RNA Synthesis by Reverse Direction Process: Phosphoramidites and High Purity RNAs and Introduction of Ligands, Chromophores, and Modifications at 3′‐End

Suresh C. Srivastava1, Divya Pandey1, Naveen P. Srivastava1, Satya P. Bajpai1

1 ChemGenes Corp., Wilmington, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.20
DOI:  10.1002/0471142700.nc0320s45
Online Posting Date:  June, 2011
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Abstract

We have synthesized and studied the coupling properties of 3′‐DMT‐5′‐CE phosphoramidites. The coupling efficiency per step surpasses 99% in the reverse‐direction synthesis methodology, leading to high‐purity RNA in a large number of 20‐ to 21‐mers and long‐chain oligonucleotides. Our data show that 5′→3′ direction synthesis has a distinct advantage compared to the conventional method. As a result, this method of RNA synthesis is expected to be a very useful and practical method of choice for therapeutic‐grade RNA. The phosphoramidites, Rev‐A‐n‐bz, Rev‐C‐n‐bz, Rev‐C‐n‐ac, Rev‐G‐n‐ac, and Rev‐rU are routinely produced with an HPLC purity of greater than 98% and 31P NMR purity greater than 99.5%. Curr. Protoc. Nucleic Acid Chem. 45:3.20.1‐3.20.39. © 2011 by John Wiley & Sons, Inc.

Keywords: oligonucleotide synthesis; reverse RNA synthesis; RNA phosphoramidites; reverse RNA phosphoramidites; 3′‐DMT‐2′‐TBDMS ribonucleoside 5′‐amidites; synthetic RNA 3′‐end modification and labeling

     
 
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Table of Contents

  • Introduction
  • Oligonucleotide Synthesis by 5′→3′ Technology
  • Basic Protocol 1: Synthesis of Sequence ID No. 1.1.19
  • Basic Protocol 2: Synthesis of Sequence ID No. 1.1.20
  • Basic Protocol 3: Synthesis of Sequence ID No. 1.1.21
  • Basic Protocol 4: Synthesis of Sequence ID No. 1.1.22
  • Basic Protocol 5: Synthesis of Sequence ID No. 1.1.23
  • Basic Protocol 6: Synthesis of Sequence ID No. 1.1.24
  • Basic Protocol 7: Synthesis of Sequence ID No. 1.1.25
  • Support Protocol 1: Oligonucleotide Deprotection
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • 5′‐O‐Bz‐N6‐Benzoyl‐2′‐O‐TBDMS‐3′‐O‐DMT‐adenosine ( S.16a; ChemGenes)
  • Pyridine, anhydrous (Caledon Laboratories, http://www.caledonlabs.com/)
  • Methanol
  • 2 M sodium hydroxide
  • 2 M HCl
  • Chloroform
  • Saturated NaCl solution
  • Na 2SO 4, anhydrous
  • Hexane
  • Acetone
  • Stirrer plate and magnetic bar
  • Rotary evaporator with vacuum pump
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F254
  • 2.5 × 25‐cm chromatography column
  • 254‐nm UV lamp for TLC
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 2:

  Materials
  • N6‐Benzoyl‐2′‐O‐TBDMS‐3′‐O‐DMT‐adenosine ( S.17a; see protocol 1)
  • 2,4,6‐collidine, distilled (Honeywell)
  • Tetrahydrofuran (THF), distilled
  • N‐methylimidazole (Sigma‐Aldrich)
  • 2‐cyanoethyl‐N,N, diisopropyl phosphonamidic chloride (ChemGenes)
  • Argon source
  • Ethyl acetate
  • Saturated sodium bicarbonate solution
  • Saturated NaCl solution
  • Na 2SO 4, anhydrous
  • Hexane
  • Triethylamine
  • Stirrer plate and magnetic bar
  • Rotary evaporator with vacuum pump
  • 2.5 × 25‐cm chromatography column
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F254
  • 254‐nm UV lamp for TLC
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 3:

