PMID: 8608452Mar 1, 1996Paper

RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation

RNA
J Lapham, D M Crothers

Abstract

Large quantities of RNA for study by NMR and X-ray crystallography can be produced by transcription reactions in vitro using T7 bacteriophage RNA polymerase. A limitation on producing RNA with this polymerase has been the strong dependence of the yield of the transcription reaction on the sequence at the 5' end of the RNA produced. We report a procedure for obtaining large quantities of enzymatically synthesized RNA from T7 RNA polymerase that has no dependence on the 5' end sequence of the target RNA. Ribonuclease H has been shown previously (Inoue H, Hayase Y, Iwai S, Ohtsuka E, 1987, FEBS Lett 215:327-330) to cleave RNA site specifically using 2'-O-methyl RNA/DNA chimeras to direct the cleavage site. We show that 2'-O-methyl RNA nucleotides on the 5'-side of the DNA nucleotides in the chimera are not essential for site-specific cleavage. This allowed us to design the method such that the same 2'-O-methyl chimera may be used to process any RNA sequence. We have adapted this reaction to the cleavage of NMR-scale quantities of RNA at high yield. RNA is synthesized using T7 RNA polymerase with a 15-nt high-yielding leader sequence at the 5' end, and then this sequence is cleaved off with the RNase H cleavage reaction. The cleave...Continue Reading

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