Solid‐Phase Chemical Synthesis of 5′‐Triphosphate DNA, RNA, and Chemically Modified Oligonucleotides

Ivan Zlatev1, Muthiah Manoharan2, Jean‐Jacques Vasseur3, François Morvan3

1 Ontorii, Inc., Boston, Massachusetts, 2 Department of Drug Discovery, Alnylam Pharmaceuticals, Cambridge, Massachusetts, 3 IBMM, University of Montpellier, Montpellier, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.28
DOI:  10.1002/0471142700.nc0128s50
Online Posting Date:  September, 2012
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Abstract

A chemical method for the solid‐phase synthesis of 5′‐triphosphate oligonucleotides is described. The full‐length oligonucleotides are first constructed using standard solid‐phase DNA/RNA synthesis, and then efficient implementation of a sequential 4‐steps synthetic procedure, executed either manually or in a fully automated fashion, affords the corresponding solid‐supported 5′‐triphosphate oligonucleotides. Using this synthetic procedure, the full‐length 5′‐hydroxyl oligonucleotides are initially transformed into the corresponding 5′‐H‐phosphonate mono esters, subsequently oxidized in the presence of imidazole to the activated 5′‐phosphorimidazolidates, and finally reacted with pyrophosphate on the solid support. The method uses safe, stable, and inexpensive reagents, and the process is scalable and readily applicable to automated synthesis compatible with the current commercially available DNA/RNA synthesizers. After cleavage from the solid support and deprotection, a range of DNA, RNA, and chemically modified 5′‐triphosphate oligonucleotides are obtained in a convenient and efficient manner and isolated in good yields after HPLC purification. Curr. Protoc. Nucleic Acid Chem. 50:1.28.1‐1.28.16. © 2012 by John Wiley & Sons, Inc.

Keywords: RNA; DNA; 5′‐triphosphate; automated synthesis; solid‐phase

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Automated Solid‐Phase Synthesis of Solid‐Supported 5′‐Hydroxyl Oligonucleotides on DNA/RNA Synthesizer
  • Basic Protocol 2: Automated Solid‐Phase Synthesis of Solid‐Supported 5′‐Triphosphate Oligonucleotides on DNA/RNA Synthesizer
  • Alternate Protocol 1: Manual Sequential Solid‐Phase Synthesis of Solid‐Supported 5′‐Triphosphate Oligonucleotides
  • Basic Protocol 3: Deprotection and Cleavage from Solid Support of 5′‐Triphosphate DNA Oligonucleotides
  • Basic Protocol 4: Deprotection and Cleavage from Solid Support of 5′‐Triphosphate RNA Oligonucleotides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Automated Solid‐Phase Synthesis of Solid‐Supported 5′‐Hydroxyl Oligonucleotides on DNA/RNA Synthesizer

  Materials
  • DNA, RNA, or 2′‐OMe RNA ultra‐mild phosphoramidites (Glen Research)
  • Acetonitrile, low water (EMD Chemicals, H 2O 10 ppm max.)
  • Controlled‐pore glass (CPG) 500 Å pore size, long chain alkyl amine (lcaa) with succinyl‐attached ultra‐mild nucleoside (Glen Research, 30 to 50 µmol/g)
  • Cap Mix A: THF/pyridine/5% Pac 2O (Glen Research)
  • Cap Mix B: 10% N‐methylimidazole/THF (Glen Research)
  • Oxidizing Solution: 0.02 M iodine in THF/pyridine/water (Glen Research)
  • Deblocking Mix: 3% dichloroacetic acid in dichloromethane (Glen Research)
  • Argon source
  • Dry amber glass bottles for the attachment of phosphoramidites and reagents to the synthesizer
  • Empty twist synthesis columns (1 or 10 µmol, Glen Research)
  • DNA/RNA synthesizer (ABI‐394, Applied Biosystems)
  • Additional reagents and equipment for oligonucleotide synthesis ( appendix 3C)

Basic Protocol 2: Automated Solid‐Phase Synthesis of Solid‐Supported 5′‐Triphosphate Oligonucleotides on DNA/RNA Synthesizer

  Materials
  • Controlled‐pore glass (CPG) 500 Å pore size, long chain alkyl amine (lcaa) with succinyl‐attached ultra‐mild 5′‐hydroxyl oligonucleotide ( protocol 1, 30 to 50 µmol/g)
  • Diphenyl phosphite (reagent 5; see recipe)
  • Triethylammonium bicarbonate buffer (reagent 6; see recipe)
  • Oxidizing reagent (reagent 7; see recipe)
  • Pyrophosphate reagent (reagent 8; see recipe)
  • Acetonitrile, low water (EMD Chemicals, H 2O 10 ppm max)
  • Argon source
  • Dry amber glass bottles for the attachment of reagents 5 to 8 to the synthesizer
  • DNA/RNA synthesizer (ABI‐394, Applied Biosystems)
  • Additional reagents and equipment for oligonucleotide synthesis ( appendix 3C)
NOTE: In the case of an uninterrupted triphosphate synthesis following the standard oligonucleotide synthesis, perform steps 1 to 4 prior to starting the synthesis from protocol 1.

