Sequence-specific cleavage of RNA using chimeric DNA splints and RNase H.
To cleave RNA molecules using E. coli RNase H in a site-specific manner, a short oligodeoxyribonucleotide (3-5 mer) linked with oligo(2'-O-methyl)ribonucleotide(s) was designed to be used as a DNA splint. Our model experiments with ribooligomer the splint duplexes (9 mers) and RNase H demonstrated that a tetradeoxynucleotide cluster seems to be sufficient for the enzyme recognition and the short DNA-containing splint directs a unique cleavage of RNA by RNase H. The method could be applied to longer ribooligonucleotide substrates. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.
Inoue
H
H
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Hayase
Y
Y
Iwai
S
S
Ohtsuka
E
E
D001483:Base Sequence; D002678:Chimera; D004247:DNA; D004722:Endoribonucleases; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012313:RNA; D016914:Ribonuclease H; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D012313:RNA; D004247:DNA; D004722:Endoribonucleases; D016914:Ribonuclease H