A DNA ENZYME WITH MG2-DEPENDENT RNA PHOSPHOESTERASE ACTIVITY()

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.