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.