Abstract
The application of messenger RNA (mRNA) beyond infectious diseases is challenged by inefficient protein production. Whereas the engineering of secondary mRNA structures has been shown to increase mRNA half-life, it remains unclear whether tertiary mRNA structures influence therapeutic efficacy. Here we develop a metal-ion-assisted RNA folding (MARF) strategy and show that, when delivered with lipid nanoparticles (LNPs), specific metals promote mRNA folding architectures that result in the amplification of protein expression by up to 7.3-fold compared with control mRNA. This effect is due to altered mechanical interactions between the mRNA LNPs and the surrounding biosystem, resulting in enhanced intracellular processing and prolonged retention of delivered mRNA in targeted cells. Administered intravenously, MARF LNPs achieved effective and durable genome editing of the clinically relevant Pcsk9 gene through treatment with a single dose. Overall, this work provides a new MARF technology for more effective mRNA therapy and highlights the potential of mechanical cues in designing nanoparticles for improved mRNA delivery.
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Data availability
The data that support the conclusions of this Article are available in Figs. 1–6 and the Supplementary Information. The raw transcriptome data used in this paper are available from the NCBI Sequence Read Archive under accession number PRJNA1364180. Source data are provided with this paper.
Code availability
The custom code for the MD simulations on the tension test, endocytosis and translocation of the LNP is available via GitHub at https://github.com/youliangzhu/pygamd-v1. The input files for the MD simulations and the custom code for property analysis are available via GitHub at https://github.com/youliangzhu/lnp.
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Acknowledgements
We thank L. Peng (CNRS) for insightful comments on our manuscript. We thank J. Li (NUS) for help with the cyro-TEM scanning, L. Jianping (NUS) for help with the animal study, Z. Zhang (NUS) for the reagent contribution, L. Ding (NUS) for data analysis of the RNA sequencing, Z. Li (Jilin University) for help with all-atom MD simulations, T. T. Thi (NUS) for providing the eGFP reporter cell line and R. Liu (NUS) for help with the polysome profiling. This research is financially supported by the National University of Singapore (NUHSRO/2022/005/Startup/02 (Q.N.)), the Singapore Ministry of Education (NUHSRO/2022/068/T1/Seed-Mar/04 (Q.N.) and MOE-T2EP30124-0009 (Q.N.)), the National Medical Research Council (MOH-001241-00 (Q.N.)), A*Star NATi N05 (H24J5a0072 (Q.N.)) and the National Natural Science Foundation of China (22171103 (G.W.)).
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Q.N. and X.C. conceived the project. Q.N. and Y.Z. designed the experiments. B.Y., B.L., M. Zhao, Y.C., X. Zhao, D.T.E.-J., Y.W., S.J., X.T. and X. Zhang performed most of the experiments, and B.Y., B.L., M. Zhang, Y.S., B.X., J.Y., G.W., Z.L., M.L., H.Y. and L.Z. analysed and interpreted the data. Q.N., X.C. and Y.Z. supervised this work. B.Y., B.L. and Q.N. wrote the paper with comments from all authors.
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X.C. is a co-founder of and holds shares in Yantai Lannacheng Biotechnology Co., Ltd. All other authors have no competing interests.
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Extended data
Extended Data Fig. 1 Cell aging impacts endocytosis of MARF LNP.
a, Heatmap of the relative expression of significantly regulated genes relevant to endocytosis in Mn-MARF LNP and LNP treated groups. (n = 3, p < 0.05, |log2FoldChange| > 1). b, Gene ontology (GO) enrichment analysis of the genes related to endocytosis from the comparison between the gene expression in Mn-MARF LNP and LNP treated groups. c, Relative cellular uptake of LNP and Mn-MARF LNP in HEK-293T cells in the presence of different inhibitors. d, GO enrichment analysis of the genes related to cell aging from the comparison between gene expression in effective and non-effective HEK-293T cell lines. e, Gene set enrichment analysis (GSEA) for comparing non-effective cells and effective cells for cell aging and cellular senescence. f, g, Representative fluorescence microscopy images (f) and relative signal intensity (g) of eGFP in aging cells or non-aging HEK-293T cells administrated with LNP and Mn-MARF LNP (n = 3). Scale bar: 50 μm. Data are shown as mean ± s.e.m. (n = 3 biologically independent samples). Statistical significance was tested using two-tailed unpaired t-test among groups. N.S., not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Supplementary information
Supplementary Information (download PDF )
Supplementary Methods, Figs. 1–49 and refs. 1–13.
Supplementary Video 1 (download MOV )
Deformable nanoparticle model with a cross-linked network to model the mechanical response under stretching for MARF LNPs.
Supplementary Video 2 (download MOV )
Deformable nanoparticle model with a cross-linked network to model the mechanical response under stretching for control LNPs.
Supplementary Video 3 (download MOV )
Coarse-grained MD simulations depicting the internalization process of Mn-MARF LNPs into cells.
Supplementary Video 4 (download MOV )
Coarse-grained MD simulations depicting the internalization process of control LNPs into cells.
Supplementary Video 5 (download MOV )
Coarse-grained MD simulations of MARF LNPs being pulled through a small, rigid pore (20 nm).
Supplementary Video 6 (download MOV )
Coarse-grained MD simulations of control LNPs being pulled through a small, rigid pore (20 nm).
Supplementary Data 1 (download XLSX )
Statistical source data for Supplementary Figs. 1, 2, 4–16, 18, 19, 21, 22, 24–41, 44–46 and 48.
Source data
Source Data Figs. 2–6 and Extended Data Fig. 1 (download XLSX )
Statistical source data.
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Yang, B., Li, B., Zhu, Y. et al. Rational design of rigid mRNA folding architecture to enhance intracellular processing and protein production. Nat. Nanotechnol. 21, 407–418 (2026). https://doi.org/10.1038/s41565-025-02114-9
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DOI: https://doi.org/10.1038/s41565-025-02114-9
