Attachment of Nitroxide Spin Labels to Nucleic Acids for EPR

Olga Romainczyk1, Xavier Elduque2, Joachim W. Engels1

1 Goethe‐Universität, Frankfurt, Germany, 2 Universitat de Barcelona, Barcelona, Spain
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.17
DOI:  10.1002/0471142700.nc0717s49
Online Posting Date:  June, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

In addition to X‐ray, NMR, and FRET, electron paramagnetic resonance (EPR) can be applied to 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 macromolecular systems. To do so, free radicals with good stability must be introduced. Here, the site‐directed spin labeling of DNA and RNA based on the Sonogashira cross‐coupling reaction is described. First, the appropriate building blocks, either 5‐iodo‐substituted pyrimidine deoxy‐ or ribo‐nucleoside phosphoramidites are prepared and incorporated by solid‐phase synthesis. Following this, the protected oligonucleotides are “on column” reacted with the acetylenic nitroxide spin labels and subsequently purified. Applications of this technique for duplexes, hairpins, and riboswitches in vitro and in cell are described. Curr. Protoc. Nucleic Acid Chem. 49:7.17.1‐7.17.40. © 2012 by John Wiley & Sons, Inc.

Keywords: site‐directed spin labeling (SDSL); nitroxide; on‐column synthesis; Sonogashira cross coupling

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 15N‐Marked Spin Label TPA
  • Basic Protocol 2: Synthesis of DNA Building Blocks for Spin Label Attachment
  • Basic Protocol 3: Synthesis of RNA Building Blocks for Spin Label Attachment
  • Basic Protocol 4: DNA Synthesis without Interruption
  • Basic Protocol 5: RNA Synthesis without Interuption
  • Alternate Protocol 1: RNA Synthesis with Interruption
  • Basic Protocol 6: Sonogashira Cross‐Coupling Reaction on the Solid Phase
  • Basic Protocol 7: Probe Preparation and EPR Measurements
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Synthesis of 15N‐Marked Spin Label TPA

  Materials
  • Sodium carbonate (Na 2CO 3)
  • Magnesium oxide (MgO)
  • Ammonium chloride 15N (NH 4Cl; 99%; Cambridge Isotope Laboratories)
  • Acetone
  • Silica gel (grade 60, 70 to 230 mesh, 60 Å; Sigma‐Aldrich)
  • Ethyl acetate (EtOAc; p.a.; Merck)
  • Methanol (MeOH)
  • n‐Hexane (p.a.; Merck), 4°C
  • Acetic acid (AcOH)
  • Bromine (Br 2)
  • Concentrated aqueous ammonia
  • KOH (sat. aqueous solution)
  • Ethanol (EtOH)
  • Methyltrioxorhenium (Sigma‐Aldrich)
  • Hydrogen peroxide (H 2O 2)
  • Chloroform
  • Anhydrous sodium sulfate (Na 2SO 4)
  • 10% aqueous NaOH
  • 2 M HCl
  • Dichloromethane (CH 2Cl 2 or DCM)
  • Disiopropylethylamine (DIPEA)
  • Argon source
  • Hydroxybenzotriazole (HOBt)
  • Diisopropylcarbodiimide (DIC)
  • N,O‐Dimethylhydroxylamine hydrochloride (Sigma‐Aldrich)
  • Tetrahydrofurane (absolute, puriss., THF; over molecular sieve; Fluka)
  • Liquid nitrogen
  • DIBAL‐H solution (1 M in hexane)
  • Ammonium chloride
  • Celite
  • Heptane
  • (Chloromethyl)triphenylphosphonium chloride (Sigma‐Aldrich)
  • n‐Butyllithium in n‐hexane (n‐BuLi, Merck)
  • Diethyl ether (Et 2O; p.a.; Merck)
  • Potassium tert‐butylate (KOtBu)
  • Autoclave
  • Büchner funnel with appropriate round filter papers
  • Vacuum/inert gas manifold
  • Rotary evaporator (Buchi)
  • 250‐mL round‐bottomed flasks equipped
  • Reflux condensers
  • Hotplate and magnetic stirrer
  • 500‐mL three‐necked round‐bottomed flasks
  • 250‐mL, 500‐mL, and 1‐liter extraction funnels
  • Rubber septa
  • 2 3/4‐in. needle and 10‐mL syringe
  • Water baths
  • Glass‐fritted funnels with medium porosity
  • Fluted filter paper
CAUTION:n‐Butyllithium is sensitive to moisture and air, and reacts violently with water, liberating extremely flammable gases (butane, hydrogen). n‐BuLi is pyrophoric (ignites in air). It is spontaneously flammable in air and causes burns. Wearing gloves during transfer of solutions containing n‐butyllithium is highly recommended.