Alternate Protocol 1: Manual Sequential Solid‐Phase Synthesis of Solid‐Supported 5′‐Triphosphate Oligonucleotides

  • 2‐mL plastic syringes with Luer fittings

Basic Protocol 3: Deprotection and Cleavage from Solid Support of 5′‐Triphosphate DNA Oligonucleotides

  Materials
  • Dry CPG solid‐supported 5′‐triphosphate DNA oligonucleotide ( protocol 2 or protocol 3)
  • 28% aqueous ammonia (J.T. Baker)
  • Sterile nuclease‐free water (Qiagen)
  • 15‐mL sterile plastic centrifuge tubes (e.g., BD Falcon)
  • 5‐mL plastic syringe
  • Syringe filter (Whatman, GD/X 25 mm, 0.2 µm)
  • Lyophilizer (or SpeedVac evaporator)
  • Additional reagents and equipment for HPLC (unit 10.5) and ESI‐MS (unit 10.2)

Basic Protocol 4: Deprotection and Cleavage from Solid Support of 5′‐Triphosphate RNA Oligonucleotides

  Materials
  • Dry CPG solid‐supported 5′‐triphosphate RNA oligonucleotide ( protocol 2 or protocol 3)
  • 28% aqueous ammonia (J.T. Baker)
  • Sterile nuclease‐free H 2O (Qiagen)
  • 1.0 M tetrabutylammonium fluoride in THF (Aldrich)
  • 15‐mL sterile plastic centrifuge tubes (e.g., BD Falcon)
  • 5‐mL plastic syringe
  • Syringe filter (Whatman, GD/X 25 mm, 0.2 µm)
  • Lyophilizer (or SpeedVac evaporator)
  • Additional reagents and equipment for HPLC (unit 10.5) and ESI‐MS (unit 10.2)
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Figures

Videos

Literature Cited

Literature Cited
   Brownlee, G.G., Fodor, E., Pritlove, D.C., Gould, K.G., and Dalluge, J.J. 1995. Solid phase synthesis of 5′‐diphosphorylated oligoribonucleotides and their conversion to capped m7Gppp‐oligoribonucleotides for use as primers for influenza A virus RNA polymerase in vitro. Nucleic Acids Res. 23:2641‐2647.
   Burgess, K. and Cook, D. 2000. Syntheses of nucleoside triphosphates. Chem. Rev. 100:2047‐2059.
   Debart, F., Vasseur, J‐J., and Lavergne, T. 2009. Process for the solid phase synthesis of RNA. WIPO Patent Application WO2009144418.
   Hornung, V., Ellegast, J., Kim, S., Brzozka, K., Jung, A., Kato, H., Poeck, H., Akira, S., Conzelmann, K.K., Schlee, M., Endres, S., and Hartmann, G. 2006. 5′‐Triphosphate RNA is the ligand for RIG‐I. Science 314:994‐997.
   Joyce, G.E. 2007. Forty years of in vitro evolution. Angew. Chem. Int. Ed. 46:6420‐6436.
   Lavergne, T., Bertrand, J‐R., Vasseur, J‐J., and Debart, F. 2008. Base‐labile group for 2′‐OH protection of ribonucleosides: A major challenge for RNA synthesis. Chem. Eur. J. 14:9135‐9138.
   Lebedev, A.V., Koukhareva, I.I., Beck, T., and Vaghefi, M.M. 2001. Preparation of oligodeoxynucleotide 5′‐triphosphates using solid support approach. Nucleosides Nucleotides Nucleic Acids 20:1403‐1409.
   Ludwig, J. and Eckstein, F. 1989. Rapid and efficient synthesis of nucleoside 5′‐O‐(1‐thiotriphosphates), 5′‐triphosphates and 2′,3′‐cyclophosphorothioates using 2‐chloro‐4H‐1,3,2‐benzodioxaphosphorin‐4‐one. J. Org. Chem. 54:631‐635.
   Olsen, D.B., Benseler, F., Cole, J.L., Stahlhut, M.W., Dempski, R.E., Darke, P.L., and Kuo, L.C. 1996. Elucidation of basic mechanistic and kinetic properties of influenza endonuclease using chemically synthesized RNAs. J. Biol. Chem. 271:7435‐7439.
   Perez, J.T., Varble, A., Sachidanandam, R., Zlatev, I., Manoharan, M., García‐Sastre, A., and tenOever, B.R. 2010. Influenza A virus‐generated small RNAs regulate the switch from transcription to replication. Proc. Natl. Acad. Sci. U.S.A. 107:11525‐11530.
   Poeck, H., Besch, R., Maihoefer, C., Renn, M., Tormo, D., Morskaya, S.S., Kirschnek, S., Gaffal, E., Landsberg, J., Hellmuth, J., Schmidt, A., Anz, D., Bscheider, M., Schwerd, T., Berking, C., Bourquin, C., Kalinke, U., Kremmer, E., Kato, H., Akira, S., Meyers, R., Hacker, G., Neuenhahn, M., Busch, D., Ruland, J., Rothenfusser, S., Prinz, M., Hornung, V., Endres, S., Tuting, T., and Hartmann, G. 2008. 5′‐Triphosphate‐siRNA: Turning gene silencing and Rig‐I activation against melanoma. Nat. Med. 14:1256‐1263.
   Wang, Y., Ludwig, J., Schuberth, C., Goldeck, M., Schlee, M., Li, H., Juranek, S., Sheng, G., Micura, R., Tuschl, T., Hartmann, G., and Patel, D.J. 2010. Structural and functional insights into 5′‐ppp RNA pattern recognition by the innate immune receptor RIG‐I. Nat. Struct. Mol. Biol. 17:781‐787.
   Zlatev, I., Lavergne, T., Debart, F., Vasseur, J.‐J., Manoharan, M., and Morvan, F. 2010. Efficient solid‐phase chemical synthesis of 5′‐triphosphates of DNA, RNA, and their analogues. Org. Lett. 12:2190‐2193.
   Zlatev, I., Morvan, F., Vasseur, J.‐J., Debart, F., and Manoharan, M. 2011. Process for triphosphate oligonucleotide synthesis. WIPO Patent Application WO2011028218.
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