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

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 sequence, in which adenosine residues were replaced by 8-azidoadenosine, crosslinks to the coat protein of the Escherichia coli phage MS2.

Chemo‐Enzymatic Synthesis of Position‐Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy

“…Solid‐phase synthesis, most widely used for the synthesis of modified oligonucleotides, has limits in the preparation of longer RNAs of high purity, a limitation that in vitro transcription by T7 RNA polymerase does not face. T7 RNA polymerase mutants allow incorporation of modified nucleotides including dNTPs, 8‐azido ATP, 2′‐fluoro and 2′‐amino modified rNTPs . However, incorporation of a modified nucleotide at a single position at will is difficult with enzymatic synthesis.…”

Studies on Transcriptional Incorporation of 5’-N-Triphosphates of 5’-Amino-5’-Deoxyribonucleosides

“…It has been previously reported that the use of Mn 2+ as a cofactor in transcription reactions is beneficial for modulating the substrate specificity of RNA polymerases [ 32 ]. Furthermore, substrate specificity is even more improved by including a combination of Mg 2+ and Mn 2+ in the reaction; thus, both ions were added in performed experiments [ 33 ]. In the presented studies, all four RNA polymerases failed to produce a full-length transcript; however, shorter intermediate products were observed ( S3A–S3E Fig ).…”

Synthesis of 2′-Fluoro RNA by Syn5 RNA polymerase

“…Manganese ions decrease the substrate discrimination for many polymerases ( 22 ). T7 RNA polymerase can use Mn 2+ instead of Mg 2+ for catalysis; the optimum concentration of Mn 2+ is 2.0–2.5 mM ( 23 ), 10-fold lower than the optimum concentration of Mg 2+ . However, even at the optimum metal concentrations, the activity of T7 RNA polymerase with Mn 2+ is much lower than that with Mg 2+ ( 13 ).…”

“…However, even at the optimum metal concentrations, the activity of T7 RNA polymerase with Mn 2+ is much lower than that with Mg 2+ ( 13 ). A mixture of Mn 2+ and Mg 2+ ions has been used successfully ( 23 , 24 ) to provide the two metal ions required for catalysis in the T7 RNA polymerase active site ( 17 ). Presumably, some T7 RNA polymerase molecules will have one Mn 2+ and one Mg 2+ in their active sites, resulting in relaxed specificity for substrate analogs.…”