Basic Protocol 2: Synthesis of DNA Building Blocks for Spin Label Attachment

  Materials
  • 3′,5′‐Bis‐O‐(tert‐butyldimethylsilyl)‐2′‐O‐deoxyguanosine (Hayakawa et al., )
  • Dichloromethane (CH 2Cl 2; puriss.; absolute; over molecular sieve; Fluka)
  • Triethylamine, purum (NEt 3; Riedel deHaën)
  • N,N‐Dimethylaminopyridine (DMAP; puriss. >99% ; Fluka)
  • 2,4,6‐Triisopropyl‐benzenesulfonic‐chloride (puriss. >99%; Fluka)
  • Sodium hydrogen carbonate (NaHCO 3; 5% w/w aqueous solution)
  • MgSO 4
  • Silica gel (grade 60, 70 to 230 mesh, 60 Å; Merck; Sigma‐Aldrich)
  • n‐Hexane; (p.a.; Merck)
  • Ethyl acetate (EtOAc; p.a.; Merck)
  • Tetrahydrofurane (THF; puriss.; absolute; over molecular sieve; Fluka)
  • Iodine (I 2; puriss. >99.5%; Fluka)
  • Copper iodide (CuI)
  • CH 2I 2 (puriss. >99%; Fluka)
  • Isopentyl nitrite (purum >97%; Fluka)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Ammonia‐saturated ethanol
  • N,N‐Dimethylformamide‐dimethylacetal (purum >95%; Fluka)
  • Dimethylformamide (DMF; puriss.; p.a. >99.8%; ACS Fluka)
  • Methanol (MeOH)
  • TEA⋅3HF (Fluka)
  • Pyridine (puriss.; absolute; over molecular sieve; Fluka)
  • 4,4′‐Dimethoxy‐trityl chloride (DMTr‐Cl; 98%; Avocado)
  • Toluene
  • DCI
  • 2‐Cyanethoxy‐di‐(di‐N,Niso‐propylamino) phosphine (99%; Aldrich)
  • Brine
  • Acetone
  • 5% TEA
  • Magnetic hotplate and stir bar
  • 250‐mL extraction funnel
  • Rotary evaporator (Buchi)
  • Three‐necked flasks with condensers
  • Rubber septa (+15.9 mm)
  • 500‐mL tight, high‐pressure tubes (schott)
  • Glass‐fritted funnels with medium porosity
  • Fluted filter paper

