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A two-residue nascent-strand steric gate controls synthesis of 2′-O-methyl- and 2′-O-(2-methoxyethyl)-RNA

Nature Chemistry volume 15, pages 91–100 (2023)Cite this article

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Abstract

Steric exclusion is a key element of enzyme substrate specificity, including in polymerases. Such substrate specificity restricts the enzymatic synthesis of 2′-modified nucleic acids, which are of interest in nucleic-acid-based drug development. Here we describe the discovery of a two-residue, nascent-strand, steric control ‘gate’ in an archaeal DNA polymerase. We show that engineering of the gate to reduce steric bulk in the context of a previously described RNA polymerase activity unlocks the synthesis of 2′-modified RNA oligomers, specifically the efficient synthesis of both defined and random-sequence 2′-O-methyl-RNA (2′OMe-RNA) and 2′-O-(2-methoxyethyl)-RNA (MOE-RNA) oligomers up to 750 nt. This enabled the discovery of RNA endonuclease catalysts entirely composed of 2′OMe-RNA (2′OMezymes) for the allele-specific cleavage of oncogenic KRAS (G12D) and β-catenin CTNNB1 (S33Y) mRNAs, and the elaboration of mixed 2′OMe-/MOE-RNA aptamers with high affinity for vascular endothelial growth factor. Our results open up these 2′-modified RNAs—used in several approved nucleic acid therapeutics—for enzymatic synthesis and a wider exploration in directed evolution and nanotechnology.

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Fig. 1: The two-residue steric gate.
Fig. 2: Site-specific RNA endonuclease catalysts composed of 2′OMe-RNA.
Fig. 3: MOE-RNA synthesis.
Fig. 4: 2′OMe/MOE-RNA aptamers and binding kinetics.
Fig. 5: Nascent strand steric gate and polymerase motifs.

Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information), except raw sequencing reads, which are available in the NCBI SRA repository, BioProject ID PRJNA847930. Source data are provided with this paper.

Code availability

Fidelity analysis of sequencing data for 2′OMe-RNA synthesis by individual polymerases was performed using the Burrows-Wheeler Aligner (BWA-0.7.17), Samtools and custom scripts, which can be found at GitHub: https://github.com/holliger-lab/fidelity-analysis.

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Acknowledgements

This work was supported by a PhD fellowship from Boehringer Ingelheim Fonds (N.F.), the Medical Research Council (MRC) programme (MC_U105178804; A.I.T., S.-Y.P.-C., P. Holliger), a research collaboration between AstraZeneca UK and the MRC (MRC–AstraZeneca Blue Sky Grant; N.S., S.A.-F.), FWO (Flanders Research Foundation) Fund of Scientific Research and the Rega Institute, KU Leuven (M.A., P. Herdewijn), and by National Institute of Health grants R35-GM128562 (B.D.F.) and K99-ES031148 (A.M.W.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. Amy M. Whitaker

    Present address: Cancer Epigenetics Institute, Fox Chase Cancer Center, Philadelphia, PA, USA

  2. These authors contributed equally: Niklas Freund, Alexander I. Taylor.

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK

    Niklas Freund, Alexander I. Taylor, Sebastian Arangundy-Franklin, Nithya Subramanian, Sew-Yeu Peak-Chew & Philipp Holliger

  2. Cambridge Institute of Therapeutic Immunology and Infectious Disease, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, University of Cambridge, Cambridge, UK

    Alexander I. Taylor

  3. Laboratory of Genome Maintenance and Structural Biology, Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA

    Amy M. Whitaker & Bret D. Freudenthal

  4. Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium

    Mikhail Abramov & Piet Herdewijn

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Contributions

N.F., S.A.-F., A.I.T. and P. Holliger conceived and designed experiments. N.F. performed polymerase studies. N.F. and S.A.-F. performed polymerase design and engineering. N.F., M.A. and P. Herdewijn synthesized MOE-nucleotides. N.F. and A.I.T. completed SPR measurements. A.I.T. performed 2′OMezyme selections and characterization. N.S. performed polymerase fidelity measurements. S.-Y.P.-C. performed MS analysis. A.M.W. and B.D.F performed and analysed steady-state kinetic measurements. All authors analysed data, discussed results and co-wrote the manuscript.

Corresponding authors

Correspondence to Alexander I. Taylor or Philipp Holliger.

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Competing interests

UK Research and Innovation has filed a UK patent priority application on behalf of the inventors N.F., S.A.-F. and P. Holliger on the 2M/3M polymerase on 25 May 2022 (application number 2207699.6).

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Supplementary information

Supplementary Information

Full materials and methods, Supplementary Figs 1–17, Tables 1–7, references.

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Supplementary Data 1

Statistical source data for graphs in Supplementary Fig. 2.

Supplementary Data 2

Statistical source data: raw Biacore SPR data that were fitted and plotted in Supplementary Fig. 9a.

Supplementary Data 3

Statistical source data: raw Biacore SPR data that were fitted and plotted in Supplementary Fig. 9b.

Source data

Source Data Fig. 1

Unprocessed gels for Fig. 1.

Source Data Fig. 2

Unprocessed gels for Fig. 2.

Source Data Fig. 2

Statistical source data for graphs in Fig. 2.

Source Data Fig. 3

Unprocessed gels for Fig. 3.

Source Data Fig. 4a

Statistical source data: raw Biacore SPR data that were fitted and plotted in Fig. 4a.

Source Data Fig. 4b

Statistical source data: raw Biacore SPR data that were fitted and plotted in Fig. 4b.

Source Data Fig. 4c

Statistical source data: raw Biacore SPR data that were fitted and plotted in Fig. 4c.

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Freund, N., Taylor, A.I., Arangundy-Franklin, S. et al. A two-residue nascent-strand steric gate controls synthesis of 2′-O-methyl- and 2′-O-(2-methoxyethyl)-RNA. Nat. Chem. 15, 91–100 (2023). https://doi.org/10.1038/s41557-022-01050-8

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