Hi Michael, If the problem is the absence of a purine on the 5' end, try using the Marc Dreyfus' mutant T7 RNA polymerase. It can accommodate any nucleotide at the 5' end.
Guillerez, J., Lopez, P. J., Proux, F., Launay, H., and Dreyfus, M. (2005) A mutation in T7 RNA polymerase that facilitates promoter clearance, Proceedings of the National Academy of Sciences of the United States of America 102, 5958-5963. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15831591 With best regards, Eric -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Martin Hällberg Sent: Sunday, March 13, 2011 9:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Synthetic RNA for Crystallization 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 <mi...@chem.ucla.edu> > 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 > > mi...@chem.ucla.edu > > > > -- > Israel Sanchez Fernandez PhD > Ramakrishnan-lab > MRC Laboratory of Molecular Biology, > Hills Road, Cambridge, CB2 0QH, UK > >
