title: Enzymatic incorporation of an azide-modified UTP analog into oligoribonucleotides for post-transcriptional chemical functionalization.
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.
authors.affiliation: Department of Chemistry, Indian Institute of Science Education and Research, Pune, India.
authors.forename: Harita
authors.initials: H
authors.lastname: Rao
authors.forename: Arun A
authors.initials: AA
authors.lastname: Tanpure
authors.forename: Anupam A
authors.initials: AA
authors.lastname: Sawant
authors.forename: Seergazhi G
authors.initials: SG
authors.lastname: Srivatsan
mesh_terms: D001386:Azides / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods*; D009843:Oligoribonucleotides / Q000737:chemistry*; D012323:RNA Processing, Post-Transcriptional; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D001386:Azides; D009843:Oligoribonucleotides; D014544:Uridine Triphosphate