Unit

UNIT 16.2 Preparation of Short Interfering RNA Containing the Modified Nucleosides 2-Thiouridine, Pseudouridine, or Dihydrouridine

  1. Barbara Nawrot1,
  2. Elzbieta Sochacka2

Published Online: 1 JUN 2009

DOI: 10.1002/0471142700.nc1602s37

Current Protocols in Nucleic Acid Chemistry

Current Protocols in Nucleic Acid Chemistry

How to Cite

Nawrot, B. and Sochacka, E. 2009. Preparation of Short Interfering RNA Containing the Modified Nucleosides 2-Thiouridine, Pseudouridine, or Dihydrouridine. Current Protocols in Nucleic Acid Chemistry. 37:16.2:16.2.1–16.2.16.

Author Information

  1. 1

    Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland

  2. 2

    Institute of Organic Chemistry, Technical University of Lodz, Lodz, Poland

Publication History

  1. Published Online: 1 JUN 2009
  2. Published Print: JUN 2009

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