UNIT 16.2 Preparation of Short Interfering RNA Containing the Modified Nucleosides 2-Thiouridine, Pseudouridine, or Dihydrouridine
Published Online: 1 JUN 2009
Copyright © 2009 by John Wiley & Sons, Inc.
Lab Protocol Title
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. 37:16.2.1–16.2.16.
- Published Online: 1 JUN 2009
- Published Print: JUN 2009
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.
- RNA synthesis;
- small interfering RNA;
- phosphoramidite synthesis