Solid‐Phase Synthesis of RNA Analogs Containing Phosphorodithioate Linkages

Xianbin Yang1

1 AM Biotechnologies, Houston, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.77
DOI:  10.1002/cpnc.40
Online Posting Date:  September, 2017
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Abstract

The oligoribonucleotide phosphorodithioate (PS2‐RNA) modification uses two sulfur atoms to replace two non‐bridging oxygen atoms at an internucleotide phosphorodiester backbone linkage. Like a natural phosphodiester RNA backbone linkage, a PS2‐modified backbone linkage is achiral at phosphorus. PS2‐RNAs are highly stable to nucleases and several in vitro assays have demonstrated their biological activity. For example, PS2‐RNAs silenced mRNA in vitro and bound to protein targets in the form of PS2‐aptamers (thioaptamers). Thus, the interest in and promise of PS2‐RNAs has drawn attention to synthesizing, isolating, and characterizing these compounds. RNA‐thiophosphoramidite monomers are commercially available from AM Biotechnologies and this unit describes an effective methodology for solid‐phase synthesis, deprotection, and purification of RNAs having PS2 internucleotide linkages. © 2017 by John Wiley & Sons, Inc.

Keywords: phosphorodithioate oligoribonucleotide; PS2‐RNA; solid‐phase synthesis; sulfur‐modified oligonucleotide; thiophosphoramidite

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Solid‐Phase Assembly of Protected RNA‐Thiophosphoramidites
  • Basic Protocol 2: Deprotection and Purification of PS2‐RNAs
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Solid‐Phase Assembly of Protected RNA‐Thiophosphoramidites

  Materials
  • RNA‐thiophosphoramidites (AM Biotechnologies)
    • Bz‐rA‐thiophosphoramidite
    • Ac‐rC‐thiophosphoramidite
    • Ac‐rG‐thiophosphoramidite
    • rU‐thiophosphoramidite
  • Normal RNA phosphoramidites (Glen Research)
    • Bz‐A‐CE phosphoramidite
    • Ac‐C‐CE phosphoramidite
    • Ac‐G‐CE phosphoramidite
    • U‐CE phosphoramidite
  • XX‐RNA‐CPG column (Glen Research)
  • Argon gas, anhydrous
  • Acetonitrile, anhydrous (Glen Research)
  • 10% (v/v) anhydrous dichloromethane in anhydrous acetonitrile
  • 0.25 M Activator 42 (Sigma‐Aldrich)
  • Cap A mix, tetrahydrofuran/acetic anhydride (THF/Ac 2O; Glen Research)
  • Cap B mix, 10% 1‐methylimidazole in tetrahydrofuran/pyridine (Glen Research)
  • 0.02 M I 2 in THF/H 2O/pyridine (oxidizing solution; Glen Research)
  • 3% trichloroacetic acid (TCA) in dichloromethane (deblocking mix; Glen Research)
  • 0.2 M 3‐ethoxy‐1,2,4‐dithiazolidine‐5‐one (EDITH; MW 163.22) in acetonitrile (sulfurization reagent; 163 mg in 20 mL acetonitrile, Carbosynth Limited)
  • Wash A (acetonitrile, anhydrous; Glen Research)
  • Synthesizer vials with caps
  • Vacuum desiccators
  • Expedite 8909 (Perseptive Biosystem) with trityl monitor

Basic Protocol 2: Deprotection and Purification of PS2‐RNAs

  Materials
  • Fully protected PS2‐RNAs attached to solid support of a synthesis column (see protocol 1)
  • Argon gas, anhydrous
  • Anhydrous ethanol (Sigma‐Aldrich)
  • Acetonitrile (HPLC grade, TEDIA)
  • Mobile phase A (1 mM EDTA, 25 mM Tris·HCl, pH 8)
  • Mobile phase B (1 mM EDTA, 25 mM Tris·HCl, 1 M NaCl, pH 8)
  • Sodium hydroxide (for adjusting pH of mobile phases; Sigma‐Aldrich)
  • >28% ammonium hydroxide or concentrated ammonium hydroxide (Sigma‐Aldrich)
  • 41% methyl amine (Fluka, cat. no. 65580)
  • Triethylamine (TEA; Sigma‐Aldrich)
  • DL‐Dithiothreitol (DDT; Sigma‐Aldrich)
  • Triethylamine trihydrofluoride, 98% (Sigma‐Aldrich, cat. no. 344648)
  • N,N‐Dimethylformamide (DMF; Sigma‐Aldrich)
  • Ammonium acetate (HPLC grade, Fluka)
  • Diethylpyrocarbonate (DEPC; Sigma‐Aldrich)
  • Loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromophenol blue, 1 mg/mL ethidium bromide in DEPC‐treated or nuclease‐free water)
  • Vials, 4 mL and sealable
  • Incubator, 55°C (temperature range: up to 100°C)
  • Sterile syringe filter, 0.2 μm cellulose acetate (VWR, cat. no. 28145‐477)
  • Dionex DNAPac PA‐100 4 × 250‐mm analytical column (Thermo Fisher Scientific, cat. no. 043010)
  • Divinyl benzene/polystyrene copolymer reverse‐phase column (Hamilton PRP‐1, cat. no. 79426, flow rate 2 mL/min or cat. no. 79425, flow rate 1 mL/min)
  • Mono Q column (GE Healthcare Life Sciences)
  • Amicon ultra‐15 centrifugal filter units, 3000 Da cut‐off (Millipore Sigma)
  • Silica TLC plates (Merck)
  • Platform shaker
  • Lyophilizer
  • SepPak C18 cartridges
Additional reagents and equipment for SDS‐PAGE (Gallagher, )
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Figures

Videos

Literature Cited

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Key References
  Yang, X., Sierant, M., Janicka, M., Peczek, L., Martinez, C., Hassell, T., Li, N., Li, X., Wang, T., & Nawrot, B. (2012). See above.
  This reference summarizes the methods for synthesis of PS2‐RNAs, as well as the synthesis of PS2‐RNA building blocks, the RNA‐thiophosphoramidites.
  Wiesler, W.T., & Caruthers, M. H. (1996). See above.
  This reference describes the detailed procedure for synthesis of DNA‐thiophosphoramidites. The procedure can be referenced for synthesis of RNA‐thiophosphoramidites.
  Yang, X., et al., (2002). See above.
  This reference describes the detailed method to purify the PS2‐DNAs. The method can be used for isolating PS2‐RNA in high purity.
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