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Crystal structure of a DNA catalyst

Abstract

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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Figure 1: Global architecture of the DNA catalyst.
Figure 2: Tertiary contacts within the catalytic domain.
Figure 3: Positioning of reactive nucleotides in the catalytic domain.
Figure 4: Active site of 9DB1.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Structural models and structure factors have been deposited in the Protein Data Bank under accession numbers 5CKI (cobalt) and 5CKK (native).

References

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

    Article  CAS  PubMed  Google Scholar 

  2. Taylor, A. I. et al. Catalysts from synthetic genetic polymers. Nature 518, 427–430 (2015)

    Article  ADS  CAS  PubMed  Google Scholar 

  3. Schlosser, K. & Li, Y. Biologically inspired synthetic enzymes made from DNA. Chem. Biol. 16, 311–322 (2009)

    Article  CAS  PubMed  Google Scholar 

  4. Coppins, R. L. & Silverman, S. K. A DNA enzyme that mimics the first step of RNA splicing. Nature Struct. Mol. Biol. 11, 270–274 (2004)

    Article  CAS  Google Scholar 

  5. Sreedhara, A., Li, Y. & Breaker, R. R. Ligating DNA with DNA. J. Am. Chem. Soc. 126, 3454–3460 (2004)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Gu, H., Furukawa, K., Weinberg, Z., Berenson, D. F. & Breaker, R. R. Small, highly active DNAs that hydrolyze DNA. J. Am. Chem. Soc. 135, 9121–9129 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Walsh, S. M., Sachdeva, A. & Silverman, S. K. DNA catalysts with tyrosine kinase activity. J. Am. Chem. Soc. 135, 14928–14931 (2013)

    Article  CAS  PubMed  Google Scholar 

  10. Chandrasekar, J. & Silverman, S. K. Catalytic DNA with phosphatase activity. Proc. Natl Acad. Sci. USA 110, 5315–5320 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  12. Chandra, M. & Silverman, S. K. DNA and RNA can be equally efficient catalysts for carbon–carbon bond formation. J. Am. Chem. Soc. 130, 2936–2937 (2008)

    Article  CAS  PubMed  Google Scholar 

  13. Nowakowski, J., Shim, P. J., Prasad, G. S., Stout, C. D. & Joyce, G. F. Crystal structure of an 82-nucleotide RNA–DNA complex formed by the 10-23 DNA enzyme. Nature Struct. Biol. 6, 151–156 (1999)

    Article  CAS  PubMed  Google Scholar 

  14. 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  PubMed  Google Scholar 

  15. 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  PubMed  CAS  Google Scholar 

  16. Vicens, Q. & Cech, T. R. A natural ribozyme with 3′,5′ RNA ligase activity. Nature Chem. Biol. 5, 97–99 (2009)

    Article  CAS  Google Scholar 

  17. Bartel, D. P. & Szostak, J. W. Isolation of new ribozymes from a large pool of random sequences [see comment]. Science 261, 1411–1418 (1993)

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Ekland, E. H., Szostak, J. W. & Bartel, D. P. Structurally complex and highly active RNA ligases derived from random RNA sequences. Science 269, 364–370 (1995)

    Article  ADS  CAS  PubMed  Google Scholar 

  19. Jaeger, L., Wright, M. C. & Joyce, G. F. A complex ligase ribozyme evolved in vitro from a group I ribozyme domain. Proc. Natl Acad. Sci. USA 96, 14712–14717 (1999)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wachowius, F., Javadi-Zarnaghi, F. & Höbartner, C. Combinatorial mutation interference analysis reveals functional nucleotides required for DNA catalysis. Angew. Chem. Int. Ed. Engl. 49, 8504–8508 (2010)

    Article  CAS  PubMed  Google Scholar 

  21. 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  PubMed  Google Scholar 

  22. Ikawa, Y., Tsuda, K., Matsumura, S. & Inoue, T. De novo synthesis and development of an RNA enzyme. Proc. Natl Acad. Sci. USA 101, 13750–13755 (2004)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. Pitt, J. N. & Ferré-D’Amaré, A. R. Structure-guided engineering of the regioselectivity of RNA ligase ribozymes. J. Am. Chem. Soc. 131, 3532–3540 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Coppins, R. L. & Silverman, S. K. Rational modification of a selection strategy leads to deoxyribozymes that create native 3′–5′ RNA linkages. J. Am. Chem. Soc. 126, 16426–16432 (2004)

