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Site-Directed Spin Labeling of RNA for Distance Measurements by EPR

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Chemical Biology of Nucleic Acids

Part of the book series: RNA Technologies ((RNATECHN))

Abstract

Electron paramagnetic resonance (EPR) is a structural method and can in addition to X-ray, nuclear magnetic resonance (NMR), and FRET elucidate the structure of different macromolecular systems and determine local surroundings of paramagnetic centers in DNA and RNA. This technique permits structural characterization as well as dynamic structural changes of the macromolecular systems. In order to do so free radicals with good stability have to be introduced, most prominently nitroxide radicals. Here, we describe the site-directed spin labeling (SDSL) of DNA and RNA based on the Sonogashira cross-coupling reaction and compare to other methods available. In our method the appropriate building blocks, either 5-iodo-substituted pyrimidine or 2-iodopurine deoxy- or ribo-nucleoside phosphoramidites, were prepared and incorporated by solid-phase synthesis. Following this the protected oligonucleotides were “on column” reacted with the acetylenic nitroxide spin labels and subsequently purified. As a result the so-called “RNA ruler” for distance measurements of double-stranded RNA was developed. We present applications of this technique for DNA duplexes, DNA/RNA hybrids, RNA hairpins, riboswitches, and aptamers in vitro and “in cell.”

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References

  • Bannwarth W, Schmidt D (1994) Oligonucleotides containing spin-labeled 2′-deoxycytidine and 5-methyl-2′-deoxycytidine as probes for structural motifs of DNA. Bioorg Med Chem Lett 4:977–980

    Article  CAS  Google Scholar 

  • Barhate N, Cekan P, Massey AP et al (2007) A nucleoside that contains a rigid nitroxide spin label: a fluorophore in disguise. Angew Chem Int Ed 46:2655–2658

    Article  CAS  Google Scholar 

  • Bats JW, Frolow O, Engels JW (2009) 4-Ethynyl-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine N-oxide. Acta Crystallogr E 65:o529

    Article  CAS  Google Scholar 

  • Beaucage SL, Caruthers MH (1981) Deoxynucleoside phosphoramidites - a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett 22:1859–1862

    Article  CAS  Google Scholar 

  • Beaucage SL, Iyer RP (1992) Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 48:2223–2311

    Article  CAS  Google Scholar 

  • Böhme S, Steinhoff HJ, Klare JP (2010) Accessing the distance range of interest in biomolecules: site-directed spin labeling and DEER spectroscopy. Spectrosc Int J 24:283–288

    Article  Google Scholar 

  • Buttner L, Seikowski J, Wawrzyniak K et al (2013) Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation. Bioorg Med Chem 21:6171–6180

    Article  PubMed  Google Scholar 

  • Cai Q, Kusnetzow AK, Hideg K et al (2007) Nanometer distance measurements in RNA using site-directed spin Labeling. Biophys J 93:2110–2117

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cheong C, Varani G, Tinoco IJ (1990) Solution structure of an unusually stable RNAhairpin, 5′GGAC(UUCG)GUCC. Nature 346:680–682

    Article  CAS  PubMed  Google Scholar 

  • Davis RH (1995) Large-scale oligoribonucleotide production. Curr Opin Biotechnol 6:213–217

    Article  CAS  PubMed  Google Scholar 

  • Dellinger DJ, Timar Z, Myerson J et al (2011) Streamlined process for the chemical synthesis of RNA using 2′-O-thionocarbamate-protected nucleoside phosphoramidites in the solid phase. J Am Chem Soc 133:11540–11556

    Article  CAS  PubMed  Google Scholar 

  • Ding P, Wunnicke D, Steinhoff HJ et al (2010) Site-directed spin-labeling of DNA by the azide-alkyne ‘click’ reaction: nanometer distance measurements on 7-deaza-2′-deoxyadenosine and 2′-deoxyuridine nitroxide conjugates spatially separated or linked to a ‘dA-dT’ base pair. Chem Eur J 16:14385–14396

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Ehrenberg M (2009) The nobel prize in chemistry 2009 - advanced information. http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2009/advanced.html

  • Frolow O, Bode BE, Engels JW (2007) The synthesis of EPR differentiable spinlabels and their coupling to uridine. Nucleosides Nucleotides Nucleic Acids 26:655–659

    Google Scholar 

  • Frolow O, Bats JW, Engels JW (2009) 3-ethynyl-2,2,5,5-tetramethyl-1-oxyl-3-pyrroline. Acta Crystallogr E 65:o1848

    Article  CAS  Google Scholar 

  • Gannett PM, Darian E, Powell JH et al (2001) A short procedure for synthesis of 4-ethynyl-2,2,6,6-tetramethyl-3,4-dehydro-piperidine-1-oxyl nitroxide. Syn Commun 31:2137–2141

