RNA ligation

Nick Theodorakis via methods%40net.bio.net (by nick.theodorakis from gmail.com)
Thu Nov 19 23:23:41 EST 2009


On Nov 19, 5:06 am, Chris McDermott-Roe
<cjmcdermott... from googlemail.com> wrote:
> Hi,
>
> Yes, they'll both be single stranded.  One will be the mRNA that I want to
> tag (at the 5' end), the other will be a ss oligo.  My concern is that the
> oligo may ligate to the 3' end of the mRNA but I guess this will depend on
> how I have the oligo modified?  Is it sensible to assume that if it (the
> oligo) is 3' hydroxylated but NOT 5' phosphorylated, then it will bond with
> the exposed 5' phosphate on the mRNA and NOT the 3' OH??
>
>

That sounds sensible. If the oligo has no terminal phosphates and the
RNA is 5' phosphorylated, then the only reactions that should happen
are ligation of the oligo to the 5'end of the RNA or circularization
on the RNA. It seems to me you might be able to prevent the latter by
pCp tailing the RNA first if you think that will be a problem.
Disclaimer: I haven't tried any of these reactions myself. There are a
couple of references mentioned in the NEB I link I posted earlier that
might possibly be of use.

Nick

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