Design of RNA enzymes distinguishing a single base mutation in RNA

Abstract

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 k_cat 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⁺