MECHANISM AND UTILITY OF AN RNA-CLEAVING DNA ENZYME

We previously reported the in vitro selection of a general-purpose RNA -cleaving DNA enzyme that exhibits a catalytic efficiency (k(cat)/K-M) exceeding that of any other known nucleic acid enzyme [Santoro, S. W. and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4262-4266]. This enzyme contains similar to 30 deoxynucleotides and can cleave al most any RNA substrate under simulated physiological conditions, recog nizing the substrate through two Watson-Crick binding domains. The kin etics of cleavage under conditions of varying pH, choice of divalent m etal cofactor, and divalent metal concentration are consistent with a chemical mechanism involving metal-assisted deprotonation of a 2'-hydr oxyl of the substrate, leading to substrate cleavage. Kinetic measurem ents reveal that the enzyme strongly prefers cleavage of the substrate over ligation of the two cleavage products and that the enzyme's cata lytic efficiency is limited by the rate of substrate binding. The enzy me displays a high level of substrate specificity, discriminating agai nst RNAs that contain a single base mismatch within either of the two substrate-recognition domains. With appropriate design of the substrat e-recognition domains, the enzyme exhibits a potent combination of hig h substrate sequence specificity and selectivity, high catalytic effic iency, and rapid catalytic turnover.