex structures in human cells. Nature Chemistry, 5(3), 182-186. doi:10.1038/nchem.1548Paeschke, K., Simonsson, T., Postberg, J., Rhodes, D., & Lipps, H. J. (2005). Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nature Structural & Molecular Biology, 12(10), 847-854. doi:10.1038/nsmb982Hwang, H., Buncher, N., Opresko, P. L., & Myong, S. (2012). POT1-TPP1 Regulates Telomeric Overhang Structural Dynamics. Structure, 20(11), 1872-1880. doi:10.1016/j.str.2012.08.018Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E., & Lingner, J. (2007). Telomeric Repeat Containing RNA and RNA Surveillance Factors at Mammalian Chromosome Ends. Science, 318(5851), 798-801. doi:10.1126/science.1147182Schoeftner, S., & Blasco, M. A. (2007). Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II. Nature Cell Biology, 10(2), 228-236. doi:10.1038/ncb1685Porro, A., Feuerhahn, S., Reichenbach, P., & Lingner, J. (2010). Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils the Presence of Distinct and Multiple Regulatory Pathways. Molecular and Cellular Biology, 30(20), 4808-4817. doi:10.1128/mcb.00460-10Deng, Z., Norseen, J., Wiedmer, A., Riethman, H., & Lieberman, P. M. (2009). TERRA RNA Binding to TRF2 Facilitates Heterochromatin Formation and ORC Recruitment at Telomeres. Molecular Cell, 35(4), 403-413. doi:10.1016/j.molcel.2009.06.025De Silanes, I. L., d’ Alcontres, M. S., & Blasco, M. A. (2010). TERRA transcripts are bound by a complex array of RNA-binding proteins. Nature Communications, 1(1). doi:10.1038/ncomms1032Xu, Y., Suzuki, Y., Ito, K., & Komiyama, M. (2010). Telomeric repeat-containing RNA structure in living cells. Proceedings of the National Academy of Sciences, 107(33), 14579-14584. doi:10.1073/pnas.1001177107Martadinata, H., & Phan, A. T. (2009). Structure of Propeller-Type Parallel-Stranded RNA G-Quadruplexes, Formed by Human Telomeric RNA Sequences in K+Solution. Journal of the American Chemical Society, 131(7), 2570-2578. doi:10.1021/ja806592zXu, Y., Kaminaga, K., & Komiyama, M. (2008). G-Quadruplex Formation by Human Telomeric Repeats-Containing RNA in Na+Solution. Journal of the American Chemical Society, 130(33), 11179-11184. doi:10.1021/ja8031532Collie, G. W., Haider, S. M., Neidle, S., & Parkinson, G. N. (2010). A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex. Nucleic Acids Research, 38(16), 5569-5580. doi:10.1093/nar/gkq259Collie, G. W., Parkinson, G. N., Neidle, S., Rosu, F., De Pauw, E., & Gabelica, V. (2010). Electrospray Mass Spectrometry of Telomeric RNA (TERRA) Reveals the Formation of Stable Multimeric G-Quadruplex Structures. Journal of the American Chemical Society, 132(27), 9328-9334. doi:10.1021/ja100345zMartadinata, H., Heddi, B., Lim, K. W., & Phan, A. T. (2011). Structure of Long Human Telomeric RNA (TERRA): G-Quadruplexes Formed by Four and Eight UUAGGG Repeats Are Stable Building Blocks. Biochemistry, 50(29), 6455-6461. doi:10.1021/bi200569fArora, A., & Maiti, S. (2009). Differential Biophysical Behavior of Human Telomeric RNA and DNA Quadruplex. The Journal of Physical Chemistry B, 113(30), 10515-10520. doi:10.1021/jp810638nJoachimi, A., Benz, A., & Hartig, J. S. (2009). A comparison of DNA and RNA quadruplex structures and stabilities. Bioorganic & Medicinal Chemistry, 17(19), 6811-6815. doi:10.1016/j.bmc.2009.08.043Qi, J., & Shafer, R. H. (2007). Human Telomere Quadruplex:  Refolding and Selection of Individual Conformers via RNA/DNA Chimeric Editing†. Biochemistry, 46(25), 7599-7606. doi:10.1021/bi602392uRandall, A., & Griffith, J. D. (2009). Structure of Long Telomeric RNA Transcripts. Journal of Biological Chemistry, 284(21), 13980-13986. doi:10.1074/jbc.m900631200Kumari, S., Bugaut, A., & Balasubramanian, S. (2008). Position and Stability Are Determining Factors for Translation Repression by an RNA G-Quadruplex-Forming Sequence within the 5′ UTR of theNRASProto-oncogene†. Biochemistry, 47(48), 12664-12669. doi:10.1021/bi8010797McKenna, S. A., Kim, I., Puglisi, E. V., Lindhout, D. A., Aitken, C. E., Marshall, R. A., & Puglisi, J. D. (2007). Purification and characterization of transcribed RNAs using gel filtration chromatography. Nature Protocols, 2(12), 3270-3277. doi:10.1038/nprot.2007.480Parkinson, G. N., Lee, M. P. H., & Neidle, S. (2002). Crystal structure of parallel quadruplexes from human telomeric DNA. Nature, 417(6891), 876-880. doi:10.1038/nature755Herrero-Galán, E., Fuentes-Perez, M. E., Carrasco, C., Valpuesta, J. M., Carrascosa, J. L., Moreno-Herrero, F., & Arias-Gonzalez, J. R. (2012). Mechanical Identities of RNA and DNA Double Helices Unveiled at the Single-Molecule Level. Journal of the American Chemical Society, 135(1), 122-131. doi:10.1021/ja3054755Bustamante, C., Bryant, Z., & Smith, S. B. (2003). Ten years of tension: single-molecule DNA mechanics. Nature, 421(6921), 423-427. doi:10.1038/nature01405Yu, Z., Schonhoft, J. D., Dhakal, S., Bajracharya, R., Hegde, R., Basu, S., & Mao, H. (2009). ILPR G-Quadruplexes Formed in Seconds Demonstrate High Mechanical Stabilities. Journal of the American Chemical Society, 131(5), 1876-1882. doi:10.1021/ja806782sKoirala, D., Dhakal, S., Ashbridge, B., Sannohe, Y., Rodriguez, R., Sugiyama, H., … Mao, H. (2011). A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands. Nature Chemistry, 3(10), 782-787. doi:10.1038/nchem.1126Dhakal, S., Cui, Y., Koirala, D., Ghimire, C., Kushwaha, S., Yu, Z., … Mao, H. (2013). Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions. Nucleic Acids Research, 41(6), 3915-3923. doi:10.1093/nar/gkt038De Messieres, M., Chang, J.-C., Brawn-Cinani, B., & La Porta, A. (2012). Single-Molecule Study ofG-Quadruplex Disruption Using Dynamic Force Spectroscopy. Physical Review Letters, 109(5). doi:10.1103/physrevlett.109.058101Schonhoft, J. D., Bajracharya, R., Dhakal, S., Yu, Z., Mao, H., & Basu, S. (2009). Direct experimental evidence for quadruplex–quadruplex interaction within the human ILPR. Nucleic Acids Research, 37(10), 3310-3320. doi:10.1093/nar/gkp181Carrion-Vazquez, M., Oberhauser, A. F., Fowler, S. B., Marszalek, P. E., Broedel, S. E., Clarke, J., & Fernandez, J. M. (1999). Mechanical and chemical unfolding of a single protein: A comparison. Proceedings of the National Academy of Sciences, 96(7), 3694-3699. doi:10.1073/pnas.96.7.369

