CLEAVAGE OF SHORT RNAS CONTAINING HIGHER ORDERED STRUCTURES BY HAMMERHEAD RIBOZYMES

Cleavage of two types of secondary structure-forming substrates by the ir cognate hammerhead ribozymes were studied by measuring their kineti c parameters. A substrate with a self-complementary structure (GGUCCUA GGA, CL-3) was slowly cleaved by a two-stranded ribozyme. An isomer ha ving no complementary sequence (GGUC (G) under bar UAG (C) under bar A , CL-3N) was cleaved more than 10 times faster than the self-complemen tary substrate. A newly designed ribozyme which contained a stable loo p and stem cleaved the self-complementary decamer 40 times faster than the two-stranded ribozyme. A 15 mer which derived from a ras mRNA was found to have an intermolecular base pairs and was used to design mor e efficient ribozymes. Gel mobility shift assay was employed to invest igate the binding properties of substrates to ribozymes. Investigation s of the thermodynamic stability of the ribozyme-substrate complex are essential in the design of ribozymes that efficiently cleave RNA.