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

Abstract

Modified uridine derivatives such as 2-thiouridine (s(2)U), pseudouridine (Psi), 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 s(2)U and D units (the 3'-phosphoramidite derivative of Psi 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 I(2)/pyridine/water which, if applied towards oligonucleotides containing s(2)U 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 (MeNH(2)/EtOH/DMSO) to avoid dihydrouracil ring opening, which is a reported side-reaction during the routine synthesis of dihydrouridine-containing RNA. Oligonucleotides modified with s(2)U, D, or Psi units are useful models for structure-function studies. Here, the procedure for preparation of siRNA duplexes is described.

Cytochrome cCatalyzes the Hydrogen Peroxide‐Assisted Oxidative Desulfuration of 2‐Thiouridines in Transfer RNAs

“…To elucidate further the desulfuration of 2‐thiouridine present in an RNA chain, we used an S2U‐containing RNA oligomer (S2U‐RNA‐1, 14‐mer), the anticodon stem‐loop (ASL) part of the human tRNA Lys3 (5′‐CGGCU S2U UUAACCGA‐3′) . This oligonucleotide was dissolved in 10 m m HEPES buffer (22 μ m ) and was treated with H 2 O 2 (200 μ m ) in the presence of cyt c (5 μ m ) for 1 h (or 3 h) at 37 °C.…”

“…Preparation of 5‐substituted 2‐thiouridines and S2U‐containing RNA : 2‐Thiouridine (S2U, 1 a ), 5‐methylcarboxymethyl‐2‐thiouridine (mcm5S2U, 1 b ), 5‐carbamoylmethyl‐2‐thiouridine (ncm5S2U, 1 c ), 5‐carboxymethyl‐2‐thiouridine (cm5S2U, 1 d ; Scheme ) as well as the corresponding 5‐substituted uridines 2 a – d and 4‐pyrimidinone riboside products 3 a – d (Scheme ) were obtained in our laboratory according to previously established methodology . A model S2U‐RNA‐1 of the sequence corresponding to the anticodon stem‐loop sequence (ASL) of human tRNA Lys3 (5′‐CGGCU S2U UUAACCGA‐3′, a 14‐mer used in our previous studies) as well as S2U‐RNA‐2 (17‐mer, 5′‐GUUGACU S2U UUAAUCAAC‐3′), the ASL from Escherichia coli tRNA Lys , were synthesized as described …”

Escherichia coli tRNA 2‐selenouridine synthase (SelU) converts S2U‐RNA to Se2U‐RNA via S‐geranylated‐intermediate

“…The rC(tac)‐succinyl‐CPG (Proligo) support and 0.07 m solutions of the monomers in acetonitrile (ACN) were used. The phosphoramidite derivatives of S2U, Se2U, and geS2U were prepared in‐house according to the previously described procedures . The phosphoramidities (a 10‐fold molar excess) were mixed with a BMT activator (0.25 m in ACN) and the coupling was executed for 10 min.…”

S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis ofS-geranylated-RNAs and their physicochemical characterization

“…The S2U–RNA of the 5′-GUUGACU S2U UUAAUCAAC-3′ sequence was synthesized automatically on a 0.2 μmole scale analogously to the synthesis of geS2U–RNA with the exception of the use of S2U-phosphoramidite 2 (17), and conditions for the oxidation steps, where a solution of t -BuOOH in toluene (0.25 M, 8 equiv.) was applied for 2 min.…”