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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 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.