DNAzymes have become attractive due to their potential biomedical and biotechnological applications, as well as their advantages in terms of stability, efficiency and synthetic accessibility with respect to protein or RNA catalysts. However, a lack of knowledge about the catalytic mechanisms of DNAzymes hampers further developments. Here, by means of high-level quantum mechanics/molecular mechanics simulations and biochemical studies, we determine the mechanism of RNA ligation catalysed by the 9DB1 DNAzyme. Our findings show that the mechanism consists of a single concerted asynchronous transition state where the O3′ atom of the acceptor RNA first attacks the α-phosphate group of the donor nucleotide, whereas the leftover proton from the O3′ atom is then transferred to the DNA. The mechanism involves the active participation of two Mg2+ ions, not present in the crystal structure but for which clear binding sites can be located.
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The data that support the plots within this paper and other findings of this study are available from the corresponding author on reasonable request.
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This work has been supported by the Spanish Ministry of Science (BFU2014-61670-EXP); the Catalan SGR; the Instituto Nacional de Bioinformática; the European Research Council (SimDNA); the European Union’s Horizon 2020 research and innovation program under grant agreement no. 676556; the Biomolecular and Bioinformatics Resources Platform (ISCIII PT 13/0001/0030), co-funded by the Fondo Europeo de Desarrollo Regional and the MINECO Severo Ochoa Award of Excellence (Government of Spain; awarded to IRB Barcelona). M.O. is an ICREA academia researcher. J.A. acknowledges the Spanish Ministry of Science for a Juan de la Cierva postdoctoral grant. H.G. acknowledges the Spanish Ministry of Science for a Juan de la Cierva postdoctoral grant. M.T. acknowledges the Instituto de Salud Carlos III for a Miguel Servet grant. We thank F. Eckstein, I. Brun-Heath, A. Grandas, E. Pedroso and R. Eritja for their help and valuable comments. We are indebted to J. L. Gelpí for his help in the processing of kinetic data.
The authors declare no competing interests.
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Supplementary methods, Supplementary Figs. 1–17, Supplementary Tables 1 and 2, Supplementary references.
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Reactants FES QM/MM
Transition State FES QM/MM
Final Structure MD
Initial Structure MD
Final Structure MD Post Catalytic
Initial Structure MD Post Catalytic
Final Structure MD Pre Catalytic
Initial Structure MD Pre Catalytic
Products PES QM/MM B3lyp
Reactants PES QM/MM B3lyp
Transition State PES QM/MM B3lyp
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Intermediate1 QM/MM PPI deprotonation
Products2 QM/MM PPI deprotonation
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Reactants2 QM/MM PPI deprotonation
Transition State1 QM/MM PPI deprotonation
Transition State2 QM/MM PPI deprotonation
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Aranda, J., Terrazas, M., Gómez, H. et al. An artificial DNAzyme RNA ligase shows a reaction mechanism resembling that of cellular polymerases. Nat Catal 2, 544–552 (2019). https://doi.org/10.1038/s41929-019-0290-y
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