Hi folks,
crystallized a lot of SRP RNAs - never had success with synthetic RNA,
even with small 30mers T7 RNA pol in vitro transcribed was always
extremely better - moreover: I always use a 3' hammerhead which yields
in the end 100% pure material in mg amounts - Kiyoshi Nagais protocol is
a perfect basis.
Greetings
Klemens
Martin Hällberg wrote:
Hi,
I'll second Israels's comment. Since the yield per coupling in synthesis is
lower for RNA than for DNA it gets really expensive over 30-35 nucleotides.
However, you can stitch together several 30 nt oligos using either T4 RNA ligase or T4 DNA ligase (with a DNA splint).
Regarding suppliers of synthetic RNA, Dharmacon is still very reliable in terms of actual delivered amount and quality. If you are going for very large scale then oligofactory.com is a good choice.
For a 90 nt oligo without any modifications I'd definitely go for T7 in-vitro transcription. On top of Nagai's classic that Israel referred to, I can also recommend the more recent method of native purification developed by Robert Batey and Jeffrey Kieft.
See:
Batey, R.T. & Kieft, J.S. Improved native affinity purification of RNA (2007)
RNA, 13, 1384-1389.
You are going to need milligrams of T7 RNA polymerase in the end so forget
about transcription kits, make your own.
Cheers,
Martin
On Mar 13, 2011, at 10:16 AM, Israel Sanchez wrote:
Hi Michael,
we normally produce synthetic RNAs following this classic paper if the size is more than let say 40-50 nucleotide, otherwise we buy the RNAs from Dharmacon and the quality is totally OK.
Hope it helps
J Mol Biol. 1995 Jun 2;249(2):398-408.
Crystallization of RNA-protein complexes. I. Methods for the large-scale
preparation of RNA suitable for crystallographic studies.
Price SR, Ito N, Oubridge C, Avis JM, Nagai K.
MRC Laboratory of Molecular Biology, Cambridge, UK.
Abstract
In vitro transcription using bacteriophage RNA polymerases and linearised
plasmid or oligodeoxynucleotide templates has been used extensively to produce
RNA for biochemical studies. This method is, however, not ideal for generating
RNA for crystallisation because efficient synthesis requires the RNA to have a
purine rich sequence at the 5' terminus, also the subsequent RNA is
heterogenous in length. We have developed two methods for the large scale
production of homogeneous RNA of virtually any sequence for crystallization. In
the first method RNA is transcribed together with two flanking
intramolecularly-, (cis-), acting ribozymes which excise the desired RNA
sequence from the primary transcript, eliminating the promoter sequence and
heterogeneous 3' end generated by run-off transcription. We use a combination
of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The
RNA-enzyme activity generates few sequence restrictions at the 3' terminus and
none at the 5' terminus, a considerable improvement on current methodologies.
In the second method the BsmAI restriction endonuclease is used to linearize
plasmid template DNA thereby allowing the generation of RNA with any 3' end. In
combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be
generated by in vitro transcription. This has proven to be extremely useful for
the synthesis of short RNAs.
2011/3/13 Michael Thompson <[email protected]>
Hello All,
I am looking for some advice from some experienced RNA crystallographers. I would
like to order some relatively short (<90 bases) synthetic RNAs for
crystallization trials. I was wondering if anyone could comment on the use of
synthetic RNAs for crystallization. Specifically, what is the longest synthetic
RNA that can be used for crystallization trials? I've seen some structures in the
PDB that are up to 88 bases and are reported to have been obtained with synthetic
constructs (3OWI - glycine riboswitch), but I don't really know if that's routine
or if it's an exceptional case. Also, for those who have experience with the use
of synthetic RNAs, I was wondering where people generally order their synthetic
constructs from? Our resident expert in RNA crystallography recommended a company
called Dharmacon (part of ThermoFisher), but I was hoping that I might get some
other opinions as to which companies make the best quality oligonucleotides,
provide samples with the highest purity, and have the most reasonable prices.
Thanks in advance for the help!
Mike
--
Michael C. Thompson
Graduate Student
Biochemistry & Molecular Biology Division
Department of Chemistry & Biochemistry
University of California, Los Angeles
[email protected]
--
Israel Sanchez Fernandez PhD
Ramakrishnan-lab
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 0QH, UK
