Preparation of Short Interfering RNA Containing the Modified Nucleosides 2‐Thiouridine, Pseudouridine, or Dihydrouridine

Barbara Nawrot1, Elzbieta Sochacka2

1 Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland, 2 Institute of Organic Chemistry, Technical University of Lodz, Lodz, Poland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 16.2
DOI:  10.1002/0471142700.nc1602s37
Online Posting Date:  June, 2009
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Abstract

Modified uridine derivatives such as 2‐thiouridine (s2U), pseudouridine (Ψ), and dihydrouridine (D) are naturally existing nucleoside units identified in tRNA molecules. Recently, we have shown that such base‐modified units introduced into functionally important sites of siRNA modulate thermodynamic stability of the duplex and its gene silencing activity. In this unit, we describe chemical synthesis of 3′‐phosphoramidite derivatives of s2U and D units (the 3′‐phosphoramidite derivative of Ψ is commercially available), and their use for the synthesis of RNA oligonucleotides according to the routine phosphoramidite protocol. The only exception concerns the oxidation step with I2/pyridine/water which, if applied towards oligonucleotides containing s2U units, would lead to the loss of sulfur. Therefore, to avoid this side reaction, tert‐butyl hydroperoxide is used as an oxidizing reagent. After the oligonucleotide chain assembly is completed, the resulting oligomer is deprotected under mild basic conditions (MeNH2/EtOH/DMSO) to avoid dihydrouracil ring opening, which is a reported side‐reaction during the routine synthesis of dihydrouridine‐containing RNA. Oligonucleotides modified with s2U, D, or Ψ units are useful models for structure‐function studies. Here, the procedure for preparation of siRNA duplexes is described. Curr. Protoc. Nucleic Acid Chem. 37:16.2.1‐16.2.16. © 2009 by John Wiley & Sons, Inc.

Keywords: 2‐thiouridine; dihydrouridine; pseudouridine; RNA synthesis; siRNA; small interfering RNA; phosphoramidite synthesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Dihydrouridine and 2‐Thiouridine 3′‐Phosphoramidites
  • Basic Protocol 2: Synthesis of Oligonucleotides Containing Modified 2‐Thiouridine, Pseudouridine, or Dihydrouridine, and Preparation of Short Interfering RNAs
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Dihydrouridine and 2‐Thiouridine 3′‐Phosphoramidites

  Materials
  • Modified nucleosides:
    • 5,6‐Dihydrouridine (International Laboratory Limited or Chemcube, Niederkassel, Germany)
    • 2‐Thiouridine (Berry and Associates, International Laboratory Limited, or Chemcube, Niederkassel, Germany)
  • Pyridine (anhydrous, analytical grade; Aldrich), freshly distilled over CaH 2
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl; Aldrich)
  • Silica gel (Merck, type 9385, 230 to 400 mesh)
  • Dichloromethane
  • Methanol
  • 5% aqueous sodium bicarbonate (NaHCO 3)
  • Sodium sulfate (anhydrous)
  • Toluene
  • Triethylamine (TEA, anhydrous, HPLC grade; Aldrich)
  • Tetrahydrofurane (anhydrous)
  • Silver nitrate (Aldrich)
  • tert‐ Butyldimethylsilyl chloride (TBDMS‐Cl; Aldrich)
  • Acetone
  • Hexane
  • Ethyl acetate
  • Imidazole (Aldrich)
  • Benzene
  • Argon (or nitrogen) gas, dry
  • Desiccant silica gel (Aldrich)
  • N,N‐Diisopropylethylamine (DIPEA, anhydrous; Aldrich)
  • 2‐Cyanoethyl N,N‐diisopropyl chlorophosphoramidite (Aldrich)
  • Acetic anhydride (Ac 2O)
  • N,N‐Dimethylaminopyridine (DMAP; 99%; Aldrich)
  • Ethyl ether
  • Petroleum ether
  • Rotary evaporator
  • Vacuum pump and water aspirator
  • Magnetic stir bars and plate
  • Thin‐layer chromatography (TLC) plates (Silica gel 60F, Merck)
  • 200‐mL and 100‐mL separatory funnels
  • 5.5 × 5–cm, 4.0 × 12–cm, and 1.5 × 25–cm silica gel columns
  • 25‐, 50‐ and 100‐mL round–bottom flasks
  • Rubber septa
  • Luer‐lock needles with syringes
  • 1‐mL, 500‐µL and 200‐µL syringes with needles
  • Glass capillaries
  • Rubber cork or Parafilm
  • Vacuum desiccators
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 2: Synthesis of Oligonucleotides Containing Modified 2‐Thiouridine, Pseudouridine, or Dihydrouridine, and Preparation of Short Interfering RNAs

  Materials
  • Commercial nucleoside 3′‐phosphoramidites:
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)]phosphinyl uridine and N‐protected cytidine, adenosine, and guanosine (Glen Research)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)] phosphinyl thymidine (Glen Research)
  • Modified nucleoside 3′‐phosphoramidites:
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)]phosphinyl 5,6‐dihydrouridine (S.4a prepared according to protocol 1)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)]phosphinyl 2‐thiouridine (S.4b prepared according to protocol 1)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)]phosphinyl pseudouridine (Glen Research)
  • Acetonitrile (CH 3CN), anhydrous (Fluka)
  • Tert‐butyl hydroperoxide (tBuOOH) ∼5.5 M solution in decane (over molecular sieve 4Å; Aldrich)
  • Methylamine, 33% ethanolic solution (Aldrich)
  • Dimethyl sulfoxide (DMSO; Aldrich)
  • (C 2H 5) 3N × 3HF 98% (TEA × 3HF; Aldrich, cat. no. 344648)
  • Ammonium bicarbonate
  • Sodium acetate (NaOAc), anhydrous (Sigma)
  • Acetate buffer (mixture of 50 mM NaOAc and 50 mM NaCl)
  • Trifluoroacetic acid, TFA (Fluka)
  • NaCl
  • Diethyl pyrocarbonate (DEPC; Aldrich)
  • Milli‐Q water
  • Automated oligonucleotide synthesizer (Applied Biosystems 394 or equivalent)
  • 4‐mL screw‐top vials
  • 65°C heating block
  • −20°C freezer
  • 10‐mL disposable syringes
  • SepPak cartridges (Waters)
  • Speedvac evaporator (Savant)
  • UV/VIS spectrophotometer (NanoDrop ND‐1000)
  • Autoclave (e.g., Varioklav Model 75T or 135T, H+P Labortechnik AG, Germany)
  • Microcentrifuge tubes (e.g., Eppendorf)
  • 95°C water bath
  • Additional reagents and equipment for automated oligoribonucleotide synthesis (see appendix 3C), purifying oligonucleotides using RP‐HPLC (unit 10.5) or PAGE (unit 10.4)
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Figures

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Literature Cited

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