A DNA enzyme with Mg(2+)-dependent RNA phosphoesterase activity.

Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approximately 10(-4) min-1 in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 degrees C. The Mg(2+)-dependent reaction is more difficult, with an uncatalyzed rate of approximately 10(-7) min-1 under comparable conditions. Mg(2+)-dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+)-dependent reaction. We generated a population of > 10(13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg2+, Mn2+, Zn2+ or Pb2+. Examination of individual clones from the Mg2+ lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction. This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01 min-1. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions. We have generated a Mg(2+)-dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approximately 10(5)-fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.