Influence of Cleavage Site on Global Folding of an RNA-Cleaving DNAzyme

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 Mn2+, which was consistent with previous metal-induced folding studies of 8-17. Interestingly, Mn2+ 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 [Mn2+] was increased Taken together, these results support the previously observed hierarchy of 817 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.