Design of RNA enzymes distinguishing a single base mutation in RNA.
RNA enzymes (ribozymes) which can cleave RNA by recognizing sequences of 9-15 bases are described. Substrates must contain UX (X = U, C or A). A ribozyme consisting of two oligoribonucleotides (19 mer and 15 mer) was shown to cleave a ribo 11 mer catalytically with Km and kcat values of 0.53 microM and 0.03 min-1, respectively. A non-cleavable substrate-ribozyme complex containing 2'-O-methylnucleoside was prepared and CD spectra were compared at different temperature. In order to obtain an efficient ribozyme, a one-strand RNA with a chain length of 37 was prepared. The ribozyme was shown to distinguish a single base mutation in mRNA's which were prepared by transcription of two synthetic DNA duplexes coding for positions 7-26 of c-Ha-ras protein. The mutant (Val-12) mRNA which had GUU was cleaved but the wild type mRNA which contained GGU was not changed, when treated by the ribozymes in the presence of Mg2+.
Koizumi
M
M
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Hayase
Y
Y
Iwai
S
S
Kamiya
H
H
Inoue
H
H
Ohtsuka
E
E
D001483:Base Sequence; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D016337:RNA, Catalytic; D012335:RNA, Ribosomal / Q000138:chemical synthesis / Q000378:metabolism*; D013379:Substrate Specificity
D016337:RNA, Catalytic; D012335:RNA, Ribosomal; D012313:RNA; D008274:Magnesium