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

Nature Catalysis volume 2, pages 544–552 (2019)Cite this article

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Abstract

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.

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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.

References

  1. Kruger, K. et al. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena. Cell 31, 147–157 (1982).

    Article  CAS  Google Scholar 

  2. Guerrier-Takada, C. et al. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35, 849–857 (1983).

    Article  CAS  Google Scholar 

  3. Guerrier-Takada, C. & Altman, S. Catalytic activity of an RNA molecule prepared by transcription in vitro. Science 223, 285–286 (1984).

    Article  CAS  Google Scholar 

  4. Ward, W. L., Plakos, K. & DeRose, V. J. Nucleic acid catalysis: metals, nucleobases, and other cofactors. Chem. Rev. 114, 4318–4342 (2014).

    Article  CAS  Google Scholar 

  5. Fedor, M. J. & Williamson, J. R. The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6, 399–412 (2005).

    Article  CAS  Google Scholar 

  6. Bertran, J. & Oliva, A. in Simulating Enzyme Reactivity: Computational Methods in Enzyme Catalysis (eds Tuñón, I. & Moliner, V.) 404–435 (The Royal Society of Chemistry, 2017).

  7. Tollefsbol, T. (ed.) Handbook of Epigenetics: The New Molecular and Medical Genetics (Academic Press, Cambridge, 2011).

  8. Berger, S. L. The complex language of chromatin regulation during transcription. Nature 447, 407–412 (2007).

    Article  CAS  Google Scholar 

  9. Breaker, R. R. & Joyce, G. F. A DNA enzyme that cleaves RNA. Chem. Biol. 1, 223–229 (1994).

    Article  CAS  Google Scholar 

  10. Silverman, S. K. Catalytic DNA (deoxyribozymes) for synthetic applications-current abilities and future prospects. Chem. Commun. 0, 3467–3485 (2008).

  11. Silverman, S. K. Catalytic DNA: scope, applications, and biochemistry of deoxyribozymes. Trends Biochem. Sci. 41, 595–609 (2016).

    Article  CAS  Google Scholar 

  12. Silverman, S. K. DNA as a versatile chemical component for catalysis, encoding, and stereocontrol. Angew. Chem. Int. Ed. 49, 7180–7201 (2010).

    Article  CAS  Google Scholar 

  13. Purtha, W. E., Coppins, R. L., Smalley, M. K. & Silverman, S. K. General deoxyribozyme-catalyzed synthesis of native 3′-5′ RNA linkages. J. Am. Chem. Soc. 127, 13124–13125 (2005).

    Article  CAS  Google Scholar 

  14. Flynn-Charlebois, A. et al. Deoxyribozymes with 2′-5′ RNA ligase activity. J. Am. Chem. Soc. 125, 2444–2454 (2003).

    Article  CAS  Google Scholar 

  15. Cuenoud, B. & Szostak, J. W. A. DNA metalloenzyme with DNA ligase activity. Nature 375, 611–614 (1995).

    Article  CAS  Google Scholar 

  16. Carmi, N., Balkhi, S. R. & Breaker, R. R. Cleaving DNA with DNA. Proc. Natl Acad. Sci. USA 95, 2233–2237 (1998).

    Article  CAS  Google Scholar 

  17. Wang, M. et al. In vitro selection of DNA-cleaving deoxyribozyme with site-specific thymidine excision activity. Nucleic Acids Res. 42, 9262–9269 (2014).

    Article  CAS  Google Scholar 

  18. Chandra, M., Sachdeva, A. & Silverman, S. K. DNA-catalyzed sequence-specific hydrolysis of DNA. Nat. Chem. Biol. 5, 718–720 (2009).

    Article  CAS  Google Scholar 

  19. Parker, D. J., Xiao, Y., Aguilar, J. M. & Silverman, S. K. DNA catalysis of a normally disfavored RNA hydrolysis reaction. J. Am. Chem. Soc. 135, 8472–8475 (2013).

    Article  CAS  Google Scholar 

  20. Wang, W., Billen, L. P. & Li, Y. Sequence diversity, metal specificity, and catalytic proficiency of metal-dependent phosphorylating DNA enzymes. Chem. Biol. 9, 507–517 (2002).

    Article  CAS  Google Scholar 

  21. Li, Y., Liu, Y. & Breaker, R. R. Capping DNA with DNA. Biochemistry 39, 3106–3114 (2000).

    Article  CAS  Google Scholar 

  22. Chinnapen, D. J.-F. & Sen, D. A deoxyribozyme that harnesses light to repair thymine dimers in DNA. Proc. Natl Acad. Sci. USA 101, 65–69 (2004).

    Article  CAS  Google Scholar 

  23. Li, Y. & Sen, D. A catalytic DNA for porphyrin metallation. Nat. Struct. Biol. 3, 743–747 (1996).

    Article  CAS  Google Scholar 

  24. Silverman, S. K. Pursuing DNA catalysts for protein modification. Acc. Chem. Res. 48, 1369–1379 (2015).

    Article  CAS  Google Scholar 

  25. Pradeepkumar, P. I., Höbartner, C., Baum, D. A. & Silverman, S. K. DNA-catalyzed formation of nucleopeptide linkages. Angew. Chem. Int. Ed. 47, 1753–1757 (2008).

