I will do RNA ligation by using two fragments. 5' fragment is ordered from Dharmacon; 3' fragment will be transcribled by T7 polymerase.
For the 3' fragment, there are two method to treat with the 5'-end to create mono-phosphate group which is required for ligation. 1. Do trancription by using NTP mix, (I add two GG at the end of T7 promoter on my PCR DNA template before the sequence), GTP will be added to the 5'-end of 3'fragment. So I need to do CIP to dephosphate, then do PNK reaction to add mono-phosphate group before ligation. 2. I read one paper they use T7 transcription primed with GMP to introduce an unlabeled 5' phosphate, but on detailed protocol. I am assuming they put certain percentage of GMP and GTP in the transcription. So the start G will be mono-phosphated and tri-phosphated by GMP or GTP. They can do ligation directly without CIP and PNK treatment. But the tri-phosphated G will affect the ligation efficiency.
Does anyone have a protocol how to make RNA ligation more efficient related to these two mentioned methods?
Dear Lingde
You don't need to do much special. As we showed many years ago, T7RP happily incorporates GMP as the 5' encoded G, even better than it incorporates GTP.
"T7 RNA Polymerase Does Not Interact with the 5'-Phosphate of the Initiating Nucleotide," Craig T. Martin and Joseph E. Coleman, Biochemistry 28, 2760-2762, 1989
So just do your regular protocol, but with GTP and maybe 2-fold higher concentration of GMP. With the lower Km and the 2X conc, you'll get predominantly the product you want. If that still leaves too much triphosphate RNA in the product, you can either up the GMP concentration or lower the GTP.
Sincerely, Craig Martin
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