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A modular XNAzyme cleaves long, structured RNAs under physiological conditions and enables allele-specific gene silencing

Nature Chemistry volume 14, pages 1295–1305 (2022)Cite this article

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Abstract

Nucleic-acid catalysts (ribozymes, DNA- and XNAzymes) cleave target (m)RNAs with high specificity but have shown limited efficacy in clinical applications. Here we report on the in vitro evolution and engineering of a highly specific modular RNA endonuclease XNAzyme, FR6_1, composed of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid (FANA). FR6_1 overcomes the activity limitations of previous DNA- and XNAzymes and can be retargeted to cleave highly structured full-length (>5 kb) BRAF and KRAS mRNAs at physiological Mg2+ concentrations with allelic selectivity for tumour-associated (BRAF V600E and KRAS G12D) mutations. Phosphorothioate-FANA modification enhances FR6_1 biostability and enables rapid KRAS mRNA knockdown in cultured human adenocarcinoma cells with a G12D-allele-specific component provided by in vivo XNAzyme cleavage activity. These results provide a starting point for the development of improved gene-silencing agents based on FANA or other XNA chemistries.

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Fig. 1: FR6_1 is an RNA endonuclease XNAzyme composed of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acids (FANA).
Fig. 2: FR6_1 is a modular XNAzyme and can be reprogrammed to alternative RNA targets.
Fig. 3: Reselection and modification of an FR6_1 variant yields a biostable allele-specific XNAzyme targeting KRAS G12D RNA.
Fig. 4: KRAS-targeting XNAzymes can invade and cleave long, structured mRNA in quasi-physiological conditions.
Fig. 5: The KRAS G12D-targeting XNAzyme retains catalytic activity inside RKO cells and performs G12D-specific mRNA knockdown.

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Data availability

All data generated or analysed during this study are included in this published Article and its Supplementary Information files, except raw sequencing reads, which are available in the NCBI SRA repository, BioProject ID PRJNA847751. Source data are provided with this Paper.

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Acknowledgements

We thank M. Watson (Sigma-Aldrich/Merck) for synthesis of FANA oligonucleotides, L. Stadler for assistance with preliminary experiments on the delivery of FANA, and members of the Holliger and Taylor groups for discussions and suggestions. This work was supported by the Medical Research Council (A.I.T., C.J.K.W., S.-Y.P.-C., P.H., programme no. MC_U105178804), the Biotechnology and Biological Sciences Research Council (INTENSIFY BB/M005623/1 to P.H. and A.I.T.), the AMGEN Scholars programme (C.J.K.W.), the Wellcome Trust (M.J.D., PhD studentship) and a Wellcome Trust/Royal Society Sir Henry Dale Fellowship to A.I.T. (215453/Z/19/Z).

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Authors and Affiliations

  1. Cambridge Institute of Therapeutic Immunology & Infectious Disease (CITIID), University of Cambridge, Cambridge, UK

    Alexander I. Taylor & Maria J. Donde

  2. MRC Laboratory of Molecular Biology, Cambridge, UK

    Christopher J. K. Wan, Sew-Yeu Peak-Chew & Philipp Holliger

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  1. Alexander I. Taylor
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Contributions

A.I.T. and P.H. conceived the project. A.I.T. designed and conducted experiments and analysed data with C.J.K.W., M.J.D. and S.-Y.P.-C. A.I.T. and P.H. wrote the manuscript, with comments from all authors.

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Extended data

Extended Data Fig. 1 Steady-state kinetics of FR6_1 XNAzyme-catalysed RNA cleavage under quasi-physiological conditions.

Michaelis-Menten curve determined for the cleavage of Sub_Ebo RNA by the FR6_1 XNAzyme under quasi-physiological conditions (37 °C, pH 7.4, 1 mM Mg2+).

Source data

Extended Data Fig. 2 Evaluation of RNA substrate cleavage site preferences by the FR6_1 XNAzyme.