    Article  CAS  Google Scholar 

  26. Xiang, Y. & Lu, Y. DNA as sensors and imaging agents for metal ions. Inorg. Chem. 53, 1925–1942 (2014).

    Article  CAS  Google Scholar 

  27. Shen, Z. et al. A catalytic DNA activated by a specific strain of bacterial pathogen. Angew. Chem. Int. Ed. 55, 2431–2434 (2016).

    Article  CAS  Google Scholar 

  28. Baum, D. A. & Silverman, S. K. Deoxyribozyme-catalyzed labeling of RNA. Angew. Chem. Int. Ed. 46, 3502–3504 (2007).

    Article  CAS  Google Scholar 

  29. Orbach, R., Willner, B. & Willner, I. Catalytic nucleic acids (DNAzymes) as functional units for logic gates and computing circuits: from basic principles to practical applications. Chem. Commun. 51, 4144–4160 (2015).

    Article  CAS  Google Scholar 

  30. Cai, H. et al. DNAzyme targeting c-jun suppresses skin cancer growth. Sci. Transl. Med. 4, 139ra82 (2012).

    Article  Google Scholar 

  31. Crunkhorn, S. Asthma: DNAzyme attenuates allergic asthma. Nat. Rev. Drug Discov. 14, 460–460 (2015).

    Google Scholar 

  32. Ponce-Salvatierra, A., Wawrzyniak-Turek, K., Steuerwald, U., Höbartner, C. & Pena, V. Crystal structure of a DNA catalyst. Nature 529, 231–234 (2016).

    Article  CAS  Google Scholar 

  33. Behera, A. K. et al. Enhanced deoxyribozyme-catalyzed RNA ligation in the presence of organic cosolvents. Biopolymers 99, 382–391 (2013).

    Article  CAS  Google Scholar 

  34. Büttner, L., Seikowski, J., Wawrzyniak, K., Ochmann, A. & Höbartner, C. Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation. Bioorg. Med. Chem. 21, 6171–6180 (2013).

    Article  Google Scholar 

  35. Barducci, A., Bussi, G. & Parrinello, M. Well-tempered metadynamics: a smoothly converging and tunable free-energy method. Phys. Rev. Lett. 100, 020603 (2008).

    Article  Google Scholar 

  36. Genna, V. et al. Self-activated mechanism for nucleic acid polymerization catalyzed by DNA/RNA polymerases. J. Am. Chem. Soc. 138, 14592–14598 (2016).

    Article  CAS  Google Scholar 

  37. Wu, S., Jiang, M. & Liu, L. Mechanism of pyrophosphate ion release in T7 RNA polymerase revealed by free energy simulations. Comput. Theor. Chem. 1094, 127–132 (2016).

    Article  CAS  Google Scholar 

  38. Da, L. T., Wang, D. & Huang, X. Dynamics of pyrophosphate ion release and its coupled trigger loop motion from closed to open state in RNA polymerase II. J. Am. Chem. Soc. 134, 2399–2406 (2012).

    Article  CAS  Google Scholar 

  39. Forconi, M. & Herschlag, D. (ed.) in Biophysical, Chemical, and Functional Probes of RNA Structure, Interactions and Folding: Part A Vol. 468, 311–333 (Academic Press, 2009).

  40. Loverix, S., Winqvist, A., Strömberg, R. & Steyaert, J. Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond. Chem. Biol. 7, 651–658 (2000).

    Article  CAS  Google Scholar 

  41. Steltz, T. A. A mechanism for all polymerases. Nature 391, 231–232 (1998).

    Article  Google Scholar 

  42. Shechner, D. M. & Bartel, D. P. The structural basis of RNA-catalyzed RNA polymerization. Nat. Struct. Mol. Biol. 18, 1036–1042 (2011).

    Article  CAS  Google Scholar 

  43. Zhao, C. & Pyle, A. M. Structural insights into the mechanism of group II intron splicing. Trends Biochem. Sci. 42, 470–482 (2017).

    Article  CAS  Google Scholar 

  44. Galej, W. P., Toor, N., Newman, A. J. & Nagai, K. Molecular mechanism and evolution of nuclear pre-mRNA and group II intron splicing: insights from cryo-electron microscopy structures. Chem. Rev. 118, 4156–4176 (2018).

    Article  CAS  Google Scholar 

  45. Stahley, M. R. & Strobel, S. A. Structural evidence for a two-metal-ion mechanism of group I intron splicing. Science 309, 1587–1590 (2005).

