Oligoribonucleotides with 2′‐O‐(tert‐Butyldimethylsilyl) Groups

Laurent Bellon1

1 Ribozyme Pharmaceuticals, Boulder, Colorado
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.6
DOI:  10.1002/0471142700.nc0306s01
Online Posting Date:  May, 2001
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Abstract

The chemical synthesis of oligoribonucleotides on solid support is routinely performed via the phosphoramidite method. However, the additional 2‐OH function of the ribofuranosyl sugar requires suitable protection during oligoribonucleotide synthesis. This unit describes methods for 2‐OH protection using the TBDMS group.

     
 
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Table of Contents

  • Basic Protocol 1: Automated Oligoribonucleotide Synthesis
  • Basic Protocol 2: Oligoribonucleotide Deprotection with Nh4OH/Ethanol and TBAF
  • Alternate Protocol 1: Oligoribonucleotide Deprotection with Aqueous Methylamine and Triethylamine Trihydrofluoride
  • Alternate Protocol 2: “One‐Pot” Oligoribonucleotide Deprotection with Anhydrous Methylamine and Neat Triethylamine Trihydrofluoride
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Automated Oligoribonucleotide Synthesis

  Materials
  • Aminomethyl polystyrene (RNA primer solid support) derivatized with 5′‐O‐DMTr‐2′‐O‐TBDMS‐3′‐O‐succinyl ribonucleosides (Amersham Pharmacia Biotech)
  • RNA phosphoramidites (Amersham Pharmacia Biotech; diluted on the synthesizer to 0.1 M in acetonitrile, using automated protocols)
  •  5′‐O‐DMTr‐N6‐(phenoxyacetyl)‐2′‐O‐TBDMS‐adenosine‐3′‐O‐(β‐cyanoethyl‐ N,N‐diisopropyl) phosphoramidite
  •  5′‐O‐DMTr‐N2‐(isopropylphenoxyacetyl)‐2′‐O‐TBDMS‐guanosine‐3′‐O‐(β‐ cyanoethyl‐N,N‐diisopropyl) phosphoramidite
  •  5′‐O‐DMTr‐N4‐(acetyl)‐2′‐O‐TBDMS‐cytidine‐3′‐O‐(β‐cyanoethyl‐N,N‐ diisopropyl) phosphoramidite
  •  5′‐O‐DMTr‐2′‐O‐TBDMS‐uridine‐3′‐O‐(β‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite.
  • 3% (v/v) TCA in methylene chloride (PE Biosystems)
  • Cap A: 10% (v/v) acetic anhydride/10% (v/v) 2,6‐lutidine in THF (PE Biosystems)
  • Cap B: 16% (v/v) 1‐methyl imidazole in THF (PE Biosystems)
  • Iodine solution: 16.9 mM I 2/49 mM pyridine/9% (v/v) water in THF (PE Biosystems)
  • Synthesis grade acetonitrile (Burdick & Jackson)
  • Activator (prepare in acetonitrile): 0.25 M 5‐ethylthio‐1H‐tetrazole (SET), made from solid (American International Chemical) or 0.5 M 4,5‐dicyanoimidazole solution (DCI), made from solid (Proligo) or 0.45 M 1H‐tetrazole (TET; Glen Research).
  • Synthesis columns for 0.2‐µmol‐scale syntheses (PE Biosystems)
  • ABI 394 DNA/RNA synthesizer (PE Biosystems)

Basic Protocol 2: Oligoribonucleotide Deprotection with Nh4OH/Ethanol and TBAF

  Materials
  • Oligoribonucleotide attached to solid support (see protocol 1)
  • 3:1 (v/v) 29% ammonium hydroxide (Mallinckrodt Baker)/100% ethanol (prepare immediately before use)
  • 3:1:1 (v/v/v) ethanol/acetonitrile/H 2O
  • 1.0 M n‐tetrabutylammonium fluoride (TBAF) in THF (Aldrich)
  • 50 mM and 2 M triethylammonium bicarbonate (TEAB), pH 7.8 (see recipe)
  • Heating blocks
  • 4‐mL glass screw‐top vial with Teflon lined lid (Wheaton)
  • 14‐mL centrifuge tubes (Falcon)
  • Qiagen‐tip 500 column (Qiagen)

Alternate Protocol 1: Oligoribonucleotide Deprotection with Aqueous Methylamine and Triethylamine Trihydrofluoride

  • 40% (w/v) aqueous methylamine (Aldrich)
  • Triethylamine trihydrofluoride/NMP/TEA solution (see recipe)
  • 3 M aqueous sodium acetate (e.g., Fluka)
  • n‐butanol
  • 70% aqueous ethanol

Alternate Protocol 2: “One‐Pot” Oligoribonucleotide Deprotection with Anhydrous Methylamine and Neat Triethylamine Trihydrofluoride

  Additional Materials (also see protocol 2)
  • 1:1 (v/v) mixture of 33% ethanolic methylamine and anhydrous DMSO
  • 1.5 M ammonium bicarbonate, pH 7.5 (see recipe)
  • Acetonitrile
  • 1:1:1 (v/v/v) acetonitrile/methanol/H 2O
  • RNase‐free H 2O/DEPC‐treated
  • C 18 SepPak cartridges (Waters)
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Figures

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Literature Cited

Literature Cited
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