PMID:7524687

Citation	Uchiyama, Y, Inoue, H, Ohtsuka, E, Nakai, C, Kanaya, S, Ueno, Y and Ikehara, M DNA-linked RNase H for site-selective cleavage of RNA. Bioconjug. Chem. 5:327-32
Abstract	ADNA-linked RNase H (Hybrid Enz-1) (Kanaya et al. (1992) J. Biol. Chem. 267, 8492-8498), in which dGTCATCTCC was attached to E. coli RNase H via a covalent linker of 21 A, was altered to improve the site-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 A, the axial rise of the DNA/RNA hybrid, to give 18-, 24-, and 27-A lengths. The conjugate with a size of A was 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 longer linkers produced higher Km, kcat, and kcat/Km values, and the kcat/Km value of the conjugate with the 27-A linker reached 83% of that of the wild-type RNase H. Hybrid Enz-4 was found to be useful as an RNA restriction endonuclease.
Links	PubMed
Keywords	Base Sequence; DNA/chemistry; DNA Restriction Enzymes/metabolism; Kinetics; Molecular Sequence Data; Nucleic Acid Hybridization; RNA/chemistry; RNA/metabolism; Ribonuclease H/chemistry; Ribonuclease H/metabolism
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