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Enzymatic incorporation of an azide-modified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization.

Nature protocols (2012-05-12)
Harita Rao, Arun A Tanpure, Anupam A Sawant, Seergazhi G Srivatsan
ABSTRACT

This protocol describes the detailed experimental procedure for the synthesis of an azide-modified uridine triphosphate analog and its effective incorporation into an oligoribonucleotide by in vitro transcription reactions. Furthermore, procedures for labeling azide-modified oligoribonucleotides post-transcriptionally with biophysical probes by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) and Staudinger reactions are also provided. This post-transcriptional chemical modification protocol is simple and modular, and it affords labeled oligonucleotides in reasonable amounts for biophysical assays. The procedure for enzymatic incorporation of the monophosphate of azide-modified UTP into an oligoribonucleotide transcript takes ∼2 d, and subsequent post-transcriptional chemical functionalization of the transcript takes about 2 d.

MATERIALS
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Brand
Product Description

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