Basic Protocol 3: Synthesis of RNA Building Blocks for Spin Label Attachment

  Materials
  • Cytidine
  • Pyridine (puriss., absolute; over molecular sieve; Fluka)
  • Markiewicz‐protecting group (C 12H 28OSi 2Cl 2)
  • Toluene
  • Silica gel (grade 60, 70 to 230 mesh, 60 Å; Merck or Sigma‐Aldrich)
  • Ethyl acetate (EtOAc; p.a.; Merck)
  • Methanol (MeOH)
  • N,N,N′,N′‐Tetramethylethylendiamine (TEMED; puriss. 98%; Fluka)
  • N‐Iodosuccinimide (98%; Avocado)
  • Dimethylformamide (puriss.; p.a. >99.8%; ACS Fluka)
  • Dichloromethane (CH 2Cl 2; puriss.; absolute; over molecular sieve; Fluka)
  • Saturated sodium thiosulfate solution
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Isopropanol (i‐Pro)
  • N,N‐Dimethylformamide‐dimethylacetal (purum >95%; Fluka)
  • n‐Hexane (p.a.; Merck)
  • Acetone
  • Tris(2‐acetoxyethyl)orthoformate (Dharmacon)
  • Pyridinium p‐toluenesulfonate (puriss. >99%; Fluka)
  • 4‐tert‐Butyl‐dimethyl‐siloxy‐3‐penten‐2‐one (Fluka)
  • Acetonitrile
  • Hydrofluoric acid
  • Diisopropylamine (DIPA; puriss.; p.a. >99.0%; Fluka)
  • Benzhydroxy‐bis(trimethylsilyloxy)chlorosilane (BzHCl; Dharmacon)
  • Sodium hydrogencarbonate (5% w/w NaHCO 3 aqueous solution)
  • Brine
  • Triethylamine (Et 3N; purum; Riedel deHaeën)
  • Methyl‐N,N,N′,N′‐tetraisopropylphosphordiamidite (puriss.; p.a. >99.0%; Fluka)
  • 1H‐Tetrazole (0.45 M in ACN for DNA synthesis; Fluka)
  • Ethanol
  • Magnetic stir plate and stir bar
  • Rotary evaporator (Buchi)
  • Vacuum/inert gas manifold
  • 50‐ and 100‐mL flask with reflux condenser
  • Rubber septa (+15.9 mm)
  • Glass‐fritted funnels with medium porosity
  • Fluted filter paper
  • Büchner funnel with appropriate round paper filter

Basic Protocol 4: DNA Synthesis without Interruption

  Materials
  • Reagents for synthesis:
    • DNA phosphoramidites of the natural bases and 5‐iodouridine (Applied Biosystems), 5‐iodo‐desoxycytidine, and 2‐iodo‐desoxyadenosine (synthesize in laboratory)
    • Trichloroacetic acid (TCA) in CH 2Cl 2 (Perseptive Biosystems)
    • CAP A (N‐methyl‐imidazole; Proligo Biochemie)
    • CAP B (acetic anhydride; Proligo Biochemie)
    • Activator: dicyanoimidazole (DCI), 0.25 M in acetonitrile (Proligo Biochemie) or tetrazole, 0.45 M in acetonitrile (Biosolve), store over molecular sieve
    • Oxidizer: iodine, 4.3 g in THF/pyridine/water, 7:1:2 v/v/v (Biosolve)
    • Prepacked columns: clear pore glass (CPG) with the first base attached via a succinyl‐linker, 1 mmol, 500 Å; (Applied Biosystems)
    • Phosphate buffer (see recipe)
  • Ammonia (p.a. 32%; Grüssing) mixed with p.a. methanol in a ratio of 3:1
  • Sterile water (Millipore)
  • 0.25 M Tris⋅Cl, pH 8, HPLC‐grade
  • 1 M NaCl, HPLC‐grade
  • PD‐10 Sephadex columns (Amersham Biosciences)
  • 0.1 M triethylammonium acetate (TEAA), pH 7
  • Acetonitrile, HPLC‐grade
  • Oligonucleotide synthesizer (Perseptive Biosystems)
  • 4‐mL vials
  • Filter (Whatman Spartan 13/0.45 RC, 0.24‐µm)
  • Speed‐Vac rotary evaporator (model no. SC110, Savant)
  • Glass‐fritted funnels with medium porosity
  • Fluted filter paper
  • HPLC device (JASCO) in combination with an anion exchange column (DNAPack PA‐100, semi‐prep, 9 × 250–mm; Dionex (flow: 5 mL/min)
  • EXPEDITE synthesizer (Perseptive Biosystems)
  • Reversed‐phase HPLC column (Phenomenex, Jupiter 4µ‐Poter‐90A)

