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Biophysics of RNA Folding pp 69–87Cite as

Studying RNA Folding Using Site-Directed Spin Labeling

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Part of the book series: Biophysics for the Life Sciences ((BIOPHYS,volume 3))

Abstract

The method of site-directed spin labeling (SDSL) uses electron ­paramagnetic resonance (EPR) spectroscopy to monitor behaviors of a stable nitroxide radical attached at specific sites within an RNA or other biomacromolecules. Parameters obtained from EPR measurements, such as inter-nitroxide distances and descriptions of rotational motions of a nitroxide, can provide unique information on the local environment of the macromolecule. The SDSL strategy is capable of studying high-molecular-weight systems under physiological conditions. It serves as a unique biophysical tool for monitoring site-specific structural and dynamic features of RNA. This chapter summarizes the use of SDSL in mapping RNA conformations as well as in investigating conformational transitions during RNA folding.

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References

  • Altenbach C, Flitsch SL, Khorana HG, Hubbell WL (1989) Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines. Biochemistry 28:7806–7812

    Article  PubMed  CAS  Google Scholar 

  • Barhate N, Cekan P, Massey Archna P, Sigurdsson Snorri T (2007) A nucleoside that contains a rigid nitroxide spin label: A fluorophore in disguise. Angew chem Int Ed 46(15):2655–2658

    Article  CAS  Google Scholar 

  • Berliner LJ (ed) (1976) Spin Labeling: Theory and Applications. Academic, New York

    Google Scholar 

  • Bonora M, Pornsuwan S, Saxena S (2004) Nitroxide spin-relaxation over the entire motional range. J Phys Chem B 108:4196–4198

    Article  CAS  Google Scholar 

  • Borbat PP, Costa-Filho AJ, Earle KA, Moscicki JK, Freed JH (2001) Electron spin resonance in studies of membranes and proteins. Science 291:266–269

    Article  PubMed  CAS  Google Scholar 

  • Borbat PP, Freed JH (2000) Double-quantum ESR and distance measurements. In: Berliner LJ, Eaton GR, Eaton SS (eds) Biological magnetic resonance: Distance measurements in biological systems by EPR, vol 19. Kluwer, New York, pp 383–459

    Chapter  Google Scholar 

  • Butcher SE, Dieckmann T, Feigon J (1997) Solution structure of a GAAA tetraloop receptor RNA. EMBO J 16:7490–7499

    Article  PubMed  CAS  Google Scholar 

  • Cai Q, Kusnetzow AK, Hideg K, Price EA, Haworth IS, Qin PZ (2007) Nanometer distance ­measurements in RNA using site-directed spin labeling. Biophys J 93(6):2110–2117

    Article  PubMed  CAS  Google Scholar 

  • Cai Q, Kusnetzow AK, Hubbell WL, Haworth IS, Gacho GPC, Van Eps N, Hideg K, Chambers EJ, Qin PZ (2006) Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe. Nucl Acids Res 34:4722–4734

    Article  PubMed  CAS  Google Scholar 

  • Cate JH, Gooding AR, Podell E, Zhou K, Golden BL, Kundrot CE, Cech TR, Doudna JA (1996) Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273(5282):1678–1685

    Article  PubMed  CAS  Google Scholar 

  • Columbus L, Hubbell WL (2002) A new spin on protein dynamics. Trends Biochem Sci 27:288–295

    Article  PubMed  CAS  Google Scholar 

  • Columbus L, Hubbell WL (2004) Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA. Biochemistry 43:7273–7287

    Article  PubMed  CAS  Google Scholar 

  • Davis JH, Tonelli M, Scott LG, Jaeger L, Williamson JR, Butcher SE (2005) RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex. J Mol Biol 351(2):371–382

    Article  PubMed  CAS  Google Scholar 

  • de Lange G, Wang ZH, Riste D, Dobrovitski VV, Hanson R (2010) Universal dynamical decoupling of a single solid-state spin from a spin bath. Science 330(6000):60–63

    Article  PubMed  Google Scholar 

  • Duchardt-Ferner E, Weigand JE, Ohlenschläger O, Schmidtke SR, Suess B, Wöhnert J (2010) Highly modular structure and ligand binding by conformational capture in a minimalistic ­riboswitch. Angew Chem Int Ed 49(35):6216–6219