  Materials
  • 5′‐O‐Bz‐N4‐Acetyl‐2′‐O‐TBDMS‐3′‐O‐DMT‐cytidine ( S.16c; ChemGenes)
  • Pyridine, anhydrous (Caledon Laboratories, http://www.caledonlabs.com/)
  • Methanol
  • 2 M sodium hydroxide
  • 2 M HCl
  • Chloroform
  • Saturated NaCl solution
  • Na 2SO 4, anhydrous
  • Hexane
  • Acetone
  • Stirrer plate and magnetic bar
  • Rotary evaporator with vacuum pump
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F254
  • 2.5 × 25‐cm chromatography column
  • 254‐nm UV lamp for TLC
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 4:

  Materials
  • N4‐Acetyl‐2′‐O‐TBDMS‐3′‐O‐DMT‐cytidine ( S.17c, see protocol 3)
  • Ethylthiotetrazole (ChemGenes, cat. no. RN‐6397)
  • Diisopropylethylamine (DIPEA)
  • N‐methylimidazole (Sigma‐Aldrich)
  • Acetonitrile
  • 2‐cyanoethyl‐N,N,N1,N1‐tetraisopropylphosphane (ChemGenes)
  • Argon source
  • Ethyl acetate
  • Saturated sodium bicarbonate solution
  • Saturated NaCl solution
  • Na 2SO 4, anhydrous
  • Chloroform
  • Hexane
  • Triethylamine
  • Stirrer plate and magnetic bar
  • Rotary evaporator with vacuum pump
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F254
  • 2.5 × 25‐cm chromatography column
  • 254‐nm UV lamp for TLC
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 5:

  Materials
  • 5′‐O‐benzoyl‐N2‐Isobutyryl‐2′‐O‐TBDMS‐3′‐O‐DMT‐guanosine ( S.16d; ChemGenes)
  • Pyridine, anhydrous (Caledon Laboratories; http://www.caledonlabs.com/)
  • Methanol
  • 2 M sodium hydroxide
  • 2 M HCl
  • Chloroform
  • Saturated NaCl solution
  • Na 2SO 4, anhydrous
  • Hexane
  • Acetone
  • Stirrer plate and magnetic bar
  • Rotary evaporator with vacuum pump
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F254
  • 2.5 × 25‐cm chromatography column
  • 254‐nm UV lamp for TLC
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 6:

  Materials
  • N2‐Isobutyryl‐2′‐O‐TBDMS‐3′‐O‐DMT‐guanosine ( S.17d; see protocol 5)
  • Ethylthiotetrazole (Chem Genes, cat. no. RN‐6397)
  • Diisopropylethylamine (DIPEA)
  • N‐methylimidazole (Sigma‐Aldrich)
  • Acetonitrile
  • 2‐cyanoethyl‐N,N,N1,N1‐tetraisopropylphosphane (ChemGenes)
  • Argon source
  • Ethyl acetate
  • Saturated sodium bicarbonate solution
  • Saturated NaCl solution
  • Na 2SO 4, anhydrous
  • Chloroform
  • Hexane
  • Triethylamine
  • Stirrer plate and magnetic bar
  • Rotary evaporator with vacuum pump
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F254
  • 2.5 × 25‐cm chromatography column
  • 254‐nm UV lamp for TLC
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 7:

  Materials
  • 5′‐O‐Bz‐2′‐O‐TBDMS‐3′‐O‐DMT‐Uridine ( S.16k; ChemGenes)
  • Pyridine, anhydrous (Caledon Laboratories, http://www.caledonlabs.com/)
  • Methanol
  • 2 M sodium hydroxide
  • 2 M HCl
  • Chloroform
  • Saturated NaCl solution
  • Na 2SO 4, anhydrous
  • Hexane
  • Acetone
  • Stirrer plate and magnetic bar
  • Rotary evaporator with vacuum pump
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F254
  • 2.5 × 25‐cm chromatography column
  • 254‐nm UV lamp for TLC
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 8:

