RNA ligation
Chris McDermott-Roe
I hoping to ligate a synthesised oligonucleotide to the 5' end (uncapped but
not dephosphorylated) of an mRNA population. Can anybody tell me the best
way of doing this?
many thanks
Chris
Nick Theodorakis
Are they both single stranded? I think T4 RNA ligase will do that
reaction.
See:
<http://www.neb.com/nebecomm/products/productM0204.asp>
Nick
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Nick Theodorakis
nick_the...@hotmail.com
contact form:
http://theodorakis.net/contact.html
Chris McDermott-Roe
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??
hope this makes sense
Chris
2009/11/19 Nick Theodorakis <nick.the...@gmail.com>
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Nick Theodorakis
<cjmcdermott...@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.
Aawara Chowdhury
Chris McDermott-Roe <cjmcder...@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 will NOT work. mRNAs do not have a free 5' phosphate. As you may
recall, mRNAs are capped with a 7-methylguanosine cap which is covalently
linked to the first transcribed nucleotide by a 5'-5' triphosphate bond.
There are mammalian/eukaryotic and viral decapping enzymes, but I don't
know of any that work on all mRNAs in vitro, or in vivo.
AC
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Email: echo 36434455860060025978157675027927670979097959886449930P | dc
Aawara Chowdhury
Nick Theodorakis <nick.the...@gmail.com> wrote:
>> 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 .. [deleted]..
Won't work for mRNA - there is no free 5'phosphate.
Nick Theodorakis
wrote:
> In <63e4a4c6-9734-4886-baba-dab9a41e7...@a21g2000yqc.googlegroups.com>,
> Nick Theodorakis <nick.theodora...@gmail.com> wrote:
>
> >> 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 .. [deleted]..
>
> Won't work for mRNA - there is no free 5'phosphate.
>
He specified that his RNA population is uncapped and phosphorylated in
his original post. I don't know how it got that way, but I can imagine
at least one scenario.
Aawara Chowdhury
Nick Theodorakis <nick.the...@gmail.com> wrote:
>>
>> Won't work for mRNA - there is no free 5'phosphate.
>>
>
> He specified that his RNA population is uncapped and phosphorylated in
> his original post. I don't know how it got that way, but I can imagine
> at least one scenario.
Then it cannot be mRNA. It could be pre-mRNA or hnRNA, but mRNA needs
to be capped.
Nick Theodorakis
wrote:
> In <4e7e0a8e-85b3-4eda-9190-7740ce3fc...@p35g2000yqh.googlegroups.com>,
> Nick Theodorakis <nick.theodora...@gmail.com> wrote:
>
>
>
> >> Won't work for mRNA - there is no free 5'phosphate.
>
> > He specified that his RNA population is uncapped and phosphorylated in
> > his original post. I don't know how it got that way, but I can imagine
> > at least one scenario.
>
> Then it cannot be mRNA. It could be pre-mRNA or hnRNA, but mRNA needs
> to be capped.
>
Well, whatever. I'll just assume that the OP knows something about the
RNA he has. But consider it could be:
1) prokaryotic or viral origin
2) mRNA that was chemically decapped or partially degraded
3) he could be looking for degradation intermediates.
If you want to get nitpicky I suppose you can call 2) or 3) "mRNA-
derived RNA fragments" or something, if that will make you happy.
Chris McDermott-Roe
phosphate off with tobacco acid pyrophosphatase - it's standard to most 5'
'RACE protocols. On the question of whether its technically correct to
refer to a decapped mRNA moiety as 'mRNA', I don't see any harm since an
uncapped mRNA can still function as a messenger RNA in that it can generate
protein products (albeit reduced in the case of cap-dependent translation
but not in the case of cap-independent translation).
Nick