Local rather than global folding enables the lead-dependent activity of the 8-17 deoxyribozyme: evidence from contact photo-crosslinking.
The 8-17 deoxyribozyme is an in vitro selected enzyme capable of sequence-specific cleavage of RNA. While selected to be a magnesium and zinc-utilizing enzyme, the 8-17 DNAzyme has been shown to utilize lead for its catalysis. Fluorescence-based experiments have indicated that the magnesium- and zinc-utilizing versions of the DNAzyme-substrate complex need to form a defined tertiary structure to be active, but no such global folding is required for the lead-mediated activity. Here, we have investigated this phenomenon, including the use of contact photo-crosslinking to map the tertiary fold of the lead-dependent DNAzyme. While our results recapitulate that global folding is not required for the lead activity, they reveal strikingly distinct lead-mediated modes of activity under conditions of low versus moderate solution ionic strength. Even in very low salt buffers, where no global folding of the 8-17 DNAzyme occurs, the active site of the enzyme appears to form a distinct local fold, one that cannot be modelled easily by DNA/RNA constructs that preserve key sequence and secondary structure features of the active site.
Liu
Yong
Y
Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
Sen
Dipankar
D
D001483:Base Sequence; D003432:Cross-Linking Reagents; D021881:DNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D055668:Photochemical Processes; D052156:SELEX Aptamer Technique; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D021881:DNA, Catalytic; C407482:RNA-cleaving DNA 8-17