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Chemical and topological design of multicapped mRNA and capped circular RNA to augment translation

Nature Biotechnology (2024)Cite this article

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Abstract

Protein and vaccine therapies based on mRNA would benefit from an increase in translation capacity. Here, we report a method to augment translation named ligation-enabled mRNA–oligonucleotide assembly (LEGO). We systematically screen different chemotopological motifs and find that a branched mRNA cap effectively initiates translation on linear or circular mRNAs without internal ribosome entry sites. Two types of chemical modification, locked nucleic acid (LNA) N7-methylguanosine modifications on the cap and LNA + 5 × 2′ O-methyl on the 5′ untranslated region, enhance RNA–eukaryotic translation initiation factor (eIF4E–eIF4G) binding and RNA stability against decapping in vitro. Through multidimensional chemotopological engineering of dual-capped mRNA and capped circular RNA, we enhanced mRNA protein production by up to tenfold in vivo, resulting in 17-fold and 3.7-fold higher antibody production after prime and boost doses in a severe acute respiratory syndrome coronavirus 2 vaccine setting, respectively. The LEGO platform opens possibilities to design unnatural RNA structures and topologies beyond canonical linear and circular RNAs for both basic research and therapeutic applications.

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Fig. 1: Multidimensional chemical screening of the mRNA cap and 5′ UTR modifications.
Fig. 2: Chemotopological design and engineering of dual-capped mRNA.
Fig. 3: Mechanistic characterization of 5′ chemical and topological modifications in mRNA translation.
Fig. 4: Synthesis of m1Ψ-modified, QRNA and mechanistic probing for cap-proximal translation induction.
Fig. 5: Chemotopologically optimized mRNA exhibits enhanced protein expression in vivo.
Fig. 6: Chemically modified dual-capped mRNA enhances SARS-CoV-2 mRNA vaccine efficacy in mice.

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Data availability

Sequences and modification information of all RNA constructs used in this study are provided in Supplementary Table 1. Plasmids used in this study were deposited to Addgene. Raw MS data for SILAC pulldown experiments are available on Zenodo96. The SARS-CoV-2 vaccine lymph node STARmap and RIBOmap datasets are available on Zenodo97. Source data are provided with this paper.

Code availability

The code used for data analysis is available on GitHub (https://github.com/wanglab-broad/Multicap-analysis).

References

  1. Mulligan, M. J. et al. Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults. Nature 586, 589–593 (2020).

    Article  PubMed  CAS  Google Scholar 

  2. Morais, P., Adachi, H. & Yu, Y.-T. The critical contribution of pseudouridine to mRNA COVID-19 vaccines. Front. Cell Dev. Biol. 9, 789427 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  3. 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  PubMed  CAS  Google Scholar 

  4. Jackson, R. J., Hellen, C. U. T. & Pestova, T. V. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 11, 113–127 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. von der Haar, T., Gross, J. D., Wagner, G. & McCarthy, J. E. G. The mRNA cap-binding protein eIF4E in post-transcriptional gene expression. Nat. Struct. Mol. Biol. 11, 503–511 (2004).

    Article  PubMed  Google Scholar 

  6. Despic, V. & Jaffrey, S. R. mRNA ageing shapes the Cap2 methylome in mammalian mRNA. Nature 614, 358–366 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Züst, R. et al. Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nat. Immunol. 12, 137–143 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Ramanathan, A., Robb, G. B. & Chan, S.-H. mRNA capping: biological functions and applications. Nucleic Acids Res. 44, 7511–7526 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Grudzien‐Nogalska, E. et al. Synthesis of anti‐reverse cap analogs (ARCAs) and their applications in mRNA translation and stability. Methods Enzymol. 431, 203–227 (2007).

    Article  PubMed  Google Scholar 

  10. Stepinski, J., Waddell, C., Stolarski, R., Darzynkiewicz, E. & Rhoads, R. E. Synthesis and properties of mRNAs containing the novel ‘anti-reverse’ cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′-deoxy)GpppG. RNA 7, 1486–1495 (2001).

