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 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.
Lam
Jeffrey C F
JC
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, L8N 3Z5, Canada.
Li
Yingfu
Y
D021881:DNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011687:Purines / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D021881:DNA, Catalytic; D011687:Purines; D011743:Pyrimidines; D008345:Manganese; D012313:RNA