I am trying to dephosphorylate RNA and ligate it after. My controls seem to work fine except for a long RNA which I suspect can't dephosphorylate probably due to secondary structures. I have tried denaturing RNA at 65C(5min) followed by keeping it on ice(5min) before the respective reactions. I also add DMSO during my ligation reaction. Nothing seems to work. Has anyone worked with ssRNA's with rigid secondary structures?
Hi Anjali
I hace a few questions before answering: how are you quantifying the success of your dephosphorylation, and what size RNA are we talking about?
Jesper
I am not quantifying my dephosphorylation. My readouts are ligations. We are talking about 2kb rna when self ligated would be 4kb in length
For radiolabeling RNA, I have dephosphorylated 3 Kb pre-rRNA using NEB's calf intestine phosphatase (only this company's worked, promega's shrimp alkaline phosphatase did not work) and rnaisin (rnaisin was essential), followed by rephosphorylation with 32P-ATP, T4 PNK, In order to bind a primer, I have also tried warming up RNA to 70 C, 5 min, and then removing the heating block out allowing it to cool to room temperature, before adding the primer for reverse trancription, this seemed to work based on the transcribed fulllength DNA obtained. It all depends on the secondary structure of RNA.
Hi Anjali
I am not sure the dephosphorylation is the problem, In my experience any commercial phosphatase will work just fine and on most substrates. I do agree with Binal above that some kind of RNase inhibitor is a good idea (although most phosphatases are supposed to be RNase free). I recommend you test the extent of dephosphorylation by first doing a kinase reaction to prepare 5' monophosphate ends, then incubate with 5'monophosphate dependent exonuclease (http://www.epibio.com/enzymes/nucleases-glycosylases-dna-binding-proteins/rna-exonucleases/terminator-5--phosphate-dependent-exonuclease). The kinase reaction work just as well as the phosphatase reaction so by measuring the amount of degraded RNA you should be able to measure the level of dephosphorylation.
We have worked extensively with ligating RNAs in our group in it seems that the ligation step is often the most critical. If it is in fact the ligation reaction that is at fault you can try a number of troubleshooting steps. For example try adding DMSO, PEG or a bridge oligo. Try lowering the ligation temperature and so on.
Hope this helped and good luck
Jesper
Hi,
For dephosphorylation I use Rpph (https://www.neb.com/products/m0356-rna-5-pyrophosphohydrolase-rpph) and I use 16ul of denatured RNA with 2ul of buffer+rpph.
As my RNAs are in-vitro transcribed, I need to dephosporylate them from tri-phosphates to mono-phosphates to make them ligatable.
Anyhow for my ligations , I use 11ul(rna's to be ligated), denature it and use DMSO,ATP , PEG8000 and ligase buffer. I use RNA ligase 1 for the purpose. Also they are ligated using a stint DNA at 16C for 16 hours.
I will try using RNAsin for the dephosporylation purpose. But if your lab works more on this, any help would be appreciated.Thanks Jesper and Binal.
@Jesper the above link is broken and I cant see it.
Anjali
I will try again
http://www.epibio.com/enzymes/nucleases-glycosylases-dna-binding-proteins/rna-exonucleases/terminator-5--phosphate-dependent-exonuclease
Otherwise, just google the following: Terminator exonuclease
I do purify the dephosporylated RNA before ligation step. Also I did check it on 30mer oligonucleotides before and that worked.
Thanks very much for the help nevertheless.
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