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An RNA-cleaving threose nucleic acid enzyme capable of single point mutation discrimination

Nature Chemistry volume 14, pages 350–359 (2022)Cite this article

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Abstract

Threose nucleic acid has been considered a potential evolutionary progenitor of RNA because of its chemical simplicity, base pairing properties and capacity for higher-order functions such as folding and specific ligand binding. Here we report the in vitro selection of RNA-cleaving threose nucleic acid enzymes. One such enzyme, Tz1, catalyses a site-specific RNA-cleavage reaction with an observed pseudo first-order rate constant (kobs) of 0.016 min−1. The catalytic activity of Tz1 is maximal at 8 mM Mg2+ and remains relatively constant from pH 5.3 to 9.0. Tz1 preferentially cleaves a mutant epidermal growth factor receptor RNA substrate with a single point substitution, while leaving the wild-type intact. We demonstrate that Tz1 mediates selective gene silencing of the mutant epidermal growth factor receptor in eukaryotic cells. The identification of catalytic threose nucleic acids provides further experimental support for threose nucleic acid as an ancestral genetic and functional material. The demonstration of Tz1 mediating selective knockdown of intracellular RNA suggests that functional threose nucleic acids could be developed for future biomedical applications.

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Fig. 1: TNA enzymes capable of catalysing RNA-cleavage reactions.
Fig. 2: Biochemical characterization of RNA-cleaving TNA enzyme Tz1.
Fig. 3: Tz1 preferentially cleaves a mutant EGFR mRNA sequence with a single point mutation.
Fig. 4: Selective cleavage of EGFR T790M mRNA and downregulation of protein expression by Tz1.

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Data availability

Source data are provided with this paper. Full experimental details and data supporting the findings of this study are available within the article and its Supplementary Information, as well as the source data files.

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Acknowledgements

This work was supported by grants from the National Key Research and Development Program of China (2019YFA0904000 to H.Y. and 2016YFA0502600 to Z.L.); the National Natural Science Foundation of China (21977046 to H.Y. and 21708018 to H.Y.); the Fundamental Research Funds for the Central Universities (0213-14380192 to H.Y.); and the Program for Innovative Talents and Entrepreneur in Jiangsu (to Z.L. and H.Y.). We thank the reviewers for their critical reading of the manuscript and for their comments and suggestions.

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  1. These authors contributed equally: Yueyao Wang, Yao Wang.

Authors and Affiliations

  1. State Key Laboratory of Coordination Chemistry, Department of Biomedical Engineering, College of Engineering and Applied Sciences, Jiangsu Key Laboratory of Artificial Functional Materials, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, China

    Yueyao Wang, Yao Wang, Xin Sun & Hanyang Yu

  2. Applied Adaptome Immunology Institute, Jiangsu Industrial Technology Research Institute, Nanjing, China

    Yueyao Wang

  3. School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China

    Dongfan Song

  4. State Key Laboratory of Analytical Chemistry for Life Science, Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, Nanjing, China

    Zhe Li

  5. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, China

    Jia-Yu Chen

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Contributions

H.Y. conceived the project. Yueyao Wang performed the in vitro selection, biochemical characterization, structural probing and substrate selectivity experiments. Yao Wang performed the intracellular gene silencing experiment. D.S. analysed Tz1 and cleavage reaction products by mass spectrometry. X.S. analysed the biological stability of Tz1. Z.L. designed the Tz1 structural probing and intracellular gene silencing experiments. J.-Y.C. analysed the deep sequencing results of the structural probing experiment by the DMS method. H.Y. wrote the manuscript with input from all authors. All authors discussed the results and commented on the manuscript.

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Correspondence to Zhe Li or Hanyang Yu.

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Peer review information Nature Chemistry thanks Vitor Pinheiro, Seung Soo Oh 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 Chemical probing of Tz1 secondary structures.

(a) Tz1 and its RNA substrate were embedded in structural cassettes, which was subject to SHAPE (for RNA) and DMS (for RNA and TNA) analysis to inform secondary structure predictions. RNA, DNA and TNA regions were shown in grey, black and cyan, respectively. The RNA substrate was inactivated by a 2’-O-methyl modification (indicated by an asterisk) at cleavage site (indicated by a black inverted triangle). The reverse transcription and PCR primer binding sites were highlighted in yellow. In order to discern amplification products from DNA template contamination, a mismatch watermark adenine residue was introduced by reverse transcription primer. Amplification products from cDNA generated by reverse transcription contained adenine at position 115, and were then selected for analysis of mutation profiles at RNA substrate and Tz1 regions. (b) SHAPE reactivity of RNA substrate at each nucleotide position. Black, magenta and red bars indicate low, moderate, and high SHAPE reactivity, respectively. Black Triangle indicates cleavage site. (c) DMS reactivity at each nucleotide position. DMS reacts predominantly with adenine and cytosine bases. Positions were defined as highly reactive (orange bars) if reactivity was greater than 0.35, and as marginally reactive (pink bars) if reactivity was greater than a cut-off of one half standard deviation above the median. RNA substrate (residues 15-33) and Tz1 (residues 52-87) regions were shown. DMS reactivities of nucleotides within linker regions were shown in wheat.

Source data

Extended Data Fig. 2 Selectivity of Tz1 and deoxyribozyme 10-23 (Dz) on mutant and wild-type RNA substrates under different conditions.

(a and b) Tz1- and Dz-catalyzed RNA cleavage reactions at different enzyme:substrate ratios. The substrate concentration was maintained at 100 nM and the enzyme concentration varied. Reactions were carried out in a phosphate-buffered solution (pH 7.4) containing 1 mM KH2PO4, 3 mM Na2HPO4, 20 mM MgCl2, 155 mM NaCl at 37 °C for 3 h. (c and d) Tz1- and Dz-catalyzed RNA cleavage reactions at different magnesium concentrations. Reactions were carried out in a phosphate-buffered solution (pH 7.4) containing 1 mM KH2PO4, 3 mM Na2HPO4, 155 mM NaCl and different concentrations of MgCl2 at 37 °C for 3 h. [Enzyme] = 100 nM. [Substrate] = 100 nM. (E and F) Tz1- and Dz-catalyzed RNA cleavage reactions for different periods of time. Reactions were also carried out in a phosphate-buffered solution (pH 7.4) containing 1 mM KH2PO4, 3 mM Na2HPO4, 155 mM NaCl and 4 mM MgCl2 (for Tz1) or 16 mM MgCl2 (for Dz) at 37 °C for 3 h. [Enzyme] = 100 nM. [Substrate] = 100 nM. Error bars denote ±s.d. of the mean for n = 3 independent replicates.

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Wang, Y., Wang, Y., Song, D. et al. An RNA-cleaving threose nucleic acid enzyme capable of single point mutation discrimination. Nat. Chem. 14, 350–359 (2022). https://doi.org/10.1038/s41557-021-00847-3

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