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