Jet-Powered Molecular Hydrogen Emission from Radio Galaxies

H2 pure-rotational emission lines are detected from warm (100-1500 K) molecular gas in 17/55 (31% of) radio galaxies at redshift z<0.22 observed with the Spitzer IR Spectrograph. The summed H2 0-0 S(0)-S(3) line luminosities are L(H2)=7E38-2E42 erg/s, yielding warm H2 masses up to 2E10 Msun. These radio galaxies, of both FR radio morphological types, help to firmly establish the new class of radio-selected molecular hydrogen emission galaxies (radio MOHEGs). MOHEGs have extremely large H2 to 7.7 micron PAH emission ratios: L(H2)/L(PAH7.7) = 0.04-4, up to a factor 300 greater than the median value for normal star-forming galaxies. In spite of large H2 masses, MOHEGs appear to be inefficient at forming stars, perhaps because the molecular gas is kinematically unsettled and turbulent. Low-luminosity mid-IR continuum emission together with low-ionization emission line spectra indicate low-luminosity AGNs in all but 3 radio MOHEGs. The AGN X-ray emission measured with Chandra is not luminous enough to power the H2 emission from MOHEGs. Nearly all radio MOHEGs belong to clusters or close pairs, including 4 cool core clusters (Perseus, Hydra, A 2052, and A 2199). We suggest that the H2 in radio MOHEGs is delivered in galaxy collisions or cooling flows, then heated by radio jet feedback in the form of kinetic energy dissipation by shocks or cosmic rays.Comment: ApJ in press, 40 pages, 18 figures, 14 table