Basic Protocol 5: RNA Synthesis without Interuption

  Materials
  • Solutions for RNA synthesizer:
    • Acetonitrile for DNA synthesis (Biosolve)
    • RNA phosphoramidites of the natural bases and 5‐iodouridine (Dharmacon), 5‐iodocytidine and 2‐iodoadenosine (synthesized in laboratory)
    • Tetrahydrofurane (THF; puriss.; absolute; over molecular sieve; Fluka)
    • CAP A: N‐methylimidazole (redist. 99%; Aldrich), 10% in acetonitrile
    • CAP B: acetic anhydride (puriss. p.a. ACS >99%; Riedel‐de Haën), 10% in acetonitrile
    • 3% dichloroacetic acid (puriss. 99%; Fluka) in absolute CH 2Cl 2 over molecular sieve (Fluka)
    • 1H‐Tetrazole (0.45 M in ACN; Fluka)
    • tBuOOH (purum, 70% in water; Fluka) in toluene (ACS; Fluka) (see recipe)
    • Prepacked columns (aminomethyl‐polystyrene with the first base protected with DMT attached via a succinyl linker, 0.2 mmol; Dharmacon)
  • Ammonia
  • Methanol (MeOH)
  • DEPC‐treated water (see recipe)
  • N‐methylpyrrolidone
  • Triethylamine (Et 3N or TEA, purum, Riedel de Haën; or puriss. p.a. >99.5%; Fluka), dried with calcium hydride (3 to 5 g per 250 mL) by heating to reflux for 3 days and distilled under inert gas before use
  • Triethylamine‐hydrofluoride (Et 3N‐3HF or HF⋅TEA; see recipe)
  • n‐Butanol
  • 1 M LiCl: dissolve 42.4 g LiCl in 1 liter Millipore water
  • PD‐10 Sephadex columns (Amersham Biosciences)
  • 0.4 M disodium‐2‐carbamoyl‐2‐ cyanoethylene‐1,1‐dithiolate‐trihydrate (S 2Na 2; see recipe)
  • N,N‐dimethyl‐formamide (DMF, puriss. p.a. ACS >99.8%; Fluka)
  • 40% methylamine in water (Aldrich)
  • TEMED/acetic acid (see recipe)
  • ABI 392 synthesizer (Applied Biosystems)
  • Magnetic hotplate and stir bar
  • Speed‐Vac (SC110, Savant)
  • Refrigerated centrifuge
  • Filters (0.45‐µm pore size)
  • Buchner funnel with appropriate round paper filter
  • Anion‐exchange HPLC device (JASCO) with an anion‐exchange column (DNAPack PA‐100, semi‐prep, 9 × 250–mm, Dionex), flow 5 mL/min
  • PD‐10 sepharose columns
  • MALDI‐TOF VOYAGER DE‐PRO mass spectrometer (Applied Biosystems)
  • Vacuum manifold
  • 4‐mL vials
CAUTION: HF is extremely toxic and must not be used without prior knowledge. Upon adding HF, a little bit of smoke evolves and the reaction is slightly exothermic. Transfer this solution directly into a 500‐mL polyethylene glycol bottle (Nalgene) and install it immediately onto the synthesizer.

Alternate Protocol 1: RNA Synthesis with Interruption

  Materials
  • RNA/DNA synthesized on column
  • Copper iodide (CuI; >99%; Riedel de Haën)
  • Dichloromethane (CH 2Cl 2; puriss.; absolute; over molecular sieve; Fluka)
  • Triethylamine (Et 3N; purum; Riedel deHaeën)
  • Pd(II)(PPh 3) 2Cl 2 (purum, >98%; Fluka)
  • 2,2,5,5‐Tetramethyl‐pyrrolin‐1‐oxyl‐3‐acetylene (TPA)
  • Vacuum/inert gas manifold
  • 10‐mL one‐necked flasks (pear‐shape or round‐bottom flasks)
  • 1‐mL vials (4‐cm high, +8 mm, Carl Roth, cat. no. H‐300.1)
  • Hotplate
  • Teflon‐coated magnetic stir bars
  • Plastic polypropylene syringes
  • Reusable syringe needles (steel, 150‐mm length, +1.2 mm) and disposable syringe needles (120‐mm length,+0.8 mm)
  • Shaker