(a) Sequence of “FR6_1_NucSR”, a variant of XNAzyme FR6_1 retargeted to cleave RNA substrate “NucSR”(see reference (56)) by changing FANA residues shown in brown. (b) Urea-PAGE gels showing reactions of FR6_1_NucSR (5 uM) with all possible variants of RNA substrate NucSR (1 uM) at positions 8, 9 and 10 (shown in red in (a))(24 h, 17 °C, 10 mM Mg2+).

Source data

Extended Data Fig. 3 Engineering the KRAS G12D-targeting XNAzyme to invade long RNA substrates.

(a-c) Urea-PAGE gels showing activity of variants of XNAzyme FR6_1_KRas12 (“Fz12”)(1.25 uM) with alternative length substrate-binding arms as indicated, and FR6_1_KRas12B (“Fz12B”)(which has 10 + 10, partially FANA-PS modified binding arms, see Fig. 3), on alternative length RNA substrates (0.25 uM): (a) Sub_KRas12 (30 nt), (b) Sub_KRas12_long (68 nt) and Sub_KRas_ORF (2.1 kb), a synthetic transcript comprising the full KRAS ORF and UTRs (1.5 h, 37 °C, pH 7.4, 1 mM Mg2+). (d) Urea-PAGE gel showing Fz12B (10 nM) performing multiple turnover catalysis of Sub_KRas12 [G12D] (1 uM) RNA cleavage (37 °C, pH 7.4, 1 mM Mg2+). Substrate secondary structure diagrams were generated using RNAfold (Methods reference85,86).

Source data

Extended Data Fig. 4 Characterisation of RNase H1-dependent RNA cleavage induced by active and inactive XNAzymes and DNAzymes.

Urea-PAGE gel showing assays of human RNase H1-mediated cleavage of RNA substrate Sub_KRas12_long [G12D], induced by XNAzyme FR6_1_KRas12B (“Fz12B”), catalytically-inactive FR6_1_KRas12B[G27A] (“Fz12Bi”), or DNAzymes 10-23_KRasC [6 + 7] or 10-23_KRasC [10 + 10]. Sequences and putative secondary structures of Fz12B and target RNA show deduced locations of cleavage sites mediated by RNase H (red arrows) or the FANAzyme alone (green arrow). Note that partial alkaline hydrolysis of the RNA substrate (-OH) and XNAzyme-mediated cleavage produce 5’ RNA products that terminate in 3’ cyclic phosphate, whereas RNase H-mediated cleavage produces 3’ OH termini; the resulting difference in PAGE mobility has been taken in account when deducing cleavage sites.

Source data

Extended Data Fig. 5 The KRAS G12D-targeting XNAzyme performs G12D-specific mRNA knockdown inside PANC-1 cells.

Example droplet digital RT-qPCR (ddPCR) multiplex assays (see Supplementary Table 3) of (a) total KRAS mRNA (channel 1, blue) and reference EIF2B2 mRNA (channel 2, green), and (b) KRAS alleles G12D (c.35 G > A) mRNA (channel 1, blue) and wild-type (wt) mRNA (channel 2, green), in RKO cells (homozygous for either G12D or wt KRAS) or PANC-1 cells (heterozygous for G12D and wt KRAS). Together, these results show that the baseline level of total KRAS expression in PANC-1 cells was ~3-fold higher than in RKO KRASG12D/G12D, with the G12D allele mRNA transcribed at approximately twice the level of the wild-type KRAS mRNA. (c) Dose response curves showing (bottom graph) total KRAS mRNA levels (relative to reference EIF2B2 mRNA and normalised to cells transfected with buffer alone (yellow circles)) and (top graph) ratio of G12D: wt KRAS mRNA, in PANC-1 cells 3 h after transfection with XNAzyme “FR6_1_KRas12B” (Fz12B)(red circles and dashed line) or the catalytically inactive mutant “FR6_1_KRas12Bi” (Fz12Bi)(black triangles and dashed line) at the concentrations shown. Each datum represents an independent biological replicate and error bars show ddPCR total error. (d) Chart showing proposed interpretation of data from (c); levels of G12D (blue bars) and wt (green bars) KRAS mRNA are equally reduced by RNase H-mediated knockdown (represented as dashed blue and green bars) induced by both the active and inactive XNAzymes (although to a greater degree by the inactive XNAzyme). In the case of the inactive XNAzyme, due to the unequal baseline transcription of G12D and wt KRAS mRNA, this causes an increase in the ratio of G12D: wt KRAS mRNA. With the active XNAzyme - in addition to this effect - allele-specific XNAzyme-mediated knockdown (represented as dashed purple bar) reduces the level of G12D, but not wt KRAS mRNA, maintaining (or reducing) of the baseline ratio of G12D: wt KRAS mRNA.

