Safe Deprotection Strategy for the Tert‐Butyldimethylsilyl (TBS) Group During RNA Synthesis

Daniel Zewge1

1 Department of Process Research, Merck Research Laboratories, Rahway, New Jersey
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.21
DOI:  10.1002/0471142700.nc0321s49
Online Posting Date:  June, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes two protocols for the deprotection of 2′‐O‐TBS groups in oligoribonucleotides under mild conditions. Desilylation using ammonium fluoride is applicable to fully protected “RNA only” substrates and desilylation using potassium fluoride is applicable to “mixed RNA/non‐RNA” substrates. Characterization of products is accomplished using LC/MS, RP HPLC and SAX HPLC. Curr. Protoc. Nucleic Acid Chem. 49:3.21.1‐3.21.11. © 2012 by John Wiley & Sons, Inc.

Keywords: desilylation; oligoribonucleotides; “RNA only”; “mixed RNA/non‐RNA” substrates; siRNA

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Desilylation of 2′‐O‐TBS Ethers from RNA at 500‐nmol Scale
  • Alternate Protocol 1: Desilylation of 2′‐O‐TBS Ethers from RNA at 50‐µmol Scale
  • Alternate Protocol 2: Desilylation of 2′‐O‐TBS Ethers from RNA at 800‐µmol Scale
  • Basic Protocol 2: Desilylation of 2′‐O‐TBS Ethers from Mixed RNA/NON‐RNA Sequences on an Intermediate Scale (50 µmol)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  Materials
  • TBS‐protected RNA (21 mer), 500 nmol
  • Methylamine (MeNH 2; 40% aq.)
  • Dimethyl sulfoxide (DMSO; 98%)
  • Glycolic acid/ hydroxyacetic acid (9.2 M, 70% aq.; see recipe)
  • Ammonium fluoride (NH 4Fl; 10.8 M, 40% aq.) solution (see recipe)
  • Acetonitrile (20% aq.)
  • DNA/RNA synthesizer
  • Multi‐well plate vacuum manifold
  • 96‐well deep‐well plate (2.2‐mL capacity)
  • Agitating/heating device (37°C and 65°C)
  • 96‐well purification plate (C‐18, 200 mg/well; Orochem)
  • Lyophilizer (Genevac), optional

Basic Protocol 2: Desilylation of 2′‐O‐TBS Ethers from RNA at 500‐nmol Scale

  Materials
  • TBS‐protected RNA (21 mer), 50 µmol
  • Methylamine (MeNH 2; 40% aq.)
  • Dimethyl sulfoxide (DMSO; 98%)
  • Glycolic acid/hydroxyacetic acid (9.2 M, 70% aq.; see recipe)
  • Ammonium fluoride (NH 4F; 10.8 M, 40% aq.) solution (see recipe)
  • NaCl
  • Acetonitrile (20 % aq.)
  • DNA/RNA synthesizer
  • 40‐mL screw‐cap vials
  • 0.2‐µm filter funnel
  • Agitating/heating device (37°C and 65°C)
  • pH meter
  • C‐18 cartridge (Sep‐pak vac, 12 mL, 2 g; Waters)

Alternate Protocol 1: Desilylation of 2′‐O‐TBS Ethers from RNA at 50‐µmol Scale

  Materials
  • TBS‐protected RNA (21 mer), 800 µmol
  • Methylamine (MeNH 2; 40% aq.)
  • Dimethyl sulfoxide (DMSO; 98%)
  • Glycolic acid/hydroxyacetic acid (9.2 M, 70% aq.; see recipe)
  • Ammonium fluoride (NH 4F; 10.8 M, 40% aq.) solution (see recipe)
  • Sodium acetate (NaOAc)
  • DNA/RNA synthesizer
  • 0.2‐µm filter funnel
  • 1‐L screw‐cap bottles
  • Agitating/heating device (37°C and 65°C)
  • pH meter
  • HPLC purification station

Alternate Protocol 2: Desilylation of 2′‐O‐TBS Ethers from RNA at 800‐µmol Scale

  Materials
  • TBS‐protected RNA (21 mer), 50 µmol
  • Methylamine (MeNH 2; 40% aq.)
  • Dimethyl sulfoxide (DMSO; 98 %)
  • Malonic acid/propanedioic acid (4.8 M, aq.; see recipe)
  • Potassium fluoride(KF; 10 M, aq.; see recipe)
  • Aq. NaCl
  • Acetonitrile (20% aq.)
  • DNA/RNA synthesizer
  • 0.2‐µm filter funnel
  • 40‐mL screw‐cap vials
  • Agitating/heating device (37°C and 65°C)
  • pH meter
  • C‐18 cartridge (Sep‐pak vac, 12 mL, 2 g; Waters)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Beaucage, S.L. 2008. Solid‐phase synthesis of siRNA oligonucleotides. Curr. Opin. Drug Discov. Dev. 11:203‐216.
   Damha, M.J. and Ogilvie, K.K. 1993. Oligoribonucleotide Synthesis. In Methods in Molecular Biology, Volume 20: Protocols for Oligonucleotides and Analogs (S. Agrawal, ed.) pp. 81‐114. Humana Press, Totowa, N.J.
   Jackson, A.L., Burchard, J., Leake, D., Reynolds, A., Schelter, J., Guo, J., Johnson, J.M., Karpilow, J., Nichols, K., Marshall, W., Khvorova, A., and Linsley, P.S. 2006. Position‐specific chemical modification of siRNAs reduces “off‐target” transcript silencing. RNA 12:1197‐1205.
   Kawahara, S‐I, Wada, T., and Sekine, M. 1995. A new method for removal of the 2′‐TBDMS group under acidic conditions in chemical synthesis of RNA. Nucleic Acids Symp. 34:29‐30.
   Ogilvie, K.K. and Iwacha, D.J. 1973. Use of tert‐butyldimethylsilyl group for protecting the hydroxyl functions of nucleosides. Tetrahedron Lett. 317‐319.
   Rayner, S., Brignac, S., and Bumeister, R. 1998. MerMade: An oligodeoxyribonucleotide synthesizer for high throughput oligonucleotide production in dual 96‐well plates. Genome Res. 8:741‐747.
   Reddy, M.P. and Farooqui, N.B. 1995. Methylamine deprotection provides increased yield of oligoribonucleotides. Tetrahedron Lett. 36:8929‐8932.
   Sharma, A. and Robertson, G.P. 2007. Models of melanoma metastasis: Using a transient siRNA‐based protein inhibition strategy in mice to validate the functional relevance of pharmacological agents. Curr. Protoc. Pharmacol. 38:14.6.1‐14.6.15.
   Vargeese, C., Shaffer, C., and Bowman, K. 2004. Deprotection and purification of oligonucleotides and their derivatives. Int. Patent Application. WO 2004/007748 A2.
   Vinayak, R., Andrus, A., and Hampel, A. 1995. Rapid desilylation of oligoribonucleotides at elevated temperatures: cleavage activity in ribozyme‐substrate assays. Biomed. Pept. Proteins Nucleic Acids 1:227‐230.
   Westman, E. and Stromberg, R. 1994. Removal of t‐butyldimethylsilyl protection in RNA‐synthesis. Triethylamine trihydrofluoride (TEA⋅3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF). Nucleic Acids Res. 22:2430‐2431.
   Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., and Usman, N. 1995. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 23:2677‐2684.
   Zewge, D., Gosselin, F., Sidler, R., DiMichele, L., and Cvetovich, R.J. 2010. A safe and practical procedure for global deprotection of oligoribonucleotides. J. Org. Chem. 75:5305‐5307.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library