Nature 529, 231–234 (2016)

Credit: VLADIMIR PENA

Enzymes comprised solely of DNA have not been observed in nature, but laboratory evolution experiments have identified proficient DNA catalysts. The lack of structural data on deoxyribozymes has limited our understanding of DNA-driven catalysis, but a new study reporting the X-ray crystal structure of the 9DB1 ligase deoxyribozyme, which catalyzes phosphodiester bond formation between the terminal 3′-hydroxyl and the 5′-triphosphate of RNA substrates, changes this landscape. In their 2.8-Å-resolution structure of a 9DB1 DNA construct with a product RNA strand, Ponce-Salvatierra et al. revealed a l-shaped folded complex, which is built from partially stacked short helical domains that are held in place by long-range base-pairing. Nucleotides of the A-G RNA ligation site are organized in a duplex-like conformation at the l three-way junction by specific base-pairing interactions with the protruding J2/3 region and additional stacking and tertiary interactions, which also enforce the enzyme's 3′,5′-ligation regiochemistry. Unlike with ligase ribozymes, no catalytic metal ions were observed in the active site of 9DB1, but a phosphate oxygen of dA13 is positioned nearby and critical for ligation catalysis. Further functional group mutagenesis of key base and sugar residues validated the 9DB1 structural model, defined how the greater conformational flexibility of DNA nucleotides facilitates catalytic structure formation and informed rational design strategies for engineering 9DB1 for broader substrate specificity.