Preparation of 2′‐Deoxy‐2′‐Methylseleno‐Modified Phosphoramidites and RNA

Ronald Micura1, Claudia Höbartner1, Renate Rieder1, Christoph Kreutz1, Barbara Puffer1, Kathrin Lang1, Holger Moroder1

1 Leopold‐Franzens University, Innsbruck, Austria
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.15
DOI:  10.1002/0471142700.nc0115s27
Online Posting Date:  January, 2007
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Abstract

The derivatization of nucleic acids with selenium is a useful approach to facilitate phase determination during three‐dimensional structural analysis by X‐ray crystallography. This unit describes (1) the synthesis and characterization of 2′‐deoxy‐2′‐methylseleno (2′‐Se‐methyl) nucleosides and their corresponding 3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite derivatives, (2) the site‐specific incorporation of 2′‐Se‐methyl ribonucleosides into oligoribonucleotides by chemical RNA solid‐phase synthesis, and (3) the enzymatic ligation of Se‐containing RNA oligonucleotides to produce a biologically relevant RNA sequence.

Keywords: selenium; RNA; chemical synthesis; phosphoramidites; enzymatic ligation; X‐ray crystallography

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of 2′‐Deoxy‐2′‐Methylselenouridine Phosphoramidite
  • Alternate Protocol 1: Preparation of 2′‐Deoxy‐2′‐Methylselenocytidine Phosphoramidite
  • Alternate Protocol 2: Preparation of 2′‐Deoxy‐2′‐Methylselenoadenosine Phosphoramidite
  • Alternate Protocol 3: Preparation of 2′‐Deoxy‐2′‐Methylselenoguanosine Phosphoramidite
  • Basic Protocol 2: Synthesis of RNA Oligonucleotides Containing 2′‐Methylseleno Nucleosides
  • Basic Protocol 3: Deprotection, Purification, and Analysis of RNA Oligonucleotides Containing 2′‐Methylseleno Labels
  • Basic Protocol 4: Enzymatic Ligation of Selenium‐Modified RNA Oligonucleotides Using T4 RNA Ligase
  • Alternate Protocol 4: Enzymatic Ligation of Selenium‐Modified RNA Oligonucleotides Using T4 DNA Ligase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of 2′‐Deoxy‐2′‐Methylselenouridine Phosphoramidite

  Materials
  • Diphenyl carbonate (Fluka)
  • N,N‐Dimethylformamide (DMF)
  • Uridine (Fluka)
  • Sodium bicarbonate (NaHCO 3)
  • Methanol (MeOH)
  • Balloon of argon
  • Anhydrous pyridine
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl; Fluka)
  • Dichloromethane (CH 2Cl 2)
  • 5% (w/v) citric acid solution in water
  • Half‐saturated sodium bicarbonate solution in water
  • Sodium sulfate (Na 2SO 4)
  • Silica gel 60 (230 to 400 mesh; Fluka)
  • Triethylamine (TEA)
  • Anisaldehyde reagent (see recipe)
  • Sodium borohydride (NaBH 4; Fluka)
  • Anhydrous tetrahydrofuran (THF)
  • Dimethyl diselenide (Aldrich)
  • 30% (v/v) H 2O 2 solution
  • Anhydrous ethanol
  • 0.2 M triethylammonium acetate (TEAA) buffer, pH 7.0
  • Ethyl acetate
  • Saturated aqueous sodium chloride (NaCl) solution
  • N‐Ethyldimethylamine (Fluka)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (Aldrich)
  • Hexanes
  • Chloroform (CHCl 3)
  • 25‐mL two‐neck round‐bottom flask
  • Reflux condenser
  • Glass‐fritted Büchner funnel (pore size 3)
  • Filter paper (Macherey‐Nagel MN 615)
  • Vacuum oil pump (rotary vane vaccuum pump; for all steps requiring drying in vacuo)
  • 50‐mL one‐neck round‐bottom flasks equipped with rubber septa
  • Rotary evaporator equipped with a membrane pump (for all evaporation steps)
  • 250‐, 500‐, and 1000‐mL separatory funnels
  • 100‐mL two‐neck round‐bottom flasks with rubber septa
  • Glass filter tube
  • 1‐ and 20‐mL syringes and 21‐G needles
  • 500‐µL Hamilton syringe
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Alternate Protocol 1: Preparation of 2′‐Deoxy‐2′‐Methylselenocytidine Phosphoramidite

