DNA-linked RNase H for site-selective cleavage of RNA.
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.
Uchiyama
Y
Y
Faculty of Pharmaceutical Sciences, Science University of Tokyo, Japan.
Inoue
H
H
Ohtsuka
E
E
Nakai
C
C
Kanaya
S
S
Ueno
Y
Y
Ikehara
M
M
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism*
D012313:RNA; D004247:DNA; D004262:DNA Restriction Enzymes; D016914:Ribonuclease H