How to produce a triphosphate (3p) motif by in vitro RNA transcription?

12 October 2022 1 5K Report

I want to perform an in vitro RNA transcription to obtain an RNA strand with a 3p (Triphosphate) motif added at the 5' end. I have found some protocols but most only use a DNA template and the necessary dNTPs. How then is the 3p motif produced?

Kyle Skalenko

Just use NTPs.......

dNTPs are NOT a substrate for RNA polymerase, they are DNA nucleotides used by DNA polymerase.

Simply, mix DNA template, NTPs, and RNA polymerase (any RNA polymerase depending on your protocol) and you will generate RNAs with 5' triphosphate ends, using any typical in vitro transcription protocol

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