  Materials
  • 2′‐O‐TBDMS‐3′‐O‐DMT‐Uridine ( S.17k, see protocol 7)
  • 2,4,6‐collidine, distilled (Honeywell)
  • Tetrahydrofuran (THF), distilled
  • N‐methylimidazole (Sigma‐Aldrich)
  • 2‐cyanoethyl‐N,N,N1,N1‐tetraisopropylphosphane (ChemGenes)
  • Argon source
  • Ethyl acetate
  • Saturated sodium bicarbonate solution
  • Saturated NaCl solution
  • Na 2SO 4, anhydrous
  • Hexane
  • Triethylamine
  • Stirrer plate and magnetic bar
  • Rotary evaporator with vacuum pump
  • 2.5 × 25‐cm chromatography column
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F254
  • 254‐nm UV lamp for TLC
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 9:

  Materials
  • 2′‐O‐TBDMS‐3′‐O‐DMT‐Uridine ( S.17k, see protocol 7)
  • 4‐Dimethylaminopyridine (DMAP)
  • Pyridine, anhydrous (Caledon Laboratories, http://www.caledonlabs.com/)
  • Succinic anhydride
  • Chloroform
  • Saturated NaCl solution
  • Hexane
  • Acetone
  • Methanol
  • Pyridine, anhydrous (Caledon Laboratories, http://www.caledonlabs.com/)
  • Stirrer plate and magnetic bar
  • Rotary evaporator equipped with a vacuum pump
  • 2.5 × 25‐cm chromatography column
  • Thin‐layer chromatography (TLC) plate, EMD silica gel 60 F254
  • 254‐nm UV lamp for TLC
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 10:

  Materials
  • Native Amino‐lcaa‐CPG 1000A (Prime Synthesis Inc.)
  • 2′‐O‐TBDMS‐3′‐O‐DMT‐uridine‐5′‐succinate, pyridinium salt ( S.18k; see protocol 9)
  • N‐hydroxysuccinimide
  • Dicyclohexyl cabodiimide (DCC)
  • Dimethylformamide (DMF)
  • Acetonitrile, anhydrous
  • Diethyl ether
  • Cap A (ChemGenes, cat. no. RN‐1458)
  • Cap B (ChemGenes, cat. no. RN‐1481)
  • 30% (v/v) perchloric acid
  • Buchner funnel
  • Filter paper
  • 500‐mL flask

Basic Protocol 11: Synthesis of Sequence ID No. 1.1.19

  Materials
  • Materials for oligonucleotide synthesis (see Table 3.20.2 for clarification):
    • Rev‐rA
    • Rev‐rC
    • Rev‐rG
    • Rev‐rU
    • Rev‐dT
    • Rev‐rC‐CPG
    • B1
    • B2
    • B3
    • B4
    • B5
    • B7
  • Acetonitrile, anhydrous
  • Dichloromethane (DCM)
  • Diethyl ether
  • Diethylamine
  • Tris base
  • 1.0 M lithium chloride in water
  • Absolute ethanol
  • 2% (w/v) lithium perchlorate in acetone (ChemGenes)
  • Expedite silanized bottles for DNA synthesis (Applied Biosystems)
  • Expedite 8900 synthesizer (Applied Biosystems)
  • HPLC system (e.g., Varian Prostar)
  • Source 15Q ion‐exchange column (1.0 cm × 25 cm; GE Healthcare)
  • Spectrophotometer for measuring OD 260
  • Capillary gel electrophoresis (CE) instrument (Beckman Coulter)
  • 4 × 250–mm DNA Pac PA200 analytical column (Dionex, http://www.dionex.com)
  • Lyophilizer
  • Additional reagents and equipment for deprotection of oligonucleotides ( protocol 18) and ESI/MS (unit 10.2)
    Table 3.0.2   MaterialsMaterials for Oligonucleotides Synthesis