Basic Protocol 6: Sonogashira Cross‐Coupling Reaction on the Solid Phase

  Materials
  • Oligonucleotides
  • 10 mM sodium cacodylate
  • 0.2 mM disodium EDTA, pH 7
  • 2.5% formamide solution
  • Diethylpyrocarbonate (DEPC) water (see recipe)
  • Liquid nitrogen
  • 20% aqueous ethylene glycol solution or 20% aqueous sucrose solution
  • Heating block
  • UV‐cuvettes
  • UV‐/VIS‐600 (Jasco) spectrophotometer equipped with a Peltier thermostat
  • JASCO J‐710 spectropolarimeter with a Peltier thermostat
  • Quartz EPR tubes
  • Sterile tips (Eppendorf)
  • 80°C oven
  • Vivaspin concentrator (Sartorius Stedim Biotech)
  • Plastic polypropylene syringes
  • Reusable syringe needles (steel, 150‐mm length, +1.2 mm) and disposable syringe needles (120‐mm length, +0.8 mm)
  • Rotary evaporator (Buchi)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Böhme, S., Steinhoff, H.‐J., and Klare, J.P. 2010. Accessing the distance range of interest in biomolecules: Site‐directed spin labeling and DEER spectroscopy. Spectroscopy 24:283‐288.
   Cheong, C., Varani, G., and Tinoco, I. Jr. 1990. Solution structure of an unusually stable RNA hairpin, 5′GGAC(UUCG)GUCC. Nature 346:680‐682
   Couet, W.R., Brasch, R.C., Sosnovsky, C., Lukszo, J., Prakash, I., Gnewech, C.T., and Tozer, T.N. 1985. Influence of chemical structure of nitroxyl spin labels on their reduction by ascorbic acid. Tetrahedron 41:1165‐1172.
   Dehmlow, E.V. and Lissel, M. 1980. Anwendungen der Phasentransfer‐katalyse, 14. Darstellung von Alkinen aus Alkylhalogeniden mit festem Kalium‐tert‐butylat und Kronenether. Liebigs Ann. Chem. 1:1‐13.
   Dellinger, D.J., Timar, Z., Myerson, J., Sierzchala, A.B., Turner, J., Ferreira, F., Kupihar, Z., Dellinger, G., Hill, K.W., Powell, J.A., Sampson, J.R., and Caruthers, M.H. 2011. Streamlined process for the chemical synthesis of RNA using 2′‐O‐thionocarbamate‐protected nucleoside phosphoramidites in the solid phase. J. Am. Chem. Soc. 113:11540‐11556.
   Ding, P., Wunnicke, D., Steinhoff, H.‐J., and Seela, F. 2010. Site‐directed spin‐labeling of DNA by the azide‐slkyne “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.
   Duchardt‐Ferner, E., Weigand, J.E., Ohlenschläger, O., Schmidtke, S.R., Suess, B., and Wöhnert, J. 2010. Highly modular structure and ligand binding by conformational capture in a minimalistic riboswitch. Angew. Chem. Int. Ed. 49:6216‐6219.
   Frolow, O., Bode, B.E., Prisner, T.F., and Engels, J.W. 2007. The synthesis of EPR differentiable spinlabels and their coupling to uridine. Nucleosides Nucleotides Nucleic Acids 26:655‐659.
   Frolow, O., Bats, J.W., and Engels, J.W. 2009. 3‐Ethynyl‐2,2,5,5‐tetramethyl‐1‐oxyl‐3‐pyrroline. Acta Crystallographica E65:o1848.
   Gannett, P.M., Darian, E., Powell, J.H, and Johnson, E.M. 2001. A short procedure for synthesis of 4‐ethynyl‐2,2,6,6‐tetramethyl‐3,4‐dehydroxy‐piperidine‐1‐oxyl nitroxyde. Synth. Commun. 31:2137‐2141.
   Godt, A., Franzen, C., Veit, S., Enkelmann, V., Pannier, M., and Jeschke, G. 2000. EPR probes with well defined, long distances between two or three unpaired electrons. J. Org. Chem. 65:7575‐7582.