    Article  CAS  Google Scholar 

  • Earle KA, Budil DE (2006) Calculating slow-motion ESR spectra of spin-labeled polymers. In: schlick S (ed) Advanced ESR methods in polymer reaserch. John wiley and sons, New York, pp 53–83

    Chapter  Google Scholar 

  • Eaton GR, Eaton SS, Berliner LJ (eds) (2000) Distance measurements in biological systems by EPR, vol 19. Biol. Mag. Res, Kluwer, New York, NY

    Google Scholar 

  • Edwards TE, Okonogi TM, Sigurdsson ST (2002) Investigation of RNA-protein and RNA-metal ion interactions by electron paramagnetic resonance spectroscopy: The HIV TAR-Tat motif. Chem Biol 9:699–706

    Article  PubMed  CAS  Google Scholar 

  • Edwards TE, Robinson BH, Sigurdsson ST (2005) Identification of amino acids that promote specific and rigid TAR RNA-Tat protein complex formation. Chem Biol 12:329–337

    Article  PubMed  CAS  Google Scholar 

  • Edwards TE, Sigurdsson ST (2007) Site-specific incorporation of nitroxide spin-labels into 2′-positions of nucleic acids. Nat Protoc 2:1954–1962

    Article  PubMed  CAS  Google Scholar 

  • Fajer PG (2000) Electron spin resonance spectroscopy labeling in proteins and peptides analysis. In: Meyers R (ed) Encyclopedia of analytical chemistry. John Wiley & Sons, Chichester, pp 5725–5761

    Google Scholar 

  • Fanucci GE, Cafiso DS (2006) Recent Advances and applications of site-directed spin labeling. Curr Opin Struct Biol 16:644–653

    Article  PubMed  CAS  Google Scholar 

  • Gannett PM, Darian E, Powell J, Johnson EM, Mundoma C, Greenbaum NL, Ramsey CM, Dalal NS, Budil DE (2002) Probing triplex formation by EPR spectroscopy using a newly synthesized spin label for oligonucleotides. Nucleic Acids Res 30:5328–5337

    Article  PubMed  CAS  Google Scholar 

  • Gesteland RF, Atkins JF, Cech TR (eds) (2006) RNA world, 3rd edn. Cold spring harbor laboratory press, cold spring harbor, New York

    Google Scholar 

  • Grant GPG, Boyd N, Herschlag D, Qin PZ (2009) Motions of the substrate recognition duplex in a group I intron assessed by site-directed apin labeling. J Am Chem Soc 131(9):3136–3137. doi:doi:10.1021/ja808217s

    Article  PubMed  CAS  Google Scholar 

  • Grant GPG, Qin PZ (2007) A facile method for attaching nitroxide spin labels at the 5′ terminus of nucleic acids. Nucl Acids Res 35(10):e77

    Article  PubMed  Google Scholar 

  • Hubbell WL, Altenbach C (1994) Investigation of structure and dynamics in membrane proteins using site-directed spin labeling. Curr Opin Struct Biol 4:566–573

    Article  CAS  Google Scholar 

  • Hunsicker-Wang L, Vogt M, Derose VJ (2009) EPR methods to study specific metal-ion binding sites in RNA. Methods Enzymol 468:335–367

    Article  PubMed  CAS  Google Scholar 

  • Hustedt EJ, Beth AH (1999) Nitroxide spin-spin interactions: Applications to protein structure and dynamics. Annu Rev Biophys Biomol Struct 28:129–153

    Article  PubMed  CAS  Google Scholar 

  • Hustedt EJ, Beth AH (2000) Structural information from CW-EPR spectra of dipolar coupled nitroxide spin labels. In: Berliner LJ, Eaton GR, Eaton SS (eds) Biological magnetic resonance: Distance measurements in biological systems by EPR, vol 19. Kluwer, New York, pp 155–184

    Chapter  Google Scholar 

  • Jakobsen U, Shelke SA, Vogel S, Sigurdsson ST (2010) Site-directed spin-labeling of nucleic acids by click chemistry: detection of abasic sites in duplex DNA by EPR spectroscopy. J Am Chem Soc 132(30):10424–10428

    Article  PubMed  CAS  Google Scholar 

  • Jeschke G, Polyhach Y (2007) Distance measurements on spin-labelled biomacromolecules by pulsed electron paramagnetic resonance. Phys Chem Chem Phys 9(16):1895–1910. doi:DOI: 10.1039/b614920k