    Abbreviated name Description ChemGenes cat. no.
    Rev‐rA N6‐Benzoyl‐2′‐O‐TBDMS‐3′‐O‐DMT‐adenosine‐5′‐CEP ANP‐3401
    Rev‐rC N4‐Acetyl‐2′‐O‐TBDMS‐3′‐O‐DMT‐cytidine‐5′‐CEP ANP‐3405
    Rev‐rG N2 ‐Isobutryl‐2′‐O‐TBDMS‐3′‐O‐DMT‐guanosine‐5′‐CEP ANP‐3403
    Rev‐rG‐CPG N2‐Isobutyryl‐2′‐O‐TBDMS‐3′‐O‐DMT‐guanosine‐5′‐Succinyl CPG 1000A N‐6203‐10
    Rev‐rU 2′‐O‐TBDMS‐3′‐O‐DMT‐Uridine‐5′‐CEP ANP‐3404
    Rev‐dT 3′‐O‐DMT‐Thymidine‐5′‐cyanoethyl‐N,N‐diisopropyl‐phosphoramidite ANP‐4674
    Rev‐rC‐CPG N4‐Acetyl‐2′‐O‐TBDMS‐3′‐O‐DMT‐cytidine ‐5′‐Succinyl CPG 1000A N‐6205‐10
    Rev‐rU‐CPG 2′‐O‐TBDMS‐3′‐O‐DMT‐Uridine ‐5′‐Succinyl CPG 1000A N‐6204‐10
    Rev‐rA‐CPG N6‐ Benzoyl‐2′‐O‐TBDMS‐3′‐O‐DMT‐Adenosine ‐5′‐Succinyl CPG 1000A N‐6201‐10
    Cholesterol TEG Amidite CLP‐2704
    Cholesterol TEG CPG N‐9166‐05
    PEG 2000 Amidite CLP‐2119
    HEG Amidite CLP‐9765
    Symmetrical branching CEP CLP‐5215
    Rev‐2′‐O‐Me –rA 2′‐O‐Me –rA (mA); N6‐Benzoyl‐2′‐O‐Me‐3′‐O‐DMT‐adenosine‐5′‐CEP ANP‐1012
    Puri‐pak cartridge CSS‐5232
    B1 Anhydrous Acetonitrile RN‐1447
    B2 CAP A RN‐1458
    B3 CAP B RN‐1481
    B4 DMT Removal Reagent RN‐1462
    B5 Oxidation Solution RN‐1455
    B6 Activation Reagent, 5‐Ethylthio‐1‐H‐Tetrazole (ETT) (0.35M in Acetonitrile) RN‐1466
    B7 Activation Reagent, 5‐benzylthio‐1‐H‐Tetrazole (BMT) (0.3M in Acetonitrile) RN‐1452
    B8 DDTT Solution [(0.1M Pyridine: Acetonitrile (90:10))] RN‐1689

Basic Protocol 12: Synthesis of Sequence ID No. 1.1.20

  Materials
  • Materials for oligonucleotide synthesis (see Table 3.20.2 for clarification):
    • Rev‐rA
    • Rev‐rC
    • Rev‐rG
    • Rev‐rU
    • Rev‐dT
    • Rev‐rC‐CPG
    • Cholesterol TEG Amidite
    • B1
    • B2
    • B3
    • B4
    • B5
    • B7
  • Acetonitrile, anhydrous
  • Dichloromethane (DCM)
  • Diethyl ether
  • Diethylamine
  • Triethylammonium acetate, pH 7.2
  • Ethanol
  • 2% (w/v) lithium perchlorate in acetone (ChemGenes)
  • Expedite silanized bottles for DNA synthesis (Applied Biosystems)
  • Expedite 8900 synthesizer (Applied Biosystems)
  • HPLC system (e.g., Varian Prostar)
  • C18 Hamilton RP‐HPLC column
  • Spectrophotometer for measuring OD 260
  • 4 × 250–mm DNA Pac PA200 analytical column (Dionex, http://www.dionex.com)
  • Capillary gel electrophoresis (CE) instrument (Beckman Coulter)
  • Lyophilizer
  • Additional reagents and equipment for deprotection of oligonucleotides ( protocol 18) and ESI/MS (unit 10.2)

