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An artificial DNAzyme RNA ligase shows a reaction mechanism resembling that of cellular polymerases


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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Fig. 1: The 9DB1 DNAzyme architecture and its post-catalytic active site.
Fig. 2: The prediction of two new cation binding regions in the pre-catalytic model.
Fig. 3: The pre-catalytic active site and its most important interactions along MD simulation.
Fig. 4: The reaction mechanism of RNA ligation catalysed by 9DB1 DNAzyme.
Fig. 5: Experimental validation.
Fig. 6: Stabilization of the transition state inside protein enzymes and 9DB1 active sites.

Data availability

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.

Author information

Authors and Affiliations



J.A. performed the simulations, analysed the data and discussed the experiments. M.T. performed the experiments, analysed the data and discussed the simulations. H.G. contributed in the simulations. N.V. contributed to all of the experiments and M.O. directed and supervised the research. J.A., M.T. and M.O. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Modesto Orozco.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary methods, Supplementary Figs. 1–17, Supplementary Tables 1 and 2, Supplementary references.

Reporting Summary

Supplementary Data 1

Products FES QM/MM

Supplementary Data 2

Reactants FES QM/MM

Supplementary Data 3

Transition State FES QM/MM

Supplementary Data 4

Final Structure MD

Supplementary Data 5

Initial Structure MD

Supplementary Data 6

Final Structure MD Post Catalytic

Supplementary Data 7

Initial Structure MD Post Catalytic

Supplementary Data 8

Final Structure MD Pre Catalytic

Supplementary Data 9

Initial Structure MD Pre Catalytic

Supplementary Data 10

Products PES QM/MM B3lyp

Supplementary Data 11

Reactants PES QM/MM B3lyp

Supplementary Data 12

Transition State PES QM/MM B3lyp

Supplementary Data 13

Products PES QM/MM Blyp

Supplementary Data 14

Reactants PES QM/MM Blyp

Supplementary Data 15

Transition State PES QM/MM Blyp

Supplementary Data 16

Intermediate1 QM/MM PPI deprotonation

Supplementary Data 17

Products2 QM/MM PPI deprotonation

Supplementary Data 18

Reactants1 QM/MM PPI deprotonation

Supplementary Data 19

Reactants2 QM/MM PPI deprotonation

Supplementary Data 20

Transition State1 QM/MM PPI deprotonation

Supplementary Data 21

Transition State2 QM/MM PPI deprotonation

Supplementary Video 1

Reaction Mechanism

Supplementary Video 2


Supplementary Video 3

Transition State

Supplementary Video 4


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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).

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