Rational design of rigid mRNA folding architecture to enhance intracellular processing and protein production

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Rational design of rigid mRNA folding architecture to enhance intracellular processing and protein production

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.

References

  1. Kulkarni, J. A. et al. The current landscape of nucleic acid therapeutics. Nat. Nanotechnol. 16, 630–643 (2021).

    Article  CAS  PubMed  Google Scholar 

  2. Mendes, B. B. et al. Nanodelivery of nucleic acids. Nat. Rev. Methods Primers 2, 24 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hogan, M. J. & Pardi, N. mRNA vaccines in the COVID-19 pandemic and beyond. Annu. Rev. Med. 73, 17–39 (2022).

    Article  CAS  PubMed  Google Scholar 

  4. Liu, C. et al. mRNA-based cancer therapeutics. Nat. Rev. Cancer 23, 526–543 (2023).

    Article  PubMed  Google Scholar 

  5. Kowalski, P. S., Rudra, A., Miao, L. & Anderson, D. G. Delivering the messenger: advances in technologies for therapeutic mRNA delivery. Mol. Ther. 27, 710–728 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Karikó, K., Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175 (2005).

    Article  PubMed  Google Scholar 

  7. Rohner, E., Yang, R., Foo, K. S., Goedel, A. & Chien, K. R. Unlocking the promise of mRNA therapeutics. Nat. Biotechnol. 40, 1586–1600 (2022).

    Article  CAS  PubMed  Google Scholar 

  8. Rappaport, A. R. et al. Low-dose self-amplifying mRNA COVID-19 vaccine drives strong protective immunity in non-human primates against SARS-CoV-2 infection. Nat. Commun. 13, 3289 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Qu, L. et al. Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell 185, 1728–1744 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Leppek, K. et al. Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. Nat. Commun. 13, 1536 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhang, H. et al. Algorithm for optimized mRNA design improves stability and immunogenicity. Nature 621, 396–403 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wojtczak, B. A. et al. 5′-Phosphorothiolate dinucleotide cap analogues: reagents for messenger RNA modification and potent small-molecular inhibitors of decapping enzymes. J. Am. Chem. Soc. 140, 5987–5999 (2018).

    Article  CAS  PubMed  Google Scholar 

  13. Holtkamp, S. et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108, 4009–4017 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Chen, H. et al. Chemical and topological design of multicapped mRNA and capped circular RNA to augment translation. Nat. Biotechnol. https://doi.org/10.1038/s41587-024-02393-y (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Chen, H. et al. Branched chemically modified poly(A) tails enhance the translation capacity of mRNA. Nat. Biotechnol. https://doi.org/10.1038/s41587-024-02174-7 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Liu, B. & Pan, T. Building better mRNA for therapeutics. Nat. Biotechnol. https://doi.org/10.1038/s41587-024-02424-8 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Pyle, A. Metal ions in the structure and function of RNA. J. Biol. Inorg. Chem. 7, 679–690 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Tinoco, I. Jr & Bustamante, C. How RNA folds. J. Mol. Biol. 293, 271–281 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Draper, D. E., Grilley, D. & Soto, A. M. Ions and RNA folding. Annu. Rev. Biophys. Biomol. Struct. 34, 221–243 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Palermo, G. et al. Catalytic metal ions and enzymatic processing of DNA and RNA. Acc. Chem. Res. 48, 220–228 (2015).

    Article  CAS  PubMed  Google Scholar 

  21. Zoroddu, M. A. et al. The essential metals for humans: a brief overview. J. Inorg. Biochem. 195, 120–129 (2019).

    Article  CAS  PubMed  Google Scholar 

  22. Guth-Metzler, R. et al. Goldilocks and RNA: where Mg2+ concentration is just right. Nucleic Acids Res. 51, 3529–3539 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bepperling, A. & Richter, G. Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation. Eur. Biophys. J. 52, 393–400 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Smith, S. B., Cui, Y. & Bustamante, C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271, 795–799 (1996).

    Article  CAS  PubMed  Google Scholar 

  25. Baumann, C. G., Smith, S. B., Bloomfield, V. A. & Bustamante, C. Ionic effects on the elasticity of single DNA molecules. Proc. Natl Acad. Sci. USA 94, 6185–6190 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li, Z. et al. Mechano-boosting nanomedicine antitumour efficacy by blocking the reticuloendothelial system with stiff nanogels. Nat. Commun. 14, 1437 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hui, Y. et al. Nanoparticle elasticity regulates phagocytosis and cancer cell uptake. Sci. Adv. 6, eaaz4316 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhu, Y.-L. et al. GALAMOST: GPU-accelerated large-scale molecular simulation toolkit. J. Comput. Chem. 34, 2197–2211 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Uroda, T. et al. Visualizing the functional 3D shape and topography of long noncoding RNAs by single-particle atomic force microscopy and in-solution hydrodynamic techniques. Nat. Protoc. 15, 2107–2139 (2020).

    Article  CAS  PubMed  Google Scholar 

  30. Miao, L. et al. Synergistic lipid compositions for albumin receptor mediated delivery of mRNA to the liver. Nat. Commun. 11, 2424 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sun, J. et al. Tunable rigidity of (polymeric core)–(lipid shell) nanoparticles for regulated cellular uptake. Adv. Mater. 27, 1402–1407 (2015).

    Article  CAS  PubMed  Google Scholar 

  32. Shen, Z., Ye, H., Yi, X. & Li, Y. Membrane wrapping efficiency of elastic nanoparticles during endocytosis: size and shape matter. ACS Nano 13, 215–228 (2019).

