title: RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation.
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 cleaved RNA has 3'-hydroxyl and 5'-phosphate ends, so that the products can be used directly as substrates for ligation by T4 DNA ligase. We show that the cleavage reaction occurs efficiently in solution and on a solid streptavidin/agarose matrix. We report an example in which we are able to improve transcription yield by more than five-fold using this technique in the synthesis of a 15N isotopically labeled hairpin found in the Crithidia fasciculata spliced leader RNA. We are able to obtain a 0.5-mM NMR sample from this inherently poorly transcribing sequence, while minimizing the amount of isotopically labeled rNTPs used to produce it. The NMR spectroscopic results are consistent with the predicted RNA secondary structure.
authors.affiliation: Department of Chemistry, Yale University, New Haven, Connecticut 06511, USA.
authors.forename: J
authors.initials: J
authors.lastname: Lapham
authors.forename: D M
authors.initials: DM
authors.lastname: Crothers
mesh_terms: D000818:Animals; D001483:Base Sequence; D001710:Biotin; D016831:Crithidia fasciculata; D011088:DNA Ligases; D004274:DNA, Recombinant / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008745:Methylation; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics; D016914:Ribonuclease H; D012265:Ribonucleotides / Q000737:chemistry; D014158:Transcription, Genetic; D014764:Viral Proteins
chemical_list: D004274:DNA, Recombinant; D009587:Nitrogen Isotopes; D012333:RNA, Messenger; D016053:RNA, Protozoan; C039919:RNA, recombinant; D012265:Ribonucleotides; D014764:Viral Proteins; D012313:RNA; D001710:Biotin; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H; D011088:DNA Ligases