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Nucleic Acids Research, 1989, Vol. 17, No. 17 7059-7069
© 1989

ENZYMOLOGY

Design of RNA enzymes distinguishing a single base mutation in RNA

Makoto Koizumi, Yoji Hayase, Shigenori Iwai, Hiroyuki Kamiya, Hideo Inoue and Eiko Ohtsuka

Faculty of Pharmaceutical Sciences, Hokkaido University Sapporo 060, Japan

Received June 29, 1989. Revised August 4, 1989. Accepted August 4, 1989.

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 µM 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 rot changed, when treated by the ribozymes in the presence of Mg2+


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