Solid‐Phase Synthesis of Oligodeoxynucleotide Analogs Containing Phosphorodithioate Linkages

Xianbin Yang1

1 AM Biotechnologies, LLC, Houston, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.71
DOI:  10.1002/cpnc.13
Online Posting Date:  September, 2016
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Abstract

The oligodeoxynucleotide phosphorodithioate modification (PS2‐ODN) uses two sulfur atoms to replace two non‐bridging oxygen atoms at an internucleotide phosphordiester backbone linkage. Like a natural phosphodiester ODN backbone linkage, a PS2‐modified backbone linkage is achiral at phosphorus. PS2‐ODNs are highly stable to nucleases and numerous in vitro assays have demonstrated their biological activity. For example, PS2‐ODNs activated RNase H in vitro, strongly inhibited human immunodeficiency virus (HIV) reverse transcriptase, induced B‐cell proliferation and differentiation, and bound to protein targets in the form of PS2‐aptamers (thioaptamers). Thus, the interest in and promise of PS2‐ODNs has spawned a variety of strategies for synthesizing, isolating, and characterizing this compounds. ODN‐thiophosphoramidite monomers are commercially available from either AM Biotechnologies or Glen Research and this unit describes an effective methodology for solid‐phase synthesis, deprotection, and purification of ODNs having PS2 internucleotide linkages. © 2016 by John Wiley & Sons, Inc.

Keywords: solid‐phase synthesis; phosphorodithioate oligodeoxynucleotide; PS2‐ODN; sulfur‐modified oligonucleotide; thiophosphoramidite

     
 
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Table of Contents

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

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

  Materials
  • ODN‐thiophosphoramidites or DNA‐thiophosphoramidites (AM Biotechnologies or Glen Research):
    • dA‐thiophosphoramidite
    • dC‐thiophosphoramidite or Ac‐dC‐thiophosphoramidite
    • dG‐thiophosphoramidite
    • dT‐thiophosphoramidite
  • Normal phosphoramidites (Glen Research):
    • dA‐CE phosphoramidite
    • dC‐CE phosphoramidite or Ac‐dC‐CE Phosphoramidite
    • dG‐CE Phosphoramidite
    • dT‐CE Phosphoramidite
  • Argon gas
  • Activator:
    • 0.45 M tetrazole in acetonitrile (Glen Research)
    • 0.25 M DCI (4,5‐dicyanoimidazole) in acetonitrile (Sigma‐Aldrich)
    • 0.25 M activator‐42 (Sigma‐Aldrich)
  • Diluent: acetonitrile, anhydrous (Glen Research) containing 10% anhydrous dichloromethane (DCM)
  • Helium gas, anhydrous
  • Cap A: tetrahydrofuran/acetic anhydride (THF/ Ac 2O) (Glen Research)
  • Cap B: 10% 1‐methyllimidazole in tetrahydrofuran/ pyridine (Glen Research)
  • Oxidizing solution: 0.02 M I 2 in THF/H 2O/pyridine (Glen Research)
  • Deblocking mix: 3% trichloroacetic acid in dichloromethane (Glen Research)
  • Sulfurization reagent: 3‐ethoxy‐1,2,4‐dithiazolidine‐5‐one (EDITH), 0.2 M (163 mg in 20 mL acetonitrile), molecular weight: 163.22 (Carbosynth Limited)
  • Synthesizer vials with caps
  • Vacuum desiccators
  • LCA‐CPG column (Glen Research)
  • Expedite 8909, Perseptive Biosystem with trityl monitor

Basic Protocol 2: Deprotection and Purification of PS2‐ODNs

  Materials
  • Argon gas
  • Fully protected ODNs attached to the solid support of a synthesis column (see protocol 1)
  • Anhydrous ethanol (Sigma‐Aldrich)
  • 28% ammonium hydroxide or concentrated ammonium hydroxide (Sigma‐Aldrich)
  • DL‐Dithiothreitol (Sigma‐Aldrich)
  • Ammonium acetate (HPLC grade, Fluka)
  • Deionized water
  • 24% denaturing polyacrylamide gel
  • Loading buffer (Promega, cat. no. G1881)
  • Acetonitrile (HPLC grade, TEDIA)
  • Trizma hydrochloride (Sigma‐Aldrich), pH 8
  • EDTA (Sigma‐Aldrich)
  • Sodium hydroxide NaCl; Sigma‐Aldrich)
  • Monobeads
  • 4.0‐mL sealable vial
  • 55°C incubator
  • 15‐mL conical tubes
  • −70°C freezer
  • Lyophilizer
  • 1000‐μL UV cuvettes
  • Spectrophotometer
  • 0.75‐mm analytical gel plate cassettes
  • Electrophoresis apparatus
  • Plastic wrap
  • TLC Merck silica plate
  • Hand‐held 254‐nm UV lamp
  • Camera with a UV filter
  • Razor blades
  • Spatula
  • Platform shaker
  • 0.22‐μm hydrophilic fluid filter
  • SepPak C18 cartridges
  • DIONEX DNAPac PA‐100 4 × 250 mm analytical column (cat. no. 043010)
  • Mono‐Q columns
  • A divinyl benzene/polystyrene copolymer reverse‐phase column (Hamilton PRP‐1): Part No. 79426, Flow = 2 ml/min or Part No. 79425, Flow = 1 mL/min
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Figures

Videos

Literature Cited

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Key References
  Yang and Mierzejewsk, 2010. See above.
  This reference summarizes most of the methods (if not all) for synthesis of PS2‐ODNs, as well as the synthesis of PS2‐ODN building block.
  Wiesler and Caruthers, 1996. See above.
  This reference describes the detailed procedure for synthesis of ODN‐thiophosphoramidites and their application.
  Yang et al., 2002b. See above.
  This reference describes the detailed method to purify the PS2‐ODNs.
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