Mesoscopic model for DNA G-quadruplex unfolding

[EN] Genomes contain rare guanine-rich sequences capable of assembling into four-stranded helical structures, termed G-quadruplexes, with potential roles in gene regulation and chromosome stability. Their mechanical unfolding has only been reported to date by all-atom simulations, which cannot dissect the major physical interactions responsible for their cohesion. Here, we propose a mesoscopic model to describe both the mechanical and thermal stability of DNA G-quadruplexes, where each nucleotide of the structure, as well as each central cation located at the inner channel, is mapped onto a single bead. In this framework we are able to simulate loading rates similar to the experimental ones, which are not reachable in simulations with atomistic resolution. In this regard, we present single-molecule force-induced unfolding experiments by a high-resolution optical tweezers on a DNA telomeric sequence capable of adopting a G-quadruplex conformation. Fitting the parameters of the model to the experiments we find a correct prediction of the rupture-force kinetics and a good agreement with previous near equilibrium measurements. Since G-quadruplex unfolding dynamics is halfway in complexity between secondary nucleic acids and tertiary protein structures, our model entails a nanoscale paradigm for non-equilibrium processes in the cell.Work supported by the Spanish Ministry of Economy and Competitiveness (MINECO), grant No. FIS2014-55867, co-financed by FEDER funds. We also thank the support of the Aragon Government and Fondo Social Europeo to FENOL group. Work in J.R.A.-G. laboratory was supported by a grant from MINECO, No. MAT2015-71806-R).Bergues-Pupo, A.; Gutiérrez, I.; Arias-Gonzalez, JR.; Falo, F.; Fiasconaro, A. (2017). Mesoscopic model for DNA G-quadruplex unfolding. Scientific Reports. 7:1-13. https://doi.org/10.1038/s41598-017-10849-2S1137Arias-Gonzalez, J. R. Single-molecule portrait of DNA and RNA double helices. Integr. 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Single-molecule study of G-quadruplex disruption using dynamic force spectroscopy. Phys. Rev. Lett. 109, 058101 (2012).Koirala, D. et al. A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands. Nat. Chem. 3, 782 (2011).Long, X. et al. Mechanical unfolding of human telomere G-quadruplex DNA probed by integrated fluorescence and magnetic tweezers spectroscopy. Nucleic Acids Res. 41, 2746 (2013).Ghimire, C. et al. Direct Quantification of Loop Interaction and pi-pi Stacking for G-Quadruplex Stability at the Submolecular Level. J. Am. Chem. Soc. 136, 15544 (2014).Garavís, M. et al. Mechanical Unfolding of Long Human Telomeric RNA (TERRA). Chem. Commun. 49, 6397 (2013).Fonseca Guerra, C., Zijlstra, H., Paragi, G. & Bickelhaupt, F. M. Telomere structure and stability: covalency in hydrogen bonds, not resonance assistance, causes cooperativity in guanine quartets. Chemistry-A European Journal 17, 12612 (2011).Yurenko, Y. 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Nucleic Acids Res. 41, 7128 (2013).Li, H., Cao, E. & Gisler, T. Force-induced unfolding of human telomeric G-quadruplex: a steered molecular dynamics simulation study. Biochem. Bioph. Res. Co. 379, 70 (2009).Yang, C., Jang, S. & Pak, Y. Multiple stepwise pattern for potential of mean force in unfolding the thrombin binding aptamer in complex with Sr2+. J. Chem. Phys. 135, 225104 (2011).Bergues-Pupo, A. E., Arias-Gonzalez, J. R., Morón, M. C., Fiasconaro, A. & Falo, F. Role of the central cations in the mechanical unfolding of DNA and RNA G-quadruplexes. Nucleic Acids Res. 43, 7638 (2015).Linak, M. C., Tourdot, R. & Dorfman, K. D. Moving beyond Watson-Crick models of coarse grained DNA dynamics. J. Chem Phys. 135, 205102 (2011).Rebi, M., Mocci, F., Laaksonen, A. & Ulin, J. Multiscale simulations of human telomeric G-quadruplex DNA. J. Phys. Chem. B 119, 105 (2014).Stadlbauer, P. et al. 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D25V apolipoprotein C-III variant causes dominant hereditary systemic amyloidosis and confers cardiovascular protective lipoprotein profile

Apolipoprotein C-III deficiency provides cardiovascular protection, but apolipoprotein C-III is not known to be associated with human amyloidosis. Here we report a form of amyloidosis characterized by renal insufficiency caused by a new apolipoprotein C-III variant, D25V. Despite their uremic state, the D25V-carriers exhibit low triglyceride (TG) and apolipoprotein C-III levels, and low very-low-density lipoprotein (VLDL)/high high-density lipoprotein (HDL) profile. Amyloid fibrils comprise the D25V-variant only, showing that wild-type apolipoprotein C-III does not contribute to amyloid deposition in vivo. The mutation profoundly impacts helical structure stability of D25V-variant, which is remarkably fibrillogenic under physiological conditions in vitro producing typical amyloid fibrils in its lipid-free form. D25V apolipoprotein C-III is a new human amyloidogenic protein and the first conferring cardioprotection even in the unfavourable context of renal failure, extending the evidence for an important cardiovascular protective role of apolipoprotein C-III deficiency. Thus, fibrate therapy, which reduces hepatic APOC3 transcription, may delay amyloid deposition in affected patients