    Article  CAS  PubMed  Google Scholar 

  33. Yi, X., Shi, X. & Gao, H. Cellular uptake of elastic nanoparticles. Phys. Rev. Lett. 107, 098101 (2011).

    Article  PubMed  Google Scholar 

  34. Wang, Y. et al. Age-associated disparity in phagocytic clearance affects the efficacy of cancer nanotherapeutics. Nat. Nanotechnol. 19, 255–263 (2024).

    Article  CAS  PubMed  Google Scholar 

  35. Wang, L. et al. Primary human breast cancer-associated endothelial cells favor interactions with nanomedicines. Adv. Mater. https://doi.org/10.1002/adma.202403986 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Pattipeiluhu, R. et al. Anionic lipid nanoparticles preferentially deliver mRNA to the hepatic reticuloendothelial system. Adv. Mater. 34, 2201095 (2022).

    Article  CAS  Google Scholar 

  37. Nakamura, T., Yamada, K., Sato, Y. & Harashima, H. Lipid nanoparticles fuse with cell membranes of immune cells at low temperatures leading to the loss of transfection activity. Int. J. Pharm. 587, 119652 (2020).

    Article  CAS  PubMed  Google Scholar 

  38. Chatterjee, S., Kon, E., Sharma, P. & Peer, D. Endosomal escape: a bottleneck for LNP-mediated therapeutics. Proc. Natl Acad. Sci. USA 121, e2307800120 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Akinc, A. et al. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther. 18, 1357–1364 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ezratty, E. J., Bertaux, C., Marcantonio, E. E. & Gundersen, G. G. Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells. J. Cell Biol. 187, 733–747 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Yin, B. et al. Sub-10 nm substrate roughness promotes the cellular uptake of nanoparticles by upregulating endocytosis-related genes. Nano Lett. 21, 1839–1847 (2021).

    Article  CAS  PubMed  Google Scholar 

  42. Wang, X. et al. Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nat. Protoc. 18, 265–291 (2023).

    Article  CAS  PubMed  Google Scholar 

  43. Cheng, Q. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol. 15, 313–320 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.)).

Author information

Author notes

  1. These authors contributed equally: Bowei Yang, Benhao Li.

Authors and Affiliations

  1. Departments of Diagnostic Radiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Bowei Yang, Benhao Li, Mengyao Zhao, Miao Zhang, Xianglong Tang, Xuanbo Zhang & Qianqian Ni

  2. Nanomedicine Translational Research Program, Immunology Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Bowei Yang, Benhao Li, Mengyao Zhao, Miao Zhang, Xianglong Tang, Xuanbo Zhang & Qianqian Ni

  3. State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, China

    Youliang Zhu, Shuang Jin, Yibin Sun & Guanglu Wu

  4. Collaborative Innovation Center of Advanced Microstructures, State Key Laboratory of Solid State Microstructures, Department of Physics, Nanjing University, Nanjing, China

    Yuanqi Cheng & Bin Xue

  5. Department of Physics, Mechanobiology Institute, National University of Singapore, Singapore, Singapore

    Xiaodan Zhao & Jie Yan

  6. Department of Biological Sciences, National University of Singapore, Singapore, Singapore

    Deryn Teoh En-Jie, Zhewang Lin & Min Luo

  7. Department of Biochemistry, Precision Medicine TRP, Cardiovascular and Metabolic Disease TRP, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Yifan Wang & Haojie Yu

  8. Mechanobiology Institute, National University of Singapore, Singapore, Singapore

    Jie Yan

  9. Department of Medical Imaging, Jinling Hospital, Medical School of Nanjing University, Nanjing, China

    Longjiang Zhang

  10. Shandong Provincial Key Laboratory of Precision Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China

    Xiaoyuan Chen

Authors

  1. Bowei Yang
  2. Benhao Li
  3. Youliang Zhu
  4. Mengyao Zhao
  5. Yuanqi Cheng
  6. Xiaodan Zhao
  7. Deryn Teoh En-Jie
  8. Yifan Wang
  9. Miao Zhang
  10. Xianglong Tang
  11. Shuang Jin
  12. Yibin Sun
  13. Xuanbo Zhang
  14. Bin Xue
  15. Jie Yan
  16. Guanglu Wu
  17. Zhewang Lin
  18. Min Luo
  19. Haojie Yu
  20. Longjiang Zhang
  21. Xiaoyuan Chen
  22. Qianqian Ni

Contributions

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.

Corresponding authors

Correspondence to Youliang Zhu, Xiaoyuan Chen or Qianqian Ni.

Ethics declarations

Competing interests

X.C. is a co-founder of and holds shares in Yantai Lannacheng Biotechnology Co., Ltd. All other authors have no competing interests.

Peer review

Peer review information

Nature Nanotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–49 and refs. 1–13.

Reporting Summary

Supplementary Video 1

Deformable nanoparticle model with a cross-linked network to model the mechanical response under stretching for MARF LNPs.

Supplementary Video 2

Deformable nanoparticle model with a cross-linked network to model the mechanical response under stretching for control LNPs.

Supplementary Video 3

Coarse-grained MD simulations depicting the internalization process of Mn-MARF LNPs into cells.

Supplementary Video 4

Coarse-grained MD simulations depicting the internalization process of control LNPs into cells.

Supplementary Video 5

Coarse-grained MD simulations of MARF LNPs being pulled through a small, rigid pore (20 nm).

Supplementary Video 6

Coarse-grained MD simulations of control LNPs being pulled through a small, rigid pore (20 nm).

Supplementary Data 1

Statistical source data for Supplementary Figs. 1, 2, 4–16, 18, 19, 21, 22, 24–41, 44–46 and 48.

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. (2026). https://doi.org/10.1038/s41565-025-02114-9

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  • DOI: https://doi.org/10.1038/s41565-025-02114-9

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