Basic Protocol 13: Synthesis of Sequence ID No. 1.1.21

  Materials
  • Materials for oligonucleotide synthesis (see Table 3.20.2 for clarification):
    • Rev‐rA
    • Rev‐rC
    • Rev‐rG
    • Rev‐rU
    • Rev‐dT
    • Rev‐rC‐CPG
    • PEG 2000 AmiditeB1
    • B2
    • B3
    • B4
    • B5
    • B7
  • Acetonitrile, anhydrous
  • Dichloromethane (DCM)
  • Diethyl ether
  • Diethylamine
  • Triethylammonium acetate, pH 7.2
  • Absolute ethanol
  • 2% (w/v) lithium perchlorate in acetone (ChemGenes)
  • Expedite silanized bottles for DNA synthesis (Applied Biosystems)
  • Expedite 8900 synthesizer (Applied Biosystems)
  • HPLC system (e.g., Varian Prostar)
  • C18 Hamilton RP‐HPLC column
  • Spectrophotometer for measuring OD 260
  • 4 × 250–mm DNA Pac PA200 analytical column (Dionex, http://www.dionex.com)
  • Capillary gel electrophoresis (CE) instrument (Beckman Coulter)
  • Lyophilizer
  • Additional reagents and equipment for deprotection of oligonucleotides ( protocol 18) and ESI/MS (unit 10.2)

Basic Protocol 14: Synthesis of Sequence ID No. 1.1.22

  Materials
  • Materials for oligonucleotide synthesis (see Table 3.20.2 for clarification):
    • Rev‐rA
    • Rev‐rC
    • Rev‐rG
    • Rev‐rU
    • Rev‐dT
    • Rev‐rC‐CPG
    • HEG amidite
    • B1
    • B2
    • B3
    • B4
    • B5
    • B7
  • Acetonitrile, anhydrous
  • Dichloromethane (DCM)
  • Diethyl ether
  • Diethylamine
  • Triethylammonium acetate, pH 7.2
  • Expedite silanized bottles for DNA synthesis (Applied Biosystems)
  • Expedite 8900 synthesizer (Applied Biosystems)
  • HPLC system (e.g., Varian Prostar)
  • C18 Hamilton RP‐HPLC column
  • Spectrophotometer for measuring OD 260
  • 4 × 250–mm DNA Pac PA200 analytical column (Dionex, http://www.dionex.com)
  • Capillary gel electrophoresis (CE) instrument (Beckman Coulter)
  • Lyophilizer
  • Additional reagents and equipment for deprotection of oligonucleotides ( protocol 18) and ESI/MS (unit 10.2)

Basic Protocol 15: Synthesis of Sequence ID No. 1.1.23

  Materials
  • Materials for oligonucleotide synthesis (see Table 3.20.2 for clarification):
    • Rev‐rA
    • Rev‐rC
    • Rev‐rG
    • Rev‐rU
    • Rev‐rU‐CPG
    • Rev‐2′‐O‐Me –rA
    • B1
    • B2
    • B3
    • B4
    • B6
    • B8
  • Acetonitrile, anhydrous
  • Dichloromethane (DCM)
  • Diethyl ether
  • Diethylamine
  • Methylamine
  • 2% (w/v) lithium perchlorate in acetone (ChemGenes)
  • 12% tetraethyl ammonium fluoride (Galaxy Chemicals) in DMSO
  • n‐butanol
  • Ethanol
  • Tris base
  • 1 M lithium chloride
  • Expedite silanized bottles for DNA synthesis (Applied Biosystems)
  • Expedite 8900 synthesizer (Applied Biosystems)
  • 65° and 45°C water bath
  • 2‐mL screw‐cap vials
  • HPLC system (e.g., Varian Prostar)
  • Source 15Q ion‐exchange column (1.0 cm × 25 cm; GE Healthcare)
  • Spectrophotometer for measuring OD 260
  • 4 × 250–mm DNA Pac PA200 analytical column (Dionex, http://www.dionex.com)
  • Capillary gel electrophoresis (CE) instrument (Beckman Coulter)
  • Lyophilizer
  • Additional reagents and equipment for deprotection of oligonucleotides ( protocol 18) and ESI/MS (unit 10.2)