    Article  CAS  Google Scholar 

  • Gesteland RF, Cech TR, Atkins JF (eds) (2006) The RNA world, 3rd edn. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  • Giordano C, Fratini F, Attanasio D et al (2001) Preparation of spin-labeled 2-amino-dA, dA, dC and 5-methyl-dC phosphoramidites for the automatic synthesis of EPR active oligonucleotides. Synth Stuttg 2001:565–572

    Article  Google Scholar 

  • Godt A, Franzen C, Veit S et al (2000) EPR probes with well-defined, long distances between two or three unpaired electrons. J Org Chem 65:7575–7582

    Article  CAS  PubMed  Google Scholar 

  • Grinstaff MW, Khan SI (1999) Palladium(0)-catalyzed modification of oligonucleotides during automated solid-phase synthesis. J Am Chem Soc 121:4704–4705

    Article  Google Scholar 

  • Höbartner C, Wachowius F (2010) Chemical synthesis of modified RNA. In: Mayer G (ed) The chemical biology of nucleic acids, vol 1. Wiley, Chichester, UK, pp 1–37

    Chapter  Google Scholar 

  • Höbartner C, Sicoli G, Wachowius F et al (2012) Synthesis and characterization of RNA containing a rigid and nonperturbing cytidine-derived spin label. J Org Chem 77:7749–7754

    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 

  • Hubbell WL, Cafiso DS, Altenbach C (2000) Identifying conformational changes with site-directed spin labeling. Nat Struct Biol 7:735–739

    Article  CAS  PubMed  Google Scholar 

  • Hubbell WL, Lopez CJ, Altenbach C et al (2013) Technological advances in site-directed spin labelling of proteins. Curr Opin Struct Biol 23:1–9

    Article  Google Scholar 

  • Jakobsen U, Shelke SA, Vogel S et al (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 132:10424–10428

    Article  CAS  PubMed  Google Scholar 

  • Jeschke G, Pannier M, Godt A et al (2000) Dipolar spectroscopy and spin alignment in electron paramagnetic resonance. Chem Phys Lett 331:243–252

    Article  CAS  Google Scholar 

  • Jeschke G, Zimmermann H, Godt A (2006) Isotope selection in distance measurements between nitroxides. J Magn Reson 180:137–146

    Article  CAS  PubMed  Google Scholar 

  • Klose D, Klare JP, Grohmann D et al (2012) Simulation vs. reality: a comparison of in silico distance predictions with DEER and FRET measurements. PLoS ONE 7:e39492

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Krstić I, Frolow O, Sezer D et al (2010) PELDOR spectroscopy reveals preorganization of the neomycin-responsive riboswitch tertiary structure. J Am Chem Soc 132:1454–1455

    Article  PubMed  Google Scholar 

  • Krstić I, Hänsel R, Romainczyk O et al (2011) Long-range distance measurements on nucleic acids in cells by pulsed EPR spectroscopy. Angew Chem Int Ed 50:5070–5074

    Article  Google Scholar 

  • Krstić I, Endeward B, Margraf D et al (2012) Structure and dynamics of nucleic acids. Top Curr Chem 321:159–198

    Article  PubMed  Google Scholar 

  • Mandal M, Breaker RR (2004) Gene regulation by riboswitches. Nat Rev Mol Cell Biol 5:451–463

    Article  CAS  PubMed  Google Scholar 

  • Milov AD, Salikohov KM, Shirov MD (1981) Application of endor in electron-spin echo for paramagnetic center space distribution in solids. Sov Phys Solid State 23:975–982

    CAS  Google Scholar 

  • Muth A, Engels JW (1995) Force field calculations of RNA-tetraloops. J Mol Model 1:97–98

    Google Scholar 

  • Nozinovic S, Fürtig B, Jonker HRA et al (2010) High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA. Nucleic Acids Res 38:683–694

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Piton N, Schiemann O, Mu YG et al (2005) Synthesis of spin-labeled RNAs for long range distance measurements by PELDOR. Nucleosides Nucleotides Nucleic Acids 24:771–775

    Article  CAS  PubMed  Google Scholar 

  • Piton N, Mu YG, Stock G et al (2007) Base-specific spin-labeling of RNA for structure determination. Nucleic Acids Res 35:3128–3143

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pitsch S, Weiss PA, Jenny L et al (2001) Reliable chemical synthesis of oligoribonucleotides (RNA) with 2′-O-[(triisopropylsilyl)oxy]methyl(2′-O-tom)-protected phosphoramidites. Helv Chim Acta 84:3773–3795

    Article  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  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Rist M, Amann N, Wagenknecht HA (2003) Preparation of 1-ethynylpyrene-modified DNA via Sonogashira-type solid-phase couplings and characterization of the fluorescence properties for electron-transfer studies. Eur J Org Chem 2003:2498–2504

    Article  Google Scholar 

  • Romainczyk O, Endeward B, Prisner TF et al (2011) The RNA-DNA hybrid structure determined by EPR, CD and RNase H1. Mol Biosyst 7:1050–1052

    Article  CAS  PubMed  Google Scholar 

  • Romainczyk O, Elduque X, Engels JW (2012) Attachment of nitroxide spin labels to nucleic acids for EPR. Curr Protoc Nucleic Acid Chem 49:7.17.1–7.17.40

