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

Nature Protocols volume 7, pages 1097–1112 (2012)Cite this article

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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.

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Figure 1
Figure 2: Incorporation of the monophosphate of 7 into an oligoribonucleotide by in vitro transcription reaction.
Figure 3: Post-transcriptional chemical modification of transcript 10.
Figure 4: UV shadow (short wave UV, 254 nm) of polyacrylamide gels of oligoribonucleotide products obtained from click reactions between azide-modified oligoribonucleotide 10 and alkyne substrates 11 and 13.
Figure 5: Rubber bladder filled with hydrogen gas.
Figure 6
Figure 7: Emission profile (2 μM) of click product 12.

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Acknowledgements

This research is supported by the Department of Science and Technology, India (SR/S1/OC-51/2009). A.A.T. and A.A.S. are thankful to the Council of Scientific and Industrial Research, India, for a graduate research fellowship.

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  1. Department of Chemistry, Indian Institute of Science Education and Research, Pune, India

    Harita Rao, Arun A Tanpure, Anupam A Sawant & Seergazhi G Srivatsan

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Contributions

S.G.S. designed and directed the research. H.R. and A.A.T. synthesized the triphosphate analog and optimized the conditions for Staudinger reduction of azide-modified RNA. A.A.S. performed post-transcriptional click reactions on RNA. All authors discussed the results and wrote the manuscript.

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Correspondence to Seergazhi G Srivatsan.

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Rao, H., Tanpure, A., Sawant, A. et al. Enzymatic incorporation of an azide-modified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization. Nat Protoc 7, 1097–1112 (2012). https://doi.org/10.1038/nprot.2012.046

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