Basic Protocol 16: Synthesis of Sequence ID No. 1.1.24

  Materials
  • Materials for oligonucleotide synthesis (see Table 3.20.2 for clarification):
    • Rev‐rA
    • Rev‐rC
    • Rev‐rG
    • Rev‐rU
    • Rev‐rU‐CPG
    • PEG 2000 Amidite
    • Symmetrical branching amidite
    • B1
    • B2
    • B3
    • B4
    • B5
    • B7
  • Acetonitrile, anhydrous
  • Dichloromethane (DCM)
  • Diethyl ether
  • Diethylamine
  • Triethylammonium acetate
  • Expedite silanized bottles for DNA synthesis (Applied Biosystems)
  • Expedite 8900 synthesizer (Applied Biosystems)
  • HPLC system (e.g., Varian Prostar)
  • C18 Hamilton RP‐HPLC column
  • Spectrophotometer for measuring OD 260
  • 4 × 250–mm DNA Pac PA200 analytical column (Dionex, http://www.dionex.com)
  • Capillary gel electrophoresis (CE) instrument (Beckman Coulter)
  • Lyophilizer
  • Additional reagents and equipment for deprotection of oligonucleotides ( protocol 18) and ESI/MS (unit 10.2)

Basic Protocol 17: Synthesis of Sequence ID No. 1.1.25

  Materials
  • Materials for oligonucleotide synthesis (see Table 3.20.2 for clarification):
    • Rev‐rA
    • Rev‐rC
    • Rev‐rG
    • Rev‐rU
    • Rev‐rA‐CPG
    • B1
    • B2
    • B3
    • B4
    • B5
    • B7
  • Acetonitrile, anhydrous
  • Dichloromethane (DCM)
  • Diethyl ether
  • Diethylamine
  • Triethylammonium acetate
  • Expedite silanized bottles for DNA synthesis (Applied Biosystems)
  • Expedite 8900 synthesizer (Applied Biosystems)
  • HPLC system (e.g., Varian Prostar)
  • C18 Hamilton RP‐HPLC column
  • Spectrophotometer for measuring OD 260
  • 4 × 250–mm DNA Pac PA200 analytical column (Dionex, http://www.dionex.com)
  • Capillary gel electrophoresis (CE) instrument (Beckman Coulter)
  • Lyophilizer
  • Additional reagents and equipment for deprotection of oligonucleotides ( protocol 18) and ESI/MS (unit 10.2)

Support Protocol 1: Oligonucleotide Deprotection

  • Solid support from oligonucleotide synthesis (i.e., from step 7 in protocol 11 or analogous step in subsequent protocols)
  • 2‐mL screw‐cap vials
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Literature Cited

