Enzymatic synthesis and reverse transcription of RNAs incorporating 2′-O-carbamoyl uridine triphosphate

Abstract

Enzymatic synthesis and the reverse transcription of RNAs containing 2'-O-carbamoyl uridine were evaluated. A mild acidic deprotection procedure allowed the synthesis of 2'-O-carbamoyl uridine triphosphate (UTP). UTP was incorporated correctly into long RNAs, and its fidelity during reverse transcription using SuperScript III was sufficient for RNA aptamer selection.

Synthesis of Deoxypseudouridine 5′-Triphosphate Bearing the Photoremovable Protecting Group at the N1 Position Capable of Enzymatic Incorporation to DNA

“…Subsequently, the 5′-OH group was converted to 5′-triphosphate according to the previous method utilizing 2-chloro-4 H -1,3,2-benzodioxaphosphorin-4-one and subsequent reactions with pyrophosphate, aqueous iodine, and hydrolysis. The TBDMS group was removed by treatment with a weak acidic buffer (pH 2.5) as previously reported . Thus, 1a was obtained as 25% yield from 5a .…”

“…The TBDMS group was removed by treatment with a weak acidic buffer (pH 2.5) as previously reported. 21 Thus, 1a was obtained as 25% yield from 5a. In a similar procedure, 1b was synthesized using the NPOM group instead of the NB group.…”

Future strategies for the discovery of therapeutic aptamers

“…Other chemically modified nucleotides such as 2'-deoxy-2'-fluoroarabinonucleotide (FANA) [11], 2'-O,4'-C-methylene bridged/locked nucleic acid (2',4'-BNA/LNA)[12], and C2'-O-methyl(C2'-OMe)/ C2'-Fluorine (C2'-F) [13] have been incorporated into aptamers with engineered polymerase. The 2'-O-carbamoyl uridine (U cm ) is successfully incorporated by a wild-type T7 RNA polymerase [14]. …”

“…To increase serum stability, in-SELEX and post-SELEX have been applied to incorporate modified nucleotide into aptamers. So far, it is very encouraging that 2′F, 2'OMe, 2'NH 2 [1], and newly developed modified nucleotides [11–14] have been successfully incorporated with the engineered polymerases in the in-SELEX. Currently available modified nucleotides are modified at the 2′-position of ribose.…”

“…Currently available modified nucleotides are modified at the 2′-position of ribose. As the 2'-modifiction can change RNA structure flexibility [18], the structural stability of aptamers incorporating with recently developed modified nucleotides [11–14] remains to be validated.…”