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A biologically stable DNAzyme that efficiently silences gene expression in cells

Nature Chemistry volume 13, pages 319–326 (2021)Cite this article

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Matters Arising to this article was published on 18 July 2022

Abstract

Efforts to use RNA-cleaving DNA enzymes (DNAzymes) as gene-silencing agents in therapeutic applications have stalled due to their low efficacy in clinical trials. Here we report a xeno-nucleic-acid-modified version of the classic DNAzyme 10–23 that achieves multiple-turnover activity under cellular conditions and resists nuclease digestion. The new reagent, X10–23, overcomes the problem of product inhibition, which limited previous 10–23 designs, using molecular chemotypes with DNA, 2′-fluoroarabino nucleic acid and α-l-threofuranosyl nucleic acid backbone architectures that balance the effects of enhanced biological stability with RNA hybridization and divalent metal ion coordination. In cultured mammalian cells, X10–23 facilitates persistent gene silencing by efficiently degrading exogenous and endogenous messenger RNA transcripts. Together, these results demonstrate that new molecular chemotypes can improve the activity and stability of DNAzymes, and may provide a new route for nucleic acid enzymes to reach the clinic.

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Fig. 1: Kinetic analysis of F10–23.
Fig. 2: Engineering of the X10–23 nucleic acid enzyme.
Fig. 3: Functional activity and biostability of X10–23.
Fig. 4: Alternative 10–23 designs.
Fig. 5: GFP inhibition activity of X10–23 in HEK293 cells.
Fig. 6: Targeting endogenous oncogene KRAS by X10–23 in cancer cells.

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Data availability

The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

This work was supported by the W. M. Keck Foundation. Y.W. was supported by a postdoctoral fellowship from the Simons Collaboration on the Origins of Life.

Author information

Author notes

  1. These authors contributed equally: Yajun Wang, Kim Nguyen.

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of California, Irvine, CA, USA

    Yajun Wang, Kim Nguyen, Robert C. Spitale & John C. Chaput

  2. Department of Chemistry, University of California, Irvine, CA, USA

    Robert C. Spitale & John C. Chaput

  3. Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA

    Robert C. Spitale & John C. Chaput

Authors
  1. Yajun Wang
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Contributions

J.C.C. and R.C.S. conceived the project and designed the experiments. Y.W. and K.N. performed the experiments. J.C.C. wrote the manuscript with drafts from Y.W. and K.N. All the authors reviewed and commented on the manuscript.

Corresponding author

Correspondence to John C. Chaput.

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Competing interests

The authors and the University of California-Irvine have filed a patent application on the X10–23 reagent.

Additional information

Peer review information Nature Chemistry thanks Yingfu Li, Chuanzheng Zhou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Mechanistic analysis of X10-23.

Representative gels showing RNA cleavage activity in the presence and absence of RNase H for an internal segment of GFP (a-c) and the first exon segment of KRAS RNA (d-f). Color code: RNA (red), DNA (black), FANA (orange), and TNA (blue). (a,d) X10-23 with an active catalytic core. (b,e) X10-23 with an inactive catalytic core. (c,f) X10-23 with an active catalytic core that does not hybridize to the RNA target. All assays were performed in buffer containing 0.5 mM MgCl2 and 150 mM NaCl at 37 °C (pH 7.5) with 1 μM substrate and 1 μM enzyme. Nuclease reactions included 0.1 unit/μL of RNase H. S: full-length substrate, P: 5’ cleavage product. Molecular weight markers indicated to the right of the gel.

Source data.

Supplementary information

Supplementary Information

Supplementary Figs. 1–11, Tables 1 and 2, and Source Data.

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Source data

Source Data Fig. 2

Uncropped gel from Fig. 2.

Source Data Fig. 3

Uncropped gels from Fig. 3a.

Source Data Fig. 4

Uncropped gels from Fig. 4c.

Source Data Extended Data Fig. 1

Uncropped gels from Extended Data Fig. 1.

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Wang, Y., Nguyen, K., Spitale, R.C. et al. A biologically stable DNAzyme that efficiently silences gene expression in cells. Nat. Chem. 13, 319–326 (2021). https://doi.org/10.1038/s41557-021-00645-x

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