   Groesbeek, M. and Lugtenburg, J. 1995. Synthesis of nitroxide containing polyenes: Two commercially modified retinals and their interaction with bacetriorhodopsin. Recl. Trav. Chim. Pays‐Bas. 114:403‐409.
   Hayakawa, Y., Hirose, M., and Noyori, R. 1993. O‐Allyl protection of guanine and thymine residues in oligodeoxyribonucleotides. J. Org. Chem. 58:5551‐5555.
   Hubbell, W., Cafiso, D.S., and Altenbach, C. 2000. Identifying conformational changes with site‐directed spin labeling. Nature Struct. Biol. 7:735‐739.
   Janeba, Z., Francon, P., and Robins, M.J. 2003. Efficient syntheses of 2‐chloro‐2′‐deoxyadenosine (cladribine) from 2′‐deoxyguanosine. J. Org. Chem. 68:989‐992.
   Jeschke, G., Pannier, M., Godt, A., and Spiess, H.W. 2000. Dipolar spectroscopy and spin alignment in electron paramagnetic resonance. Chem. Phys. Lett. 331:243‐252.
   Jeschke, G., Zimmermann, H., and Godt, A. 2006. Isotope selection in distance measurements between nitroxides J. Magn. Reson. 180:137‐146.
   Kelemen, P., Lugtenburg, J., and Klumperman, B.J. 2003. 15N NMR spectroscopy of labeled alkoxyamines. 15N‐labeled model compounds for nitroxide‐trapping studies in free‐radical (Co)polymerization. J. Org. Chem. 68:7322‐7328.
   Krstic, I., Frolow, O., Sezer, D., Endeward, B., Weigand, J.E., Suess, B., Engels, J.W., and Prisner, T.F. 2010. PELDOR spectroscopy reveals preorganization of the neomycin‐responsive riboswitch tertiary structure. J. Am. Chem. Soc. 132:1454‐1455.
   Krstic, I., Haensel, R., Romainczyk, O., Engels, J.W., Doetsch, V., and Prisner, T.F. 2011. Long‐range distance measurement on nucleic acids in cells by pulsed EPR spectroscopy. Angew. Chem. Int. Ed. 50:6782‐6785
   Margraf, D., Bode, B.E., Marko, A., Schiemann, O., and Prisner, T.F. 2007. Conformational flexibility of nitroxide biradicals determined by X‐band PELDOR experiments. Mol. Phys. 105:2153‐2160.
   Matteucci, M.D. and Caruthers, M.H. 1981. Studies on nucleotide chemistry IV. Synthesis of deoxyoligonucleotides on a polymer support. J. Am. Chem. Soc. 103:3185‐3191.
   Milov, A.D., Salikhov, K.M., and Shirov, M.D. 1981. Application of the double resonance method to electron spin echo in a study of the spatial distribution of paramagnetic centers in solids. Sov. Phys. Solid State 23:975‐982.
   Murray, R.W., Iyanar, K., Chen, J., and Wearing, J. 1996. Oxidation of organonitrogen compounds by the methyltrioxorhenium‐hydrogen peroxide system. Tetrahedron Lett. 37:805‐808.
   Muth, A. and Engels, J.W. 1995. Force field calculations of RNA‐tetraloops. J. Mol. Model. 1:97‐98.
   Nesvadba, P., Bugnon, L., and von Büren, M. 2004. Process for the oxidation of secondary amines into the corresponding nitroxides. Patent no. WO 2004/085397.
   Nozinovic, S., Fürtig, B., Jonker, H.R.A., Richter, C., and Schwalbe, H. 2010. High‐resolution NMR structure of an RNA model system: The 14‐mer cUUCGg tetraloop hairpin RNA. Nucleic Acids Res. 38:683‐694.
   Obeid, S., Yulikov, M., Jeschke, G., and Marx, A. 2008. Enzymatic synthesis of multiple spin‐labeled DNA. Angew. Chem. Int. Ed. 47:6782‐6785.
