Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes)1 or synthetic genetic polymers2. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage3. DNA-catalysed reactions include RNA and DNA ligation in various topologies4,5, hydrolytic cleavage6,7 and photorepair of DNA8, as well as reactions of peptides9,10 and small molecules11,12. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold13. Here we report the crystal structure of the RNA-ligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms.
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This work was supported by the Max Planck Society. We thank J. Ludwig for a gift of 5′-triphosphorylated RNAs, J. Seikowski for assistance with RNA and DNA synthesis, F. Wachowius and B. Samanta for initial crystallization samples, P. Afonine and members of the Pena and Höbartner laboratories for discussions, and the beamline staff at the Swiss Light Source, Villigen, Switzerland for assistance with data collection.
The authors declare no competing financial interests.
Extended data figures and tables
Nucleotides important for the crystal contacts are labelled accordingly.
Extended Data Figure 3 Distribution of pseudorotation phase angels of 9DB1 deoxyribozyme in comparison with twister46 and L1 ligase ribozyme30.
Left: deoxyribozyme 9DB1; middle: class I ribozyme; right: L1 ligase ribozyme. Figure was generated using Protein Data Bank (PDB) accession numbers 5CKK (9DB1), 3R1L (class I) and 2OIU (L1).
Extended Data Figure 5 Positioning of the reactive nucleotides in the active centre of ligase deoxyribozyme 9DB1 in comparison to ribozymes.
A schematic of the three enzymes is shown before and after the crystal structure determination (left and central columns, respectively). Strands that were deliberately chosen are depicted in red and blue, while the ones that resulted from random selection are shown in black. Nucleotides at the ligation junction are coloured in cyan, stacking nucleotides are depicted in light red. Nucleotides shown as white-filled circles are indicated for orientation purposes. Recognition between reactive and pairing nucleotides is shown as dashed lines (left column). Structures of product-bound nucleic acid enzymes are compared (PDB accessions 5CKK (9DB1), 3HHN (class I) and 2OIU (L1), right column).
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Ponce-Salvatierra, A., Wawrzyniak-Turek, K., Steuerwald, U. et al. Crystal structure of a DNA catalyst. Nature 529, 231–234 (2016). https://doi.org/10.1038/nature16471
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