  • S.3 (see protocol 1)
  • Imidazole (Fluka)
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl; Fluka)
  • 4‐(N,N‐Dimethylamino)pyridine (DMAP; Fluka)
  • 2,4,6‐Triisopropylbenzenesulfonyl chloride (TPS‐Cl; Fluka)
  • Saturated sodium bicarbonate solution in water
  • 32% (v/v) aqueous ammonia
  • Acetic anhydride (Fluka)
  • Tetrabutylammonium fluoride trihydrate (TBAF; Fluka)
  • Acetic acid (AcOH)
  • 25‐mL one‐neck round‐bottom flasks equipped with rubber septa

Alternate Protocol 2: Preparation of 2′‐Deoxy‐2′‐Methylselenoadenosine Phosphoramidite

  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐{[(triisopropylsilyl)oxy]methyl}adenosine (S.11; Pitsch et al., ; unit 2.9)
  • 4‐(N,N‐Dimethylamino)pyridine (DMAP; Fluka)
  • Trifluoromethanesulfonyl chloride (Tf‐Cl; Fluka)
  • Saturated sodium bicarbonate solution in water
  • Anhydrous toluene
  • N‐Ethyldiisopropylamine (Fluka)
  • Potassium trifluoroacetate (Fluka)
  • 18‐Crown‐6‐ether (Fluka)
  • Tetrabutylammonium fluoride trihydrate (TBAF; Fluka)
  • Acetic acid (AcOH)
  • 25‐ and 100‐mL one‐neck round‐bottom flasks equipped with rubber septa

Alternate Protocol 3: Preparation of 2′‐Deoxy‐2′‐Methylselenoguanosine Phosphoramidite

  • 9‐[β‐D‐Arabinofuranosyl]guanine (S.18; Metkinen Oy)
  • 1,3‐Dichloro‐1,1,3,3‐tetraisopropyldisiloxane (TIPSCl 2; Fluka)
  • Acetic anhydride (Fluka)
  • Triphenylphosphine (PPh 3; Fluka)
  • 2‐(4‐Nitrophenyl)ethanol (NPE‐OH; Fluka)
  • Anhydrous dioxane
  • Diisopropyl azodicarboxylate (DIAD; Aldrich)
  • Saturated sodium bicarbonate solution in water
  • 0.5 M sodium hydroxide (NaOH) solution in water
  • Acetic acid
  • 4‐(N,N‐Dimethylamino)pyridine (DMAP; Fluka)
  • Trifluoromethanesulfonyl chloride (Tf‐Cl; Fluka)
  • Tetrabutylammonium fluoride trihydrate (TBAF; Fluka)
  • 250‐ and 500‐mL one‐neck round‐bottom flasks
  • 500‐, 1000‐, and 2000‐mL separatory funnels

Basic Protocol 2: Synthesis of RNA Oligonucleotides Containing 2′‐Methylseleno Nucleosides

  Materials
  • 2′‐Methylseleno phosphoramidites (S.4, S.10, S.17, S.27; see protocol 1 and Alternate Protocols protocol 21 through protocol 43)
  • 2′‐O‐TOM‐phosphoramidites (Glen Research; also see units 2.9& 3.8)
  • 5‐Benzylthio‐1H‐tetrazole (BTT; Glen Research)
  • Anhydrous acetonitrile (CH 3CN; <30 ppm water; Biosolve)
  • 4‐Å molecular sieves (optional)
  • Detritylation solution (see recipe)
  • Oxidation solution (see recipe)
  • Capping solutions A and B (see recipes)
  • 100 mM DTT solution (see recipe)
  • 1,2‐Dichloroethane (Fluka)
  • Solid support: e.g., long‐chain alkylamine controlled‐pore glass (LCAA‐CPG; 500 Å for <40 nt; 1000 Å for >40 nt) derivatized with 5′‐O‐(4,4′‐dimethoxytritylated) nucleosides (Glen Research) or polystyrene base supports (PS200; GE Biosciences)
  • Automated DNA synthesizer (e.g., Gene Assembler; Amersham Pharmacia Biotech)
  • Syringes
  • Synthesis column for 1‐µmol scale
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C)

