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Engineering a DNA polymerase for modifying large RNA at specific positions

Nature Chemistry volume 17, pages 382–392 (2025)Cite this article

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An Author Correction to this article was published on 16 February 2025

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Abstract

The synthesis of large RNA with precise modifications at specific positions is in high demand for both basic research and therapeutic applications, but efficient methods are limited. Engineered DNA polymerases have recently emerged as attractive tools for RNA labelling, offering distinct advantages over conventional RNA polymerases. Here, through semi-rational designs, we engineered a DNA polymerase variant and used it to precisely incorporate a diverse range of modifications, including base modifications, 2′-ribose modifications and backbone modifications, into desired positions within RNA. We achieved efficiencies exceeding 85% in the majority of modification cases, demonstrating success in introducing 2′-O-methyl, phosphorothioate, N4-acetylcytidine and a fluorophore to specific sites in eGFP and Firefly luciferase messenger RNA. Our mRNA products with N4-acetylcytidine, 2′-O-methyl and/or phosphorothioate have demonstrated the ability to enhance stability and affect protein production. This method presents a promising tool for the comprehensive functionalization of RNA, enabling the introduction of plentiful modifications irrespective of RNA lengths and sequences.

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Fig. 1: Schematic diagram of PORDVA for position-specific labelling of RNA.
Fig. 2: Semi-rational engineering of DNAP for PORDVA.
Fig. 3: Application of PORDVA-generated RNA for smFRET.
Fig. 4: Application of PORDVA in incorporating multiple modifications into crRNA.
Fig. 5: PORDVA fluorescent labelling of lncRNA.
Fig. 6: PORDVA position-specific labelling of 867 nt and 1,800 nt mRNA.

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The data generated and analysed during this study are included in this published article, the Supplementary Information and Source data. Source data are provided with this paper.

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Acknowledgements

We thank the Student Innovation Center at Shanghai Jiao Tong University for assistance in FRET data collection. This work was supported by the National Key Research and Development Program of China (grant 2021YFA0910300) and the National Natural Science Foundation of China (grant 32471342).

Author information

Authors and Affiliations

  1. State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

    Dian Chen, Zhanghui Han, Xiaoge Liang & Yu Liu

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  1. Dian Chen
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  2. Zhanghui Han
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  3. Xiaoge Liang
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  4. Yu Liu
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Contributions

D.C. and Y.L. conceived and designed experiments. D.C. performed the polymerase design, engineering, sample preparation and characterization. Z.H. and X.L. assisted with protein purification. D.C. and Y.L. wrote the manuscript. All authors discussed the results.

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Correspondence to Yu Liu.

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The authors declare no competing interests.

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Nature Chemistry thanks Marcel Hollenstein, Masayasu Kuwahara 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 Applications of hybrid solid-liquid phase PORDVA in synthesizing RNA with 5′-proximal and internal modifications.

(a) Synthesis schemes for P10 and P11 with Cy3 and 2′-OMe, respectively, at the 5′-end using the hybrid-phase PORDVA. (b) Secondary structures of P10-P13 synthesized via the hybrid-phase PORDVA. (c) Synthesis schemes for P12 and P13 labeled with internal m6A and m7G, respectively, using the hybrid-phase PORDVA. (d) Denaturing PAGE images of P10-P13 (Lanes 1 to 4) under UV and fluorescence excitation. The 45 nt unmodified RNA was loaded to Lane 5 as a control. (e) MS spectra of P10, P11, P12, and P13. (f) Native agarose gel image of DNA cleavage by Cas12a in the presence of unmodified RNA (Lane 2), P10 (Lane 3), P11 (Lane 4), P12 (Lane 5) and P13 (Lane 6). The target DNA was loaded to Lane 1 as a control. The DNA and RNA sequences used in the hybrid-phase PORDVA synthesis are listed in Supplementary Table 21.

Source data

Extended Data Fig. 2 Application of hybrid solid-liquid phase PORDVA in synthesizing RNA with multiple modifications at discrete positions.

(a) Synthesis scheme for P14 with an internal m7G (at residue 26) and Cy3 (at residue 39) using the hybrid-phase PORDVA. (b) Images of denaturing PAGE of 10 µL reaction mixture collected from steps 1 to 4 after treatment with DNase I and proteinase K at 37 °C for 30 min. 25 nt LRNA and 45 nt unmodified RNA were loaded into Lanes 1 and 6, respectively, as controls. (c) LC-MS of P14. The DNA and RNA sequences used in the hybrid-phase PORDVA synthesis are listed in Supplementary Table 21.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–27, Methods, Figs. 1–20 and HPLC chromatogram mass spectra.

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Supplementary Data 1

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Chen, D., Han, Z., Liang, X. et al. Engineering a DNA polymerase for modifying large RNA at specific positions. Nat. Chem. 17, 382–392 (2025). https://doi.org/10.1038/s41557-024-01707-6

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