DNA-LINKED RNASE-H FOR SITE-SELECTIVE CLEAVAGE OF RNA

A DNA-linked RNase H (Hybrid Enz-1) (Kanaya et al. (1992) J. Biol. Che m. 267, 8492-8498), in which dGTCATCTCC was attached to E. coli RNase H via a covalent linker of 21 angstrom, was altered to improve the sit e-specific RNA cleavage by increasing the linker length. The sizes of the linkers on these hybrid enzymes (Hybrid Enz-2,-3, and -4) differed by 3 angstrom, the axial rise of the DNA/RNA hybrid, to give 18-, 24- , and 27-angstrom lengths. The conjugate with a size of 27 angstrom wa s able to cleave a synthetic 22mer RNA (5'-rAAGAUGUCUACGGAGAUGACCA-3') , containing the complementary 9mer RNA sequence (underlined), at one position, A16-U17. The kinetic parameters of Hybrid Enz-1, -2, -3, and -4 were examined using a 9mer RNA target. The results showed that lon ger linkers produced higher K(m), k(cat), and k(cat)/K(m) values, and the k(cat)/K(m) value of the conjugate with the 27-angstrom linker rea ched 83% of that of the wild-type RNase H. Hybrid Enz-4 was found to b e useful as an RNA restriction endonuclease.