Background: Previously we demonstrated that DNA can act as an enzyme in the Pb2+-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of ∼10−4 min−1 in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23°C. The Mg2+-dependent reaction is more difficult, with an uncatalyzed rate of ∼10−7 min−1 under comparable conditions. Mg2+-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 Mg2+-dependent reaction.
Results: We generated a population of > 103 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. 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.
Conclusions: We have generated a Mg2+-dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate ∼105-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.
