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Reversible 2′-OH acylation enhances RNA stability

Nature Chemistry volume 15, pages 1296–1305 (2023)Cite this article

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Abstract

The presence of a hydroxyl group at the 2′-position in its ribose makes RNA susceptible to hydrolysis. Stabilization of RNAs for storage, transport and biological application thus remains a serious challenge, particularly for larger RNAs that are not accessible by chemical synthesis. Here we present reversible 2′-OH acylation as a general strategy to preserve RNA of any length or origin. High-yield polyacylation of 2′-hydroxyls (‘cloaking’) by readily accessible acylimidazole reagents effectively shields RNAs from both thermal and enzymatic degradation. Subsequent treatment with water-soluble nucleophilic reagents removes acylation adducts quantitatively (‘uncloaking’) and recovers a remarkably broad range of RNA functions, including reverse transcription, translation and gene editing. Furthermore, we show that certain α-dimethylamino- and α-alkoxy- acyl adducts are spontaneously removed in human cells, restoring messenger RNA translation with extended functional half-lives. These findings support the potential of reversible 2′-acylation as a simple and general molecular solution for enhancing RNA stability and provide mechanistic insights for stabilizing RNA regardless of length or origin.

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Fig. 1: RNA polyacylation (cloaking) inhibits thermal degradation of RNA.
Fig. 2: Structurally diverse acylimidazole reagents enhance RNA stability in solution.
Fig. 3: RNA cloaking suppresses enzymatic RNA degradation by RNases and biofluids.
Fig. 4: Nucleophilic reagents remove 2′-polyacylation to uncloak RNA.
Fig. 5: Nucleophile-promoted RNA uncloaking restores RNA functions.
Fig. 6: Spontaneous RNA uncloaking restores mRNA translation with extended functional half-lives in human cells.

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Data availability

All relevant data supporting the findings of this study are available within the article and supplementary information. The characterization data of all organic compounds are provided within the supplementary information. All reagents generated in this study are available from the corresponding author upon reasonable request. Data used for this paper are also available via figshare at https://figshare.com/articles/dataset/NCHEM-22030547_source_data/19555132. Source data are provided with this paper.

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Acknowledgements

This work was supported by a grant from the US National Institutes of Health GM127295 to E.T.K. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank K. Fukui and other staff at the Stanford PAN facility for performing BioAnalyzer QC. We thank Stanford Shared FACS Facility (SSFF) for the flow cytometry analysis. We thank T. McLaughlin in the Vincent Coates Foundation Mass Spectrometry Laboratory, Stanford University Mass Spectrometry (RRID:SCR_017801) for acquiring the HRMS data. We also thank T. Trinh for performing circular dichroism measurements and L. Zheng for the helpful discussion.

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Authors and Affiliations

  1. Department of Chemistry, Stanford University, Stanford, CA, USA

    Linglan Fang, Lu Xiao, Yong Woong Jun, Yoshiyuki Onishi & Eric T. Kool

  2. Sarafan ChEM-H Institute, Stanford University, Stanford, CA, USA

    Eric T. Kool

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Contributions

L.F. and E.T.K. conceived the project and designed the experiments. L.F. performed the experiments and data analysis. L.X. performed the Cas9-mediated DNA cleavage assay. Y.W.J. collected and analysed the confocal microscopy data. E.T.K. supervised the work. L.F. and E.T.K. wrote the paper, with input from all authors.

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Correspondence to Eric T. Kool.

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Competing interests

Stanford University has filed a patent application (PCTUS2023/010686) based on the RNA cloaking and uncloaking reagents and methods described in this work, in which L.F. and E.T.K. are named as inventors. The other authors declare no competing interests.

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Nature Chemistry thanks Ashwani Sharma, Joanna Sztuba-Solinska, Wen Zhang 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 Reaction conditions of RNA cloaking with acylimidazole reagents.

A heatmap shows reaction conditions for each acylimidazole reagent (1-7) required to introduce the desired level of RNA cloaking using a model 18nt tRF-3005 RNA. The X-axis indicates the number of 2′-acyl adducts per RNA molecule identified by MALDI-TOF. The color intensity of each cell represents the mass intensity of the acyl adduct peak (numbers at bottom show numbers of acyl adducts for the model RNA strand). See Supplementary Table 1 for quantification.

Source data

Extended Data Fig. 2 One-step synthesis of acylimidazoles for selectively 2′-OH acylation.

a, Schematic for one-step synthesis of acylimidazole reagents. b-c, Comparisons of RNA and DNA with the same sequence (tRF-3005-DNA and tRF-3005-DNA-Phos) after reacting with 1-7 show that reaction occurs entirely or almost entirely at 2′-OH groups rather than nucleobases or phosphodiester bonds. b, Number of acyl adducts on tRF-3005-DNA (18nt) upon treatment with acylimidazole reagents 1-7. tRF-3005-DNA contains an unmodified 3′-OH (Sequence: 5′-ATC CTG CCG ACT ACG CCA-3′-OH). c, Number of acyl adducts on tRF-3005-DNA-Phos (18nt) upon treatment with acylimidazole reagents 1, 5, 6, and 7. tRF-3005-DNA-Phos contains a blocked 3′-end by 3′-phosphorylation (Sequence: 5′-ATC CTG CCG ACT ACG CCA-3′-phosphate). The X-axis shows the number of acyl adducts per DNA identified by MALDI-TOF. The color intensity of each cell represents the mass intensity of the acyl adduct peak.

Source data

Extended Data Fig. 3 Sensitive acylimidazole-nucleophile pairs.

A summary of sensitive acylimidazole-nucleophile pairs that afford >50% hydrolysis of 2′-acyl adducts within 2 hours. The electrophilic centers are coloured orange.

Extended Data Fig. 4 Spontaneous RNA uncloaking restores mRNA translation in human cells.

a, Representative single-blind fluorescence micrographs of HeLa cells transfected with 1-cloaked eGFP-mRNA showing strongly inhibited mRNA translation after 24 or 48 h. Each condition was imaged at areas that contain more than 50 cells over two independent experiments. b, Time-course plot showing 7-cloaked eGFP-mRNA (~50% of 2′-hydroxyls acylated) translates in HeLa cells. Its translation kinetics were compared to unprotected or equimolarly 4-cloaked eGFP-mRNA. Data represent mean ± s.e.m., n = 3 per group from three biologically independent experiments.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–4, Figs. 1–13, Synthesis protocol, Uncropped gels for supplementary information and NMR/HRMS data.

Reporting Summary

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 2

Statistical source data and unprocessed gels.

Source Data Fig. 3

Statistical source data and unprocessed gels.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

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Fang, L., Xiao, L., Jun, Y.W. et al. Reversible 2′-OH acylation enhances RNA stability. Nat. Chem. 15, 1296–1305 (2023). https://doi.org/10.1038/s41557-023-01246-6

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