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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 <javascript:AL_get(this,%20'jour',%20'J%20Mol%20Biol.');> J Mol Biol. <javascript:AL_get(this,%20'jour',%20'J%20Mol%20Biol.');> 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<http://www.ncbi.nlm.nih.gov/pubmed?term=%22Price%20SR%22%5BAuthor%5D>, Ito N <http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ito%20N%22%5BAuthor%5D>, Oubridge C <http://www.ncbi.nlm.nih.gov/pubmed?term=%22Oubridge%20C%22%5BAuthor%5D>, Avis JM <http://www.ncbi.nlm.nih.gov/pubmed?term=%22Avis%20JM%22%5BAuthor%5D>, Nagai K <http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nagai%20K%22%5BAuthor%5D>. 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 <[log in to unmask]> > 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 > > [log in to unmask] > -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK