Abstract
Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic genetic polymers. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage. DNA-catalysed reactions include RNA and DNA ligation in various topologies, hydrolytic cleavage and photorepair of DNA, as well as reactions of peptides and small molecules. 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 fold. 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.
Highlights
Catalysis in biology is restricted to RNA and protein enzymes, but synthetic biomolecular catalysts can be made of DNA[1] or synthetic genetic polymers[2]
The deoxyribozyme 9DB1 catalyses the regioselective formation of a native phosphodiester bond between the 3′-hydroxyl and the 5′-triphosphate termini of two RNA strands, using divalent metal ions (Mg2+ or Mn2+) as cofactors[14,15]
The 9DB1 DNA enzyme is composed of a central catalytic domain flanked by two arms that hybridize to the RNA substrates by canonical Watson–Crick base pairing (Fig. 1a, b), leaving only the two nucleotides embracing the ligation junction unpaired
Summary
Almudena Ponce-Salvatierra[1,2], Katarzyna Wawrzyniak-Turek[1,3], Ulrich Steuerwald[2], Claudia Höbartner1,3 & Vladimir Pena[2]. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage[3]. Attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold[13]. The 9DB1 DNA enzyme is composed of a central catalytic domain flanked by two arms that hybridize to the RNA substrates by canonical Watson–Crick base pairing (Fig. 1a, b), leaving only the two nucleotides embracing the ligation junction unpaired. The model built in the experimental map was refined against a 2.8 Å resolution data set collected from a native crystal (Fig. 1c). The RNA nucleotides A−1 and G1 encompass the ligation junction and form extensive tertiary contacts with the DNA catalyst (Fig. 1d, e). The 3′-overhanging DNA and RNA nucleotides form a semi-continuous duplex in the crystal lattice a
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