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

Nature Protocols
Harita RaoSeergazhi 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.

References

Jan 1, 1989·Methods in Enzymology·J F Milligan, O C Uhlenbeck
Jul 6, 2002·Science·Kinneret KerenErez Braun
Aug 22, 2003·Organic & Biomolecular Chemistry·Evdoxia Coutouli-ArgyropoulouCatherine Raptopoulou
Oct 27, 2005·Journal of the American Chemical Society·Stefan JägerMichael Famulok
Feb 24, 2007·Langmuir : the ACS Journal of Surfaces and Colloids·Joseph M Kinsella, Albena Ivanisevic
Apr 5, 2007·Nature Protocols·Nicholas J Greco, Yitzhak Tor
May 4, 2007·Chemical Communications : Chem Comm·Samuel H Weisbrod, Andreas Marx
Jun 16, 2007·Nature Protocols·Seergazhi G Srivatsan, Yitzhak Tor
Aug 19, 2007·Nucleic Acids Research·Michiko KimotoIchiro Hirao
Oct 24, 2007·Molecular Biotechnology·Nadrian C Seeman
Mar 5, 2008·Biochimie·Alexander Heckel, Michael Famulok
Apr 3, 2008·Angewandte Chemie·Philipp M E GramlichThomas Carell
Sep 10, 2008·Chembiochem : a European Journal of Chemical Biology·Venkata Ramana SirivoluFrank Seela
Sep 25, 2008·Angewandte Chemie·Philipp M E GramlichThomas Carell
Nov 15, 2008·Chemical Communications : Chem Comm·Samuel H Weisbrod, Andreas Marx
Dec 17, 2008·Chemistry, an Asian Journal·Seergazhi G Srivatsan, Yitzhak Tor
Apr 22, 2009·Journal of the American Chemical Society·Zbigniew PianowskiNicolas Winssinger
Aug 12, 2009·The Journal of Organic Chemistry·Gwladys PourceauFrançois Morvan
Sep 10, 2009·Chembiochem : a European Journal of Chemical Biology·Neil M Bell, Jason Micklefield
Nov 6, 2009·Journal of the American Chemical Society·Raphael M Franzini, Eric T Kool
Jan 14, 2010·Analytical Chemistry·Gudrun StengelRobert D Kuchta
Feb 6, 2010·Chembiochem : a European Journal of Chemical Biology·Falk Wachowius, Claudia Höbartner
Mar 18, 2010·Chemical Communications : Chem Comm·Christoph Beyer, Hans-Achim Wagenknecht
Aug 18, 2010·Proceedings of the National Academy of Sciences of the United States of America·Afaf H El-Sagheer, Tom Brown
Nov 6, 2010·Organic Letters·Pieter van DelftDmitri V Filippov
Nov 12, 2010·Journal of the American Chemical Society·Alireza OmumiRichard A Manderville
Dec 7, 2010·Chembiochem : a European Journal of Chemical Biology·Eduardo Paredes, Subha R Das
Dec 21, 2010·Chembiochem : a European Journal of Chemical Biology·Michaela AignerRonald Micura
Mar 25, 2011·Angewandte Chemie·Yamuna Krishnan, Friedrich C Simmel
Apr 13, 2011·Chemical Communications : Chem Comm·Omid Khakshoor, Eric T Kool
Jul 19, 2011·Journal of the American Chemical Society·Ashwani K Sharma, Jennifer M Heemstra

❮ Previous
Next ❯

Citations

Sep 19, 2015·Nucleic Acids Research·Anupam A SawantSeergazhi G Srivatsan
Mar 7, 2017·Bioconjugate Chemistry·Katharina SchmidMark Helm
Apr 14, 2017·Bioconjugate Chemistry·Jerrin Thomas George, Seergazhi G Srivatsan
Feb 22, 2017·Methods : a Companion to Methods in Enzymology·Jerrin Thomas George, Seergazhi G Srivatsan
Mar 26, 2014·Chemical Communications : Chem Comm·Josephin Marie HolsteinAndrea Rentmeister
Dec 1, 2015·Methods : a Companion to Methods in Enzymology·Josephin M Holstein, Andrea Rentmeister
Nov 15, 2017·Nucleic Acids Research·Meng ZhaoEva Freisinger
May 14, 2016·Organic & Biomolecular Chemistry·Anupam A SawantSeergazhi G Srivatsan
Oct 4, 2012·Chemical Communications : Chem Comm·Takumi IshizukaIchiro Hirao
Sep 7, 2016·Chembiochem : a European Journal of Chemical Biology·Sarah NainarRobert C Spitale
Oct 8, 2020·Chemical Communications : Chem Comm·Jerrin Thomas George, Seergazhi G Srivatsan
Jun 9, 2021·Chemical Reviews·Tyler K HeissJennifer A Prescher
Aug 27, 2014·Organic Letters·Maksim A FomichVadim V Shmanai
Jan 15, 2014·Chemical Reviews·Sethuramasundaram PitchiayaNils G Walter
Jul 14, 2020·Journal of the American Chemical Society·Jerrin Thomas GeorgeSeergazhi G Srivatsan
Oct 23, 2020·Bioconjugate Chemistry·Manisha B Walunj, Seergazhi G Srivatsan

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.