Influence of cleavage site on global folding of an RNA-cleaving DNAzyme

Chembiochem. 2010 Aug 16;11(12):1710-9. doi: 10.1002/cbic.201000144.

Abstract

8-17 is a DNAzyme with metal-dependent endoribonuclease activity. Recently, a variant termed 8-17NG was reported as the first nucleic acid enzyme capable of cleaving all 16 dinucleotide junctions of RNA with rate enhancements ranging from 1000- to 1,000,000,000-fold over background activity. We attributed this broad-ranging cleavage efficiency to global folding of the DNAzyme. We sought to examine the influence of dinucleotides at the cleavage site of 8-17NG on global folding by using three-color (3c) FRET. By comparing the folding of 8-17NG with all 16 possible dinucleotide junctions, we found all examined DNAzyme-substrate constructs adopted a two-step folding process in the presence of Mn(2+), which was consistent with previous metal-induced folding studies of 8-17. Interestingly, Mn(2+) titration experiments also suggest that the second folding step is dependent on dinucleotide identity: purine-purine junctions allowed 8-17NG to fold at lower concentrations than pyrimidine-pyrimidine linkages. This finding was corroborated by RNA cleavage assays, in which the largest improvement in cleavage yield was observed in pyrimidine-pyrimidine junctions when [Mn(2+)] was increased. Taken together, these results support the previously observed hierarchy of 8-17 activity for different cleavage sites. Complemented by earlier sequence and structure-function studies, this investigation allowed for the first detailed examination of crucial relationships between the structural influence and junction preferences of nucleic acid-catalyzed RNA cleavage reactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Catalytic / chemistry
  • DNA, Catalytic / metabolism*
  • Fluorescence Resonance Energy Transfer
  • Manganese / chemistry
  • Manganese / metabolism*
  • Nucleic Acid Conformation*
  • Purines / metabolism*
  • Pyrimidines / metabolism*
  • RNA / chemistry
  • RNA / metabolism*
  • Structure-Activity Relationship

Substances

  • DNA, Catalytic
  • Purines
  • Pyrimidines
  • Manganese
  • RNA