   Ogilvie, K.K., Sadana, K.L., Thompson, E.A., Qiulliam, M.A., and Westmore, J.B. 1974. The use of silyl groups in protecting the hydroxyl functions of ribonucleosides. Tetrahedron Lett. 33:2861‐2863.
   Piton, N., Schiemann, O., Mu, Y., Stock, G., Prisner, T.F., and Engels, J.W. 2005. Synthesis of spin‐labeled RNAs for long range distance measurements by PELDOR. Nucleosides Nucleotides Nucleic Acids 24:771‐775.
   Piton, N., Mu, Y., Stock, G., Prisner, T.F., Schiemann, O., and Engels, J.W. 2007. Base‐specific spin‐labeling of RNA for structure determination. Nucleic Acids Res. 35:3128‐3143.
   Romainczyk, O., Endeward, B., Prisner, T.F., and Engels, J.W. 2011. RNA‐DNA hybrid structure determined by EPR and enzyme recognition. Mol. Biosyst. 7:1050‐1052.
   Scaringe, S.A. 2000. Orthoester Protecting Groups. U.S. Patent no. 6,111,086.
   Scaringe, S.A., Kitchen, D., Kaiser, R., and Marshall, W.S. 2004. Preparation of 5′‐silyl‐2′‐othoester ribonucleosides for use in oligoribonucleotide synthesis. Curr. Protoc. Nucleic Acid Chem. 16:2.10.1‐2.10.15.
   Schiemann, O. and Prisner, T.F. 2007. Long range distance determinations in biomacromolecules by EPR spectroscopy. Q. Rev. Biophys. 40:1‐53.
   Schiemann, O., Piton, N., Mu, Y., Stock, G., Engels, J.W., and Prisner, T.F. 2004. A PELDOR‐based nanometer distance ruler for oligonucleotides. J. Am. Chem. Soc. 126:5722‐5729.
   Schiemann, O., Piton, N., Plackmeyer, J., Bode, B., Prisner, T., and Engels, J.W. 2007. Spin labeling of oligonucleotides with the nitroxid TPA and use of PELDOR, a pulse EPR method, to measure intermolecular distances. Nat. Protoc. 2:904‐923.
   Sicoli, G., Wachowius, F., Bennati, M., and Höbartner, C. 2010. Probing secondary structures of spin‐labeled RNA by pulsed EPR spectroscopy. Angew. Chem. Int. Ed. 49:6443‐6447.
   Strube, T., Schiemann, O., McMillan, F., Prisner, T., and Engels, J.W. 2001. A new facile method for spin‐labeling of oligonucleotides. Nucleosides Nucleotides Nucleic Acids 20:1271‐1274.
   Weigand, J.E., Sanchez, M., Gunnesch, E.B., Zeiher, S., Schroeder, R., and Suess, B. 2008. Screening for engineered neomycin riboswitches that control translation initiation. RNA 14:89‐97.
   Wautelet, P., Le Moigne, J., Videva, V., and Turek, P. 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.
   Woese, C.R., Winker, S., and Gutell, R.R. 1990. Architecture of ribosomal RNA: Constraints on the sequence of “tetra‐loops”. Proc. Nat. Acad. Sci.U.S.A. 87:8467‐8471.
   Wurm, J.P., Meyer, B., Bahr, U., Held, M., Frolow, O., Kötter, P., Engels, J.W., Heckel, A., Karas, M., Entian, K.D., and Wöhnert, J. 2010. The ribosome assembly factor Nep1 responsible for Bowen‐Conradi syndrome is a pseudouridine‐N1‐specific methyltransferase. Nucleic Acids Res. 38:2387‐2398.
   Yamada, K., Kinoshita, Y., Yamasaki, T., Sadasue, H, Mito, F., Nagai, M., Matsumoto, S., Aso, M., Suemune, H., Sakai, K., and Utsumi, H. 2008. Synthesis of nitroxyl radicals for overhauser‐enhanced magnetic resonance imaging. Arch. Pharm. Chem. Life Sci. 341:548‐553.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library