Sequence specific cleavage of messenger RNA by a modified ribonuclease H.
Ribonuclease H (RNase H) is an endonuclease that cleaves only the RNA strand of an RNA-DNA hybrid to produce 5'-phosphate and 3'-hydroxy termini and lacks useful sequence specific recognition properties. A mutant form of the E. coli enzyme has been prepared that is suited for selective chemical modification at a site proximal to the substrate binding region. The chemical derivatization involves the formation of a disulfide linkage to a modified octadeoxyribonucleotide. The conjugate retains only 0.3% of the normal sequence independent RNase H activity demonstrating that substrate recognition can be modulated by a covalent appendage. A beta-globin RNA transcript containing a sequence complementary to that of the octadeoxyribonucleotide was cleaved in a catalytic fashion to two products upon treatment with the conjugate. The selectivity in the phosphodiester bond cleavage mediated by the conjugate was found to be different than that displayed by the nonderivatized enzyme. These results demonstrate the potential of semi-synthetic RNase H conjugates for mechanistic studies and their application as RNA targeted diagnostic or therapeutic agents.
Ma
W P
WP
Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, IN 47907-1333.
Hamilton
S E
SE
Stowell
J G
JG
Byrn
S R
SR
Davisson
V J
VJ
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005914:Globins / Q000235:genetics; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D012329:RNA, Bacterial; D012333:RNA, Messenger; D005914:Globins; D016914:Ribonuclease H