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PROTOCOL
Enzymatic incorporation of an azide-modified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization
Harita Rao, Arun A Tanpure, Anupam A Sawant & Seergazhi G Srivatsan
Department of Chemistry, Indian Institute of Science Education and Research, Pune, India. Correspondence should be addressed to S.G.S. ([email protected]).
Published online 10 May 2012; doi:10.1038/nprot.2012.046
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.
2012 Nature America, Inc. All rights reserved.
INTRODUCTION
Modified nucleic acids have provided effective biophysical tools to investigate the structure, dynamics and recognition properties of several nucleic acid motifs14. In addition to predictable Watson-Crick base pairing property, functionalization of oligonucleotides has also allowed the construction of several nanoarchitectures that have electrical, magnetic or probe-like properties510. Modified
oligonucleotides are routinely synthesized by solid-phase chemical synthesis and enzymatic methods1113. For most applications, solid-phase synthesis on the basis of phosphoramidite chemistry is the method of choice to obtain modified oligonucleotides. However, synthesis of many modified phosphoramidite substrates involves extensive chemical manipulations, and there are also instances in which the modified phosphoramidite substrates do not survive the relatively stringent solid-phase chemical synthesis conditions. Functionalities can also be introduced into oligonucleotides under milder conditions by enzymatic methods such as primer extension of DNA oligonucleotides, in vitro transcription reactions in the presence of corresponding nucleoside triphosphates and ligation reactions using ligases1319. The major drawback of enzymatic methods is that it is difficult to control the site of incorporation20, and modified triphosphate analogs are not always accepted and effectively incorporated into the growing nucleic acid chain by polymerases21,22.
Labeled oligonucleotides can also be obtained postsynthetically by performing chemoselective reactions on oligonucleotides containing reactive functionalities13,2326. One of the regularly used postsynthetic labeling procedures is the coupling of aminoalkylor thioalkyl-modified oligonucleotides with activated esters...