Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase

RNA
Sailesh GopalakrishnaRajesh K Gaur

Abstract

UV-induced photochemical crosslinking is a powerful approach that can be used for the identification of specific interactions involving nucleic acid-protein and nucleic acid-nucleic acid complexes. 8-AzidoATP (8-N(3)ATP) is a photoaffinity-labeling agent which has been widely used to elucidate the ATP binding site of a variety of proteins. However, its true potential as a photoactivatable nucleotide analog could not be exploited due to the lack of 8-azidoadenosine phosphoramidite, a monomer used in the synthesis of RNA, and the inability of 8-N(3)ATP to serve as an efficient substrate for bacteriophage RNA polymerase. In this study, we explored the ability of SP6, T3, and T7 RNA polymerases and metal ion cofactors to catalyze the incorporation of 8-N(3)AMP into RNA. Whereas transcription buffer containing 2.0-2.5 mM Mn(2+) supports T7 RNA polymerase-mediated insertion of 8-N(3)AMP into RNA, a mixture of 2.5 mM Mn(2+) and 2.5 mM Mg(2+) further improves the yield of 8-N(3)AMP-containing transcript. In addition, both RNA transcription and reverse transcription proceed with high fidelity for the incorporation of 8-N(3)AMP and complementary residue, respectively. Finally, we show that a high-affinity MS2 coat protein binding sequenc...Continue Reading

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Citations

Aug 24, 2010·Analytical Biochemistry·Kalvin J GregoryValeri Golovlev
Apr 22, 2015·Nucleic Acids Research·Bin ZhuCharles C Richardson
Aug 4, 2011·Research in Nursing & Health·Ann K CashionThomas R Sutter
Feb 19, 2014·Journal of Cellular and Molecular Medicine·Leni MoldovanMelissa Piper
Oct 8, 2020·Chemical Communications : Chem Comm·Jerrin Thomas George, Seergazhi G Srivatsan
Jun 9, 2021·Chemical Reviews·Tyler K HeissJennifer A Prescher

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