    Article  CAS  PubMed  Google Scholar 

  25. Doudna, J. A. & Cech, T. R. The chemical repertoire of natural ribozymes. Nature 418, 222–228 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Davies, D. R. et al. Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets. Proc. Natl Acad. Sci. USA 109, 19971–19976 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Shechner, D. M. et al. Crystal structure of the catalytic core of an RNA-polymerase ribozyme. Science 326, 1271–1275 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  30. Robertson, M. P. & Scott, W. G. The structural basis of ribozyme-catalyzed RNA assembly. Science 315, 1549–1553 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  31. Pitsch, S., Weiss, P. A., Jenny, L., Stutz, A. & Wu, X. Reliable chemical synthesis of oligoribonucleotides (RNA) with 2′-O-[(Triisopropylsilyl)oxy]methyl(2′-O-TOM)-protected phosphoramidites. Helv. Chim. Acta 84, 3773–3795 (2001)

    Article  CAS  Google Scholar 

  32. Kumar, R. K., Olsen, P. & Ravikumar, V. T. An alternative advantageous protocol for efficient synthesis of phosphorothioate oligonucleotides utilizing phenylacetyl disulfide (PADS). Nucleosides Nucleotides Nucleic Acids 26, 181–188 (2007)

    Article  CAS  PubMed  Google Scholar 

  33. Frederiksen, J. K. & Piccirilli, J. A. Separation of RNA phosphorothioate oligonucleotides by HPLC. Methods Enzymol. 468, 289–309 (2009)

    Article  CAS  PubMed  Google Scholar 

  34. Koch, M. et al. Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered GTP hydrolysis. Proc. Natl Acad. Sci. USA 112, E2561–E2568 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Ludwig, J. & Eckstein, F. Rapid and efficient synthesis of nucleoside 5′-O-(1-thiotriphosphates), 5′-triphosphates and 2′,3′-cyclophosphorothioates using 2-chloro-4h-1,3,2-benzodioxaphosphorin-4-one. J. Org. Chem. 54, 631–635 (1989)

    Article  CAS  Google Scholar 

  36. Goldeck, M., Tuschl, T., Hartmann, G. & Ludwig, J. Efficient solid-phase synthesis of pppRNA by using product-specific labeling. Angew. Chem. Int. Ed. Engl. 53, 4694–4698 (2014)

    Article  CAS  PubMed  Google Scholar 

  37. Kabsch, W. X. D. S. Acta Crystallogr. D 66, 125–132 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Terwilliger, T. C. et al. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D 65, 582–601 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Keating, K. S. & Pyle, A. M. RCrane: semi-automated RNA model building. Acta Crystallogr. D 68, 985–995 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Sun, G., Voigt, J. H., Filippov, I. V., Marquez, V. E. & Nicklaus, M. C. PROSIT: pseudo-rotational online service and interactive tool, applied to a conformational survey of nucleosides and nucleotides. J. Chem. Inf. Comput. Sci. 44, 1752–1762 (2004)

    Article  CAS  PubMed  Google Scholar 

  46. Ren, A. et al. In-line alignment and Mg2+ coordination at the cleavage site of the env22 twister ribozyme. Nat. Commun. 5, 5534 (2014)

    Article  ADS  CAS  PubMed  Google Scholar 

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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

Crystallographic work was performed by A.P.-S. under the supervision of V.P., biochemical experiments were performed by A.P.-S., K.W.-T. and C.H. U.S. obtained initial crystals. A.P.-S., V.P. and C.H. designed the experiments and all authors discussed the results and contributed to the manuscript.

Corresponding authors

Correspondence to Claudia Höbartner or Vladimir Pena.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Semi-continuous helix in the crystal lattice.

Nucleotides important for the crystal contacts are labelled accordingly.

Extended Data Figure 2 Influence of 2′-modifications on mutant acceptor RNA U-1.

Extended Data Figure 3 Distribution of pseudorotation phase angels of 9DB1 deoxyribozyme in comparison with twister46 and L1 ligase ribozyme30.

Extended Data Figure 4 Active sites of RNA-ligating nucleic acid enzymes.

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).

Extended Data Table 1 Data collection and refinement statistics
Extended Data Table 2 Oligonucleotides for kinetic experiments

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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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