Basic Protocol 3: Deprotection, Purification, and Analysis of RNA Oligonucleotides Containing 2′‐Methylseleno Labels

  Materials
  • Se‐modified, protected oligoribonucleotide attached to the solid support (see protocol 5)
  • 150 mM and 2 M DTT solutions (see recipe)
  • 8 M methylamine in ethanol (Fluka)
  • 40% (v/v) aqueous methylamine (Fluka)
  • 50% (v/v) ethanol in water
  • 1 M tetrabutylammonium fluoride trihydrate (TBAF⋅3H 2O; Fluka) in dry tetrahydrofuran (THF)
  • N‐Methylpyrrolidone (NMP) or N,N‐dimethylformamide (DMF; optional)
  • 1 M triethylammonium acetate (TEAA) buffer, pH 7.4, or 1 M Tris⋅Cl buffer, pH 7.4 (RNase‐free, sterile; Fluka, for molecular biology)
  • Eluant A: 25 mM Tris⋅Cl (pH 8.0; appendix 2A)/6 M urea
  • Eluant B: 25 mM Tris⋅Cl (pH 8.0)/0.5 M NaClO 4/6 M urea
  • 0.1 M triethylammonium bicarbonate (TEAB) buffer
  • Acetonitrile (CH 3CN)
  • Ethylenediaminetetraacetic acid (EDTA)
  • Eluant C: 8.6 mM triethylamine (TEA)/100 mM 1,1,1,3,3,3‐hexafluoroisopropanol (HFIP) in H 2O, pH 8.3
  • Eluant D: MeOH
  • 1.5‐ or 2.0‐mL microcentrifuge tubes (twist‐top and/or screw‐cap)
  • 10‐mL one‐neck round‐bottom flasks
  • Rotary evaporator
  • Speedvac evaporator
  • HPLC system (e.g., Amersham ÄKTAprime or ÄKTApurifier system) with:
    • 2.6 × 10–cm Amersham HiPrep 26/10 desalting column (Sephadex G25)
    • 4 × 250– and 9 × 250–mm Dionex NucleoPac PA‐100 or 200 columns
  • C18 SepPak cartridges (Waters/Millipore)
  • Lyophilizer
  • LC‐ESI mass spectrometer (e.g., Finnigan LCQ Advantage MAX ion trap)
  • 2.1 × 100–mm Amersham µRPC C2/C18 column

Basic Protocol 4: Enzymatic Ligation of Selenium‐Modified RNA Oligonucleotides Using T4 RNA Ligase

  Materials
  • 2′‐Methylseleno‐modified RNA oligonucleotide (see protocol 6)
  • T4 RNA ligase 1 (ssRNA ligase, 20,000 U/mL) and 10× ligation buffer (New England Biolabs)
  • Phenol (water‐saturated)
  • Chloroform
  • Isoamyl alcohol
  • 1.5‐mL screw‐cap vials
  • 21° and 90°C heating block
  • HPLC and LC‐ESI‐MS systems (see protocol 6)

Alternate Protocol 4: Enzymatic Ligation of Selenium‐Modified RNA Oligonucleotides Using T4 DNA Ligase

  • 2′‐O‐Methyl RNA oligonucleotide splint
  • T4 DNA ligase (5 U/µL) and 10× ligation buffer (Fermentas)
  • 50% (w/v) polyethylene glycol (PEG) 4000 (Fermentas)
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Figures