    Article  CAS  Google Scholar 

  46. Berdis, A. J. Mechanisms of DNA polymerases. Chem. Rev. 109, 2862–2879 (2009).

    Article  CAS  Google Scholar 

  47. Mattioli, E. J., Bottoni, A. & Calvaresi, M. DNAzymes at work: a DFT computational investigation on the mechanism of 9DB1. J. Chem. Inf. Model. 59, 1547–1553 (2019).

    Article  CAS  Google Scholar 

  48. Wachowius, F. & Höbartner, C. Probing essential nucleobase functional groups in aptamers and deoxyribozymes by nucleotide analogue interference mapping of DNA. J. Am. Chem. Soc. 133, 14888–14891 (2011).

    Article  CAS  Google Scholar 

  49. Gelpí, J. L. et al. Classical molecular interaction potentials: improved setup procedure in molecular dynamics simulations of proteins. Proteins Struct. Funct. Genet. 45, 428–437 (2001).

    Article  Google Scholar 

  50. Orozco, M. & Luque, F. J. Theoretical methods for the description of the solvent effect in biomolecular systems. Chem. Rev. 100, 4187–4226 (2000).

    Article  CAS  Google Scholar 

  51. Case, D. A. et al. AMBER 14 (University of California, San Francisco, 2014).

  52. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    Article  CAS  Google Scholar 

  53. Allnér, O., Nilsson, L. & Villa, A. Magnesium ion-water coordination and exchange in biomolecular simulations. J. Chem. Theory Comput. 8, 1493–1502 (2012).

    Article  Google Scholar 

  54. Meagher, K. L., Redman, L. T. & Carlson, H. A. Development of polyphosphate parameters for use with the AMBER force field. J. Comput. Chem. 24, 1016–1025 (2003).

    Article  CAS  Google Scholar 

  55. Ivani, I. et al. Parmbsc1: a refined force field for DNA simulations. Nat. Methods 13, 55–58 (2015).

    Article  Google Scholar 

  56. Cheatham, T. E., Cieplak, P. & Kollman, P. A. A modified version of the Cornell et al. force field with improved sugar pucker phases and helical repeat. J. Biomol. Struct. Dyn. 16, 845–862 (1999).

    Article  CAS  Google Scholar 

  57. Wang, J., Cieplak, P. & Kollman, P. A. How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J. Comput. Chem. 21, 1049–1074 (2000).

    Article  CAS  Google Scholar 

  58. Pérez, A. et al. Refinement of the AMBER force field for nucleic acids: improving the description of α/γ conformers. Biophys. J. 92, 3817–3829 (2007).

    Article  Google Scholar 

  59. Zgarbová, M. et al. Refinement of the Cornell et al. nucleic acids force field based on reference quantum chemical calculations of glycosidic torsion profiles. J. Chem. Theory Comput. 7, 2886–2902 (2011).

    Article  Google Scholar 

  60. Mlýnský, V. et al. Comparison of ab Initio, DFT, and semiempirical QM/MM approaches for description of catalytic mechanism of hairpin ribozyme. J. Chem. Theory Comput. 10, 1608–1622 (2014).

    Article  Google Scholar 

  61. Torrie, G. M. & Valleau, J. P. Nonphysical sampling distributions in Monte Carlo free-energy estimation: umbrella sampling. J. Comput. Phys. 23, 187–199 (1977).

    Article  Google Scholar 

  62. Kumar, S., Rosenberg, J. M., Bouzida, D., Swendsen, R. H. & Kollman, P. A. Multidimensional free‐energy calculations using the weighted histogram analysis method. J. Comput. Chem. 16, 1339–1350 (1995).

    Article  CAS  Google Scholar 

  63. Ufimtsev, I. S. & Martinez, T. J. Quantum chemistry on graphical processing units. 3. Analytical energy gradients, geometry optimization, and first principles molecular dynamics. J. Chem. Theory Comput. 5, 2619–2628 (2009).

    Article  CAS  Google Scholar 

  64. Götz, A. W., Clark, M. A. & Walker, R. C. An extensible interface for QM/MM molecular dynamics simulations with AMBER. J. Comput. Chem. 35, 95–108 (2014).

    Article  Google Scholar 

  65. Schowen, K. B. & Schowen, R. L. Solvent isotope effects of enzyme systems. Methods Enzymol. 87, 551–606 (1982).

    Article  CAS  Google Scholar 

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Acknowledgements

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.

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Authors and Affiliations

  1. Institute for Research in Biomedicine, Barcelona Institute of Science and Technology, Barcelona, Spain

    Juan Aranda, Montserrat Terrazas, Hansel Gómez, Núria Villegas & Modesto Orozco

  2. Joint BSC–Institute for Research in Biomedicine Research Program in Computational Biology, Barcelona, Spain

    Juan Aranda, Montserrat Terrazas, Hansel Gómez, Núria Villegas & Modesto Orozco

  3. Department of Biochemistry and Biomedicine, University of Barcelona, Barcelona, Spain

    Modesto Orozco

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Contributions

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.

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Correspondence to Modesto Orozco.

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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

Reactant

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). https://doi.org/10.1038/s41929-019-0290-y

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