Background: Previously we demonstrated that DNA can act as an enzyme i
n the Pb2+-dependent cleavage of an RNA phosphoester. This is a facile
reaction, with an uncatalyzed rate for a typical RNA phosphoester of
similar to 10(-4) min(-1) in the presence of 1 mM Pb(OAc)(2) at pH 7.0
and 23 degrees C. The Mg2+-dependent reaction is more difficult, with
an uncatalyzed rate of similar to 10(-7) min(-1) under comparable con
ditions. Mg2+-dependent cleavage has special relevance to biology beca
use it is compatible with intracellular conditions. Using in vitro sel
ection, we sought to develop a family of phosphoester-cleaving DNA enz
ymes that operate in the presence of various divalent metals, focusing
particularly on the Mg2+-dependent reaction. Results: We generated a
population of >10(13) DNAs containing 40 random nucleotides and carrie
d out repeated rounds of selective amplification, enriching for molecu
les that cleave a target RNA phosphoester in the presence of 1 mM Mg2, Mn2+, Zn2+ or Pb2+. Examination of individual clones from the Mg2+ l
ineage after the sixth round revealed a catalytic motif comprised of a
three-stem junction. This motif was partially randomized and subjecte
d to seven additional rounds of selective amplification, yielding cata
lysts with a rate of 0.01 min(-1). The optimized DNA catalyst was divi
ded into separate substrate and enzyme domains and shown to have a sim
ilar level of activity under multiple turnover conditions. Conclusions
: We have generated a Mg2+-dependent DNA enzyme that cleaves a target
RNA phosphoester with a catalytic rate similar to 10(5)-fold greater t
han that of the uncatalyzed reaction. This activity is compatible with
intracellular conditions, raising the possibility that DNA enzymes mi
ght be made to operate in vivo.