    Article  PubMed  CAS  Google Scholar 

  • Keyes RS, Bobst AM (1998) Spin-labeled nucleic acids. In: Berliner LJ (ed) Biological magnetic resonance, vol 14. Plenum Press, New York, pp 283–338

    Chapter  Google Scholar 

  • Kim N, Murali A, DeRose VJ (2004) A distance ruler for RNA using EPR and site-directed spin labeling. Chem Biol 11:939–948

    Article  PubMed  CAS  Google Scholar 

  • Kim NK, Bowman MK, DeRose VJ (2010) Precise mapping of RNA tertiary structure via ­nanometer distance measurements with double electron–electron resonance spectroscopy. J Am Chem Soc 132(26):8882–8884

    Article  PubMed  CAS  Google Scholar 

  • Kim NK, Murali A, DeRose VJ (2005) Separate metal requirements for loop interactions and catalysis in the extended hammerhead ribozyme. J Am Chem Soc 127:14134–14135

    Article  PubMed  CAS  Google Scholar 

  • Klug CS, Feix JB (2008) Methods and applicants of site-directed spin labeling EPR spectroscopy. Methods Cell Biol 84:617–658

    Article  PubMed  CAS  Google Scholar 

  • Krstic I, Frolow O, Sezer D, Endeward B, Weigand JE, Suess B, Engels JW, Prisner TF (2010) PELDOR spectroscopy reveals preorganization of the neomycin-responsive riboswitch tertiary structure. J Am Chem Soc 132(5):1454–1455

    Article  PubMed  CAS  Google Scholar 

  • Krstic I, Hansel R, Romainczyk O, Engels JW, Dotsch V, Prisner TF (2011) Long-range distance measurements on nucleic acids in cells by pulsed EPR spectroscopy. Angew chem Int Ed 50(22):5070–5074

    Article  CAS  Google Scholar 

  • Leulliot V, Quevillon-Cheruel S, Graille M, van Tilbeurgh H, Leeper TC, Godin KS, Edwards TE, Sigurdsson ST, Rozenkrants N, Nagel RJ, Ares MJ, Varani G (2004) A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1p RNAse III. EMBO J 23:2468–2477

    Article  PubMed  CAS  Google Scholar 

  • Macosko JC, Pio MS, Tinoco I Jr, Shin Y-K (1999) A novel 5′ displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA. RNA 5:1158–1166

    Article  PubMed  CAS  Google Scholar 

  • Makinen MW, Mustafi D, Kasa S (1998) ENDOR of spin labels for structure determination: from small molecules to enzyme reaction intermediates. In: Berliner LJ (ed) Biological Magnetic Resonance, vol 14, biological magnetic resonance. Springer-verlag New York, LLC, pp 181–249

    Chapter  Google Scholar 

  • Marsh D (1981) Electron spin resonance: Spin labels. Mol Biol Biochem Biophys 31:51–142

    Article  PubMed  CAS  Google Scholar 

  • Mchaourab HS, Lietzow MA, Hideg K, Hubbell WL (1996) Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. Biochemistry 35:7692–7704

    Article  PubMed  CAS  Google Scholar 

  • McHaourab HS, Perozo E (2000) Determination of protein folds and conformational dynamics using spin-labeling EPR spectroscopy. In: Berliner LJ, Eaton SS, Eaton GR (eds) Biol. Mag. Res, vol 19. Kluwer, New York, pp 185–248

    Google Scholar 

  • Miller TR, Alley SC, Reese AW, Solomon MS, McCallister WV, Mailer C, Robinson BH, Hopkins PB (1995) A probe for sequence-dependent nucleic acid dynamics. J Am Chem Soc 117(36):9377–9378

    Article  CAS  Google Scholar 

  • Moro G, Freed JH (1980) Efficient computation of magnetic resonance spectra and related correlation functions from stochastic Liouville equations. J Phys Chem 84:2837–2840

    Article  CAS  Google Scholar 

  • Nguyen P, Qin PZ (2012) RNA dynamics: perspectives from spin labels. WIREs: RNA 3:62–72

    Article  CAS  Google Scholar 

  • Piton N, Mu Y, Stock G, Prisner TF, Schiemann O, Engels JW (2007) Base-specific spin-labeling of RNA for structure determination. Nucl Acids Res 35(9):3128–3143