Supplementary information

Supplementary Information

Supplementary Figs. 1–9, source data for supplementary figures, the key to source data displayed as graphs in the supplementary figures.

Reporting Summary

Supplementary Table 1

Supplementary Tables 1–3

Supplementary Data 1

Raw ddPCR data for data used in the supplementary figures.

Supplementary Table 2

Key to source data displayed as graphs in figures and extended data.

Source data

Source Data Fig. 1

Raw image of urea PAGE gel shown in Figs. 1b, 1c, 1d, 1e & 1f. Key to source data displayed as graphs is provided in: Main Fz graphed data.

Source Data Fig. 2

Raw image of urea PAGE gel shown in Figs. 2a, 2b, 2c & 2d. Key to source data displayed as graphs is provided in: Main Fz graphed data.

Source Data Fig. 3

Raw image of Urea PAGE gel shown in Figs. 3b, 3c, 3d & 3e. Key to source data displayed as graphs is provided in: Main Fz graphed data.

Source Data Fig. 4

Raw image of urea PAGE gel shown in Fig. 4a (unstained). Raw image of urea PAGE gel shown in Fig. 4a (stained with Sybr Gold). Raw image of urea PAGE gel shown in Fig. 4a.

Source Data Fig. 4, Source Data Fig. 5 and Source Data Extended Data Fig. 5

Contains raw ddPCR data used to produce graph shown in Fig. 4b, Fig. 5b, 5c & 5d, Fig. 5a, 5b & 5c.

Source Data Fig. 5

Raw image of agarose electrophoresis gel shown in Fig. 5e.

Source Data Extended Data Fig. 1

Raw images of urea PAGE gel used to produce graph shown in Extended Data Fig. 1. Key to source data displayed as graphs is provided in: Main Fz graphed data.

Source Data Extended Data Fig. 2

Raw images of urea PAGE gels shown in Extended Data Fig. 2b.

Source Data Extended Data Fig. 3

Raw image of urea PAGE gel shown in Extended Data Fig. 3a (unstained). Raw image of urea PAGE gel shown in Extended Data Fig. 3a (Sybr Gold stained). Raw image of urea PAGE gel shown in Extended Data Fig. 3b (unstained). Raw image of urea PAGE gel shown in Extended Data Fig. 3b (Sybr Gold stained). Raw image of urea PAGE gel shown in Extended Data Fig. 3c (unstained). Raw image of urea PAGE gel shown in Extended Data Fig. 3c (Sybr Gold stained). Raw image of urea PAGE gel shown in Extended Data Fig. 3d.

Source Data Extended Data Fig. 4

Raw image of urea PAGE gel shown in Extended Data Fig. 4.

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Taylor, A.I., Wan, C.J.K., Donde, M.J. et al. A modular XNAzyme cleaves long, structured RNAs under physiological conditions and enables allele-specific gene silencing. Nat. Chem. 14, 1295–1305 (2022). https://doi.org/10.1038/s41557-022-01021-z

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