    Google Scholar 

  • Scaringe SA, Wincott FE, Caruthers MH (1998) Novel RNA synthesis method using 5′-O-silyl-2′-O-orthoester protecting groups. J Am Chem Soc 120:11820–11821

    Article  CAS  Google Scholar 

  • Scaringe SA, Kitchen D, Kaiser R et al (2004) Preparation of 5′-silyl-2′-orthoester ribonucleosides for use in oligoribonucleotide synthesis. Curr Protoc Nucleic Acid Chem 16:2.10.1–2.10.16

    Google Scholar 

  • Schiemann O (2012) EPR-spektroskopie an biologischen systemen. In: Lottspeich F, Engels JW (eds) Bioanalytik, 3rd edn. Springer, Berlin, pp 509–526

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Schiemann O, Weber A, Edwards TE et al (2003) Nanometer distance measurements on RNA using PELDOR. J Am Chem Soc 125:3434–3435

    Article  CAS  PubMed  Google Scholar 

  • Schiemann O, Piton N, Mu YG et al (2004) A PELDOR-based nanometer distance ruler for oligonucleotides. J Am Chem Soc 126:5722–5729

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Google Scholar 

  • Shelke SA, Sigurdsson ST (2012) Site-directed spin labelling of nucleic acids. Eur J Org Chem 2012:2291–2301

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Somoza A (2008) Protecting groups for RNA synthesis: an increasing need for selective preparative methods. Chem Soc Rev 37:2668–2675

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Sprinzl M, Kramer E, Stehlik D (1974) Structure of phenylalanine transfer-RNA from yeast - spin-label studies. Eur J Biochem 49:595–605

    Article  CAS  PubMed  Google Scholar 

  • Strube T, Schiemann O, MacMillan F et al (2001) A new facile method for spin-labeling of oligonucleotides. Nucleosides Nucleotides Nucleic Acids 20:1271–1274

    Article  CAS  PubMed  Google Scholar 

  • Usman N, Pon RT, Ogilvie KK (1985) Preparation of ribonucleoside 3′-O-phosphoramidites and their application to the automated solid-phase synthesis of oligonucleotides. Tetrahedron Lett 26:4567–4570

    Article  CAS  Google Scholar 

  • Usman N, Ogilvie KK, Jiang MY et al (1987) Automated chemical synthesis of long oligoribonucleotides using 2′-O-silylated ribonucleoside 3′-O-phosphoramidites on a controlled-pore glass support - synthesis of a 43-nucleotide sequence similar to the 3′-half molecule of an Escherichia coli formylmethionine transfer-RNA. J Am Chem Soc 109:7845–7854

    Article  CAS  Google Scholar 

  • Wautelet P, Le Moigne J, Videva V et al (2003) Spin exchange interaction through phenylene-ethynylene bridge in diradicals based on iminonitroxide and nitronylnitroxide radical derivatives. 1. Experimental investigation of the through-bond spin exchange coupling. J Org Chem 68:8025–8036

    Article  CAS  PubMed  Google Scholar 

  • Weigand JE, Sanchez M, Gunnesch E-B et al (2008) Screening for engineered neomycin riboswitches that control translation initiation. RNA 14:89–97

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Woese CR, Winker S, Gutell RR (1990) Architecture of ribosomal RNA: constraints on the sequence of “tetra-loops”. Proc Natl Acad Sci USA 87:8467–8471

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wu XL, Pitsch S (1998) Synthesis and pairing properties of oligoribonucleotide analogues containing a metal-binding site attached to beta-D-allofuranosyl cytosine. Nucleic Acids Res 26:4315–4323

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wurm JP, Meyer B, Bahr U et al (2010) The ribosome assembly factor Nep1 responsible for Bowen–Conradi syndrome is a pseudouridine-N1-specific methyltransferase. Nucleic Acids Res 38:2387–2398

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhang Q, Stelzer AC, Fisher CK et al (2007) Visualizing spatially correlated dynamics that directs RNA conformational transitions. Nature 450:1263–1267

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Institute of Organic Chemistry and Chemical Biology, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt am Main, Germany

    Joachim W. Engels, Christian Grünewald & Lena Wicke

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  1. Joachim W. Engels
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  2. Christian Grünewald
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  3. Lena Wicke
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Correspondence to Joachim W. Engels .

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Editors and Affiliations

  1. Institute of Chemistry/Biochemistry, Free University Berlin, Berlin, Germany

    Volker A. Erdmann

  2. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland

    Wojciech T. Markiewicz

  3. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland

    Jan Barciszewski

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Engels, J.W., Grünewald, C., Wicke, L. (2014). Site-Directed Spin Labeling of RNA for Distance Measurements by EPR. In: Erdmann, V., Markiewicz, W., Barciszewski, J. (eds) Chemical Biology of Nucleic Acids. RNA Technologies. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-54452-1_21

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