DNA catalysts are single-stranded DNA molecules that fold into structures more complex than the double-helical form. Such deoxyribozymes have not been found in nature but have been synthesized and selected for specific activities in vitro. They are able to catalyze a wide range of reactions, including cleavage and ligation of DNA and RNA substrates. Despite more than two decades of biochemical study, mechanistic understanding of deoxyribozymes has been limited by the lack of any high-resolution structures of a deoxyribozyme in a productive conformation. Now, Ponce-Salvatierra et al. have presented the 2.8-Å-resolution crystal structure of the RNA-ligating deoxyribozyme 9DB1 in the postcatalytic state (Nature 529, 231–234, 2016).

Credit: Image from V. Pena

The deoxyribozyme 9DB1 consists of a central catalytic domain flanked by two arms that base-pair with the RNA substrate, leaving only the two nucleotides flanking the ligation junction unpaired. The structure of the 44-nucleotide deoxyribozyme in complex with a 15-nucleotide RNA strand reveals a double pseudoknot (pictured, with DNA in orange, RNA in blue and the ligation junction marked by a red sphere). The two nucleotides flanking the ligation junction form extensive tertiary contacts, including base pairs, with the DNA catalyst. Therefore, compensatory mutagenesis of these previously unknown base pairs allows manipulation of the substrate specificity.

There are interesting differences between the active site of 9DB1 and that of a ribozyme that catalyzes the same reaction. For example, no electron density for a catalytic metal ion is seen in the 9DB1 structure; instead, the authors have identified a putative catalytic phosphate. Compared to that of the ribozyme, the sugar-phosphate backbone of the catalytic domain of 9DB1 has greater conformational diversity, thus compensating for the lack of the 2′-hydroxyl group.

In summary, the structure confirms the idea that DNA can form complex tertiary folds that support catalysis. Perhaps single-stranded DNA molecules can also adopt complex folds with catalytic or other activities in vivo. In any case, the more detailed mechanistic understanding provided here and in future structural studies will be important for the use of deoxyribozymes in diverse applications.