    Article  PubMed  CAS  Google Scholar 

  • Price EA, Sutch BT, Cai Q, Qin PZ, Haworth IS (2007) Computation of nitroxide-nitroxide distances for spin-labeled DNA duplexes. Biopolymers 87:40–50

    Article  PubMed  CAS  Google Scholar 

  • Qin PZ, Butcher SE, Feigon J, Hubbell WL (2001) Quantitative analysis of the GAAA tetraloop/receptor interaction in solution: A site-directed spin labeling study. Biochemistry 40:6929–6936

    Article  PubMed  CAS  Google Scholar 

  • Qin PZ, Dieckmann T (2004) Application of NMR and EPR methods to the study of RNA. Curr Opin Struct Biol 14:350–359

    Article  PubMed  CAS  Google Scholar 

  • Qin PZ, Feigon J, Hubbell WL (2005) Site-directed spin labeling studies reveal solution conformational changes in a GAAA tetraloop receptor upon Mg2+-dependent docking of a GAAA tetraloop. J Mol Biol 351:1–8

    Article  PubMed  CAS  Google Scholar 

  • Qin PZ, Haworth IS, Cai Q, Kusnetzow AK, Grant GPG, Price EA, Sowa GZ, Popova A, Herreros B, He H (2007) Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe. Nat Protocols 2(10):2354–2365

    Article  CAS  Google Scholar 

  • Qin PZ, Hideg K, Feigon J, Hubbell WL (2003) Monitoring RNA base structure and dynamics using site-directed spin labeling. Biochemistry 42:6772–6783

    Article  PubMed  CAS  Google Scholar 

  • Ramos A, Varani G (1998) A new method to detect long-range protein-RNA contacts: NMR detection of electron-proton relaxation induced by nitroxide spin-labeled RNA. J Am Chem Soc 120:10992–10993

    Article  CAS  Google Scholar 

  • Schiemann O (2009) Mapping Global Folds of Oligonucleotides by Pulsed Electron–electron Double Resonance. Methods Enzymol 469:329–351

    Article  PubMed  CAS  Google Scholar 

  • Schiemann O, Cekan P, Margraf D, Prisner TF, Sigurdsson ST (2009) Relative orientation of rigid nitroxides by PELDOR: beyond distance measurements in nucleic acids. Angew Chem Int Ed 48(18):3292–3295

    Article  CAS  Google Scholar 

  • Schiemann O, Piton N, Mu Y, Stock G, Engels JW, Prisner TF (2004) A PELDOR based nanometer distance ruler for oligonucleotides. J Am Chem Soc 126:5722–5729

    Article  PubMed  CAS  Google Scholar 

  • Schiemann O, Piton N, Plackmeyer J, Bode BE, Prisner TF, Engels JW (2007) Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances. Nat Protoc 2(4):904–923

    Article  PubMed  CAS  Google Scholar 

  • Schiemann O, Prisner TF (2007) Long-range distance determinations in biomacromolecules by EPR spectroscopy. Q Rev Biophys 40:1–53

    Article  PubMed  CAS  Google Scholar 

  • Schiemann O, Weber A, Edwards TE, Prisner TF, Sigurdsson ST (2003) Nanometer distance ­measurements on RNA using PELDOR. J Am Chem Soc 125:3334–3335

    Article  Google Scholar 

  • Schweiger A, Jeschke G (2001) Principles of pulse electron paramagnetic resonance. Oxford University Press, Oxford

    Google Scholar 

  • Scott WG, Martick M, Chi Y-I (2009) Structure and function of regulatory RNA elements: Ribozymes that regulate gene expression. Biochimica et Biophysica Acta (BBA) 1789(9–10):634–641

    Article  CAS  Google Scholar 

  • Serganov A (2009) The long and the short of riboswitches. Curr Opin Struct Biol 19(3):251–259

    Article  PubMed  CAS  Google Scholar 

  • Shelke SA, Sigurdsson ST (2010) Noncovalent and site-directed spin labeling of nucleic acids. Angew Chem Int Ed 49(43):7984–7986

    Article  CAS  Google Scholar 

  • Shin YK, Hubbell WL (1992) Determination of electrostatic potentials at biological interfaces using electron–electron double resonance. Biophys J 61:1443–1453

    Article  PubMed  CAS  Google Scholar 

  • Sicoli G, Wachowius F, Bennati M, Hobartner C (2010) Probing secondary structures of spin-labeled RNA by pulsed EPR spectroscopy. Angew Chem Int Ed 49(36):6443–6447