Videos

Literature Cited

Literature Cited
   Adams, P.L., Stahley, M.R., Kosek, A.B., Wang, J., and Strobel, S.A. 2004. Crystal structure of a self‐splicing group I intron with both exons. Nature 430:45‐50.
   Arn, E.A. and Abelson, J. 1998. RNA ligases: Function, mechanism, and sequence conservation. In RNA Structure and Function (R.W. Simons and M. Grunberg‐Manago, eds.) pp. 695‐726. CSHL Press, Cold Spring Harbor, N.Y.
   Du, Q., Carrasco, N., Teplova, M., Wilds, C.J., Egli, M., and Huang, Z. 2002. Internal derivatization of oligoribonucleotides with selenium for X‐ray crystallography using MAD. J. Am. Chem. Soc. 124:24‐25.
   Ennifar, E., Carpentier, P., Ferrer, J.L., Walter, P., and Dumas, P. 2002. X‐ray‐induced debromination of nucleic acids at the Br K absorption edge and implications for MAD phasing. Acta Crystallogr. D Biol. Crystallogr. 58:1262‐1268.
   Gott, J.M., Wu, H., Koch, T.H., and Uhlenbeck, O.C. 1991. A specific, UV‐induced RNA‐protein cross‐link using 5‐bromouridine‐substituted RNA. Biochemistry 30:6290‐6295.
   Höbartner, C. and Micura, R. 2004. The chemical synthesis of selenium‐modified oligoribonucleotides and their enzymatic ligation leading to an U6 snRNA stem‐loop segment. J. Am. Chem. Soc. 126:1141‐1149.
   Höbartner, C., Rieder, R., Kreutz, C., Puffer, B., Lang, K., Polonskaia, A., Serganov, A., and Micura, R. 2005. Combined chemical and enzymatic syntheses of RNAs with up to 100 nucleotides containing site‐specific 2′‐Se‐methyl labels for use in X‐ray crystallography. J. Am. Chem. Soc. 127:12035‐12045.
   Moroder, H., Kreutz, C., Lang, K., Serganov, A., and Micura, R. 2006. Synthesis, oxidation behavior, crystallization and structure of 2′‐methylseleno guanosine containing RNAs. J. Am. Chem. Soc. 128:9909‐9918.
   Pitsch, S., Weiss, P.A., Jenny, L., Stutz, A., and Wu, X. 2001. Reliable chemical synthesis of oligoribonucleotides (RNA) with 2′‐O‐[(triisopropylsilyl)oxy]methyl (2′‐O‐TOM) protected phosphoramidites. Helv. Chim. Acta 84:3773‐3795.
   Serganov, A., Keiper, S., Malinina, L., Tereshko, V., Skripkin, E., Höbartner, C., Polonskaia, A., Phan, A.T., Wombacher, R., Micura, R., Dauter, Z., Jaschke, A., and Patel, D.J. 2005. Structural basis for Diels‐Alder ribozyme catalyzed carbon‐carbon bond formation. Nat. Struct. Mol. Biol. 12:218‐224.
   Teplova, M., Wilds, C.J., Wawrzak, Z., Tereshko, V., Du, Q., Carrasco, N., Huang, Z., and Egli, M. 2002. Covalent incorporation of selenium into oligonucleotides for X‐ray crystal structure determination via MAD: Proof of principle. Biochimie 84:849‐858.
   Wilds, C.J., Pattanayek, R., Pan, C., Wawrzak, Z., and Egli, M. 2002. Selenium‐assisted nucleic acid crystallography: Use of phosphoroselenoates for MAD phasing of a DNA structure. J. Am. Chem. Soc. 124:14910‐14916.
   Xu, Y. and Sugiyama, H. 2004. Highly efficient photochemical 2′‐deoxyribonolactone formation at the diagonal loop of a 5‐iodouracil‐containing antiparallel G‐quartet. J. Am. Chem. Soc. 126:6274‐6279.
   Zeng, Y. and Wang, Y. 2004. Facile formation of an intrastrand cross‐link lesion between cytosine and guanine upon pyrex‐filtered UV light irradiation of d(BrCG) and duplex DNA containing 5‐bromocytosine. J. Am. Chem. Soc. 126:6552‐6553.
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