Literature Cited
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   Duplaa, A., Gasparutto, D., Livache, T., Molko, D., and Teoule, R. 1996. Process for the Synthesis of Ribo Nucleic Acid (RNA) Using a Novel Deprotection Reagent. U.S. Patent 5552539.
   Gait, M.J., Pritchard, C., and Slim, G. 1991. Oligonucleotides and Their Analogs: A Practical Approach (M.J. Gait, ed.). Oxford University Press, Oxford.
   Hsiung, H.M. 1983. Process for isolating oligonuceotide product from a coupling reaction mixture. U.S. Patent 4417046.
   Laikhter, A., Martin, W., and Edgar, E. 2008. Methods for RNA Desilylation. U.S. Patent Application 2008/0119563 A1.
   Martin, A.R., Lavergne, T., Vasseur, J.J., and Debart, F. 2009. Assessment of new 2′‐O‐acetalester protecting group for regular RNA synthesis and original 2′‐modified pro RNA. Bioorg. Med. Chem. Lett. 19:4046‐4049.
   Ogilvie, K.K. 1983. Proceedings of the 5th International Round Table on Nucleosides, Nucleotides and Their Biological Applications, Rideout (J.L. Henry and L.M. Beacham III, eds.) Academic Press, London.
   Ogilvie, K.K., Sadana, K.L, Thompson, E.A., and Quilliam, M.A., and Westmore, J.B. 1974. The use of silyl groups in protecting the hydroxyl functions of ribonucleosides. Tetrahydron Lett. 15:2861‐2863.
   Ogilvie, K.K., Beaucage, S.L, Entwistle, D.W., Thompson, E.A., and Quilliam, M.A. 1976. Alkylsilyl groups in nucleoside and nucleotide chemistry. J. Carbohydr. Nucleos. Nucleot. 3:197‐227.
   Ogilvie, K.K., Theriault, N.Y., Seifert, J.‐M., Pon, R.T., and Nemer, M.J. 1980. The chemical synthesis of oligoribonucleotides. IX. A comparison of protecting groups in the dichloridite procedure. Can. J. Chem. 58:2686‐2693.
   Ogilvie, K.K., Usman, N., Nicoghosian, K., and Cedergren, R.J. 1988. Total chemical synthesis of a 77‐nucleotide‐long RNA sequence having methionine acceptance activity. Proc. Natl. Acad. Sci. U.S.A. 85:5764‐5768.
   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.
   Ravikumar, V.T., Manoharan, M., Capaldi, D., Krotz, A., Cole, D.L., and Guzaev, A. 2005. Process for synthesis of oligomeric compounds. US Patent 6858715 B2 and US Patent 7041816 B2.
   Scaringe, S.A., Wincott, F.E., and Caruthers, M.H. 1998. Novel RNA synthesis method using 5′‐silyl‐2′‐orthoester protecting groups. J. Am. Chem. Soc. 120:11820‐11821.
   Semenyuk, A., Foldesi, A., Johansson, T., Estmer‐Nilsson, C., Blomgren, P., Brannvall, M., Kirsebom, L.A., and Kwiatkowski, M. 2006. Synthesis of RNA using 2′‐O‐DTM protection. J. Am. Chem. Soc. 128:12356‐12357.
   Shiba, Y., Masuda, H., Watanabe, N., Ego, T., Takagaki, K., Ishiyama, K., Ohgi, T., and Yano, J. 2007. Chemical synthesis of a very long oligoribonucleotide with 2‐cyanoethoxymethyl (CEM) as the 2′‐O‐protecting group: Structural identification and biological activity of a synthetic 110mer precursor‐microRNA candidate. Nucleic Acids Res. 35:3287‐3296.
   Sinha, N.D. 2006. Method of preventing modification of synthetic oligonucleotides. U.S. Patent 7038027.
   Srivastava, S.C., Pandey, D., Srivastava, N.P., and Bajpai, S.P. 2008. RNA synthesis: Phosphoramidites for RNA synthesis in the reverse direction. Highly efficient synthesis and application to convenient introduction of ligands, chromophores and modifications of synthetic RNA at the 3′‐ end. Nucleic Acids Symp. Ser. 52:103‐104.
   Srivastava, S.C., Pandey, D., Srivastava, N.P., and Bajpai, S.P. 2010. Synthesis of high‐purity RNA and 3′‐end conjugates further process refinement. International Round Table on Nucleosides, Nucleotides, and Nucleic Acids‐IRT2010. 54:42‐43.
   Usman, N., Ogilvie, K.K., Jiang, M.Y., and Cedergren, R.J. 1987. Automated chemical synthesis of long oligoribonucleotides using 29‐O‐silylated ribonucleoside 39‐O‐phosphoramidites on a controlled‐pore glass support: Synthesis of a 43‐nucleotide sequence similar to the 39‐half molecule of an Escherichia coli formylmethionine tRNA. J. Am. Chem. Soc. 109:7845–7854.
   Westmore, J.B. 1974. The use of silyl groups in protecting the hydroxyl functions of ribonucleosides. Tetrahedron Lett. 15:2861‐2864.
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