    Article  CAS  Google Scholar 

  • Sigurdsson ST (2011) EPR spectroscopy for the study of RNA-ligand interactions. In: Wanunu M, Tor Y (eds.), Methods for Studying Nucleic Acid/Drug Interactions. Francis and Taylor, 223–240

    Google Scholar 

  • Sowa GZ, Qin PZ (2008) Site-directed spin labeling studies on nucleic acid structure and dynamics. Prog Nucleic Acids Res Mol Biol 82:147–197

    Article  CAS  Google Scholar 

  • Spaltenstein A, Robinson BH, Hopkins PB (1988) A rigid and nonperturbing probe for duplex DNA motion. J Am Chem Soc 110(4):1299–1301. doi:10.1021/ja00212a053

    Article  CAS  Google Scholar 

  • Ward R, Keeble DJ, El-Mkami H, Norman DG (2007) Distance determination in heterogeneous DNA model systems by pulsed EPR. Chembiochem 8:1957–1964

    Article  PubMed  CAS  Google Scholar 

  • Wu Q, Huang L, Zhang Y (2009) The structure and function of catalytic RNAs. Sci China C Life Sci 52(3):232–244

    Article  PubMed  CAS  Google Scholar 

  • Wunnicke D, Strohbach D, Weigand JE, Appel B, Feresin E, Suess B, Muller S, Steinhoff HJ (2011) Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR. RNA 17(1):182–188

    Article  PubMed  CAS  Google Scholar 

  • Xi X, Sun Y, Karim CB, Grigoryants VM, Scholes CP (2008) HIV-1 Nucleocapsid Protein NCp7 and Its RNA Stem Loop 3 Partner: Rotational Dynamics of Spin-Labeled RNA Stem Loop 3. Biochemistry 47(38):10099–10110

    Article  PubMed  CAS  Google Scholar 

  • Xiao W, Shin Y-K (2000) EPR spectroscopic ruler: the method and its applications. In: Berliner LJ, Eaton GR, Eaton SS (eds) Biological magnetic resonance: Distance measurements in biological systems by EPR, vol 19. Kluwer, New York, pp 249–276

    Chapter  Google Scholar 

  • Zhang X, Cekan P, Sigurdsson ST, Qin PZ (2009) Studying RNA using site-directed spin-labeling and continuous-wave electron paramagnetic resonance spectroscopy. Method Enzymol 469:303–328

    Article  CAS  Google Scholar 

  • Zhang X, Lee SW, Zhao L, Xia T, Qin PZ (2010a) Conformational distributions at the N-peptide/boxB RNA interface studied using site-directed spin labeling. RNA 16:2474–2483

    Article  PubMed  CAS  Google Scholar 

  • Zhang X, Tung C-S, Sowa GZ, Hatmal MM, Haworth IS, Qin PZ (2012) Global structure of a three-way junction in a phi29 packaging RNA dimer determined using site-directed spin labeling. J Am Chem Soc 134(5):2644–2652

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z, Fleissner MR, Tipikin DS, Liang Z, Moscicki JK, Earle KA, Hubbell WL, Freed JH (2010b) Multifrequency Electron Spin Resonance Study of the Dynamics of Spin Labeled T4 Lysozyme. J Phys Chem B 114(16):5503–5521

    Article  PubMed  CAS  Google Scholar 

  • Zhang Z, Xi X, Scholes CP, Karim CB (2008) Rotational dynamics of HIV-1 nucleocapsid protein NCp7 as probed by a spin label attached by peptide synthesis. Biopolymers 89(12):1125–1135

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors would like to acknowledge support from the National Institute of Health (GM069557) and the National Science Foundation (MCB 054652).

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  1. Department of Chemistry, University of Southern California, LJS-251, 840 Downey Way, Los Angeles, CA, 90089-0744, USA

    Xiaojun Zhang & Peter Z. Qin

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  1. Xiaojun Zhang
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  2. Peter Z. Qin
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Correspondence to Peter Z. Qin .

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  1. , Dept. of Chemistry and Biochemistry, University of Texas, Austin, 78712, Texas, USA

    Rick Russell

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Zhang, X., Qin, P.Z. (2013). Studying RNA Folding Using Site-Directed Spin Labeling. In: Russell, R. (eds) Biophysics of RNA Folding. Biophysics for the Life Sciences, vol 3. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-4954-6_5

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