Background

Previously we demonstrated that DNA can act as an enzyme in the Pb²⁺-dependent cleavage of an RNA posphoester. 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 reactions.

Results

We generated a population of >10¹³ DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phospheoster in the present of 1mM Mg²⁺, Mn²⁺, Zn²⁺ or Pb²⁺. Examination of individual clones from the Mg²⁺ lineage 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 seperate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover condition.

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 compativble with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.