    PubMed  PubMed Central  CAS  Google Scholar 

  11. Ishikawa, M., Murai, R., Hagiwara, H., Hoshino, T. & Suyama, K. Preparation of eukaryotic mRNA having differently methylated adenosine at the 5′-terminus and the effect of the methyl group in translation. Nucleic Acids Symp. Ser. 53, 129–130 (2009).

    Article  CAS  Google Scholar 

  12. Sikorski, P. J. et al. The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells. Nucleic Acids Res. 48, 1607–1626 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Jurga, S. & Barciszewski, J. (eds) Messenger RNA Therapeutics (Springer Nature, 2022).

  14. Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 9, 2629 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Chen, R. et al. Engineering circular RNA for enhanced protein production. Nat. Biotechnol. 41, 262–272 (2023).

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Koch, A., Aguilera, L., Morisaki, T., Munsky, B. & Stasevich, T. J. Quantifying the dynamics of IRES and cap translation with single-molecule resolution in live cells. Nat. Struct. Mol. Biol. 27, 1095–1104 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Abe, N. et al. Complete chemical synthesis of minimal messenger RNA by efficient chemical capping reaction. ACS Chem. Biol. 17, 1308–1314 (2022).

    Article  PubMed  CAS  Google Scholar 

  19. Kawaguchi, D. et al. Phosphorothioate modification of mRNA accelerates the rate of translation initiation to provide more efficient protein synthesis. Angew. Chem. Int. Ed. Engl. 59, 17403–17407 (2020).

    Article  PubMed  CAS  Google Scholar 

  20. Wojcik, R. et al. Novel N7-arylmethyl substituted dinucleotide mRNA 5′ cap analogs: synthesis and evaluation as modulators of translation. Pharmaceutics 13, 1941 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Chen, X. et al. Structure-guided design, synthesis, and evaluation of guanine-derived inhibitors of the eIF4E mRNA–cap interaction. J. Med. Chem. 55, 3837–3851 (2012).

    Article  PubMed  CAS  Google Scholar 

  22. Inagaki, M. et al. Cap analogs with a hydrophobic photocleavable tag enable facile purification of fully capped mRNA with various cap structures. Nat. Commun. 14, 2657 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Kore, A. R., Shanmugasundaram, M., Charles, I., Vlassov, A. V. & Barta, T. J. Locked nucleic acid (LNA)-modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization. J. Am. Chem. Soc. 131, 6364–6365 (2009).

    Article  PubMed  CAS  Google Scholar 

  24. Senthilvelan, A. et al. Trinucleotide cap analogue bearing a locked nucleic acid moiety: synthesis, mRNA modification, and translation for therapeutic applications. Org. Lett. 23, 4133–4136 (2021).

    Article  PubMed  CAS  Google Scholar 

  25. Aitken, C. E. & Lorsch, J. R. A mechanistic overview of translation initiation in eukaryotes. Nat. Struct. Mol. Biol. 19, 568–576 (2012).

    Article  PubMed  CAS  Google Scholar 

  26. Gu, Y., Mao, Y., Jia, L., Dong, L. & Qian, S.-B. Bi-directional ribosome scanning controls the stringency of start codon selection. Nat. Commun. 12, 6604 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Aditham, A. et al. Chemically modified mocRNAs for highly efficient protein expression in mammalian cells. ACS Chem. Biol. 17, 3352–3366 (2022).

    Article  PubMed  CAS  Google Scholar 

  28. Hellman, L. M. & Fried, M. G. Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions. Nat. Protoc. 2, 1849–1861 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Nagaraj, N. et al. Deep proteome and transcriptome mapping of a human cancer cell line. Mol. Syst. Biol. 7, 548 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Mitchell, S. F. et al. The 5′-7-methylguanosine cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and to block an alternative pathway. Mol. Cell 39, 950–962 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Balatsos, N. A. A., Maragozidis, P., Anastasakis, D. & Stathopoulos, C. Modulation of poly(A)-specific ribonuclease (PARN): current knowledge and perspectives. Curr. Med. Chem. 19, 4838–4849 (2012).

    Article  PubMed  CAS  Google Scholar 

  32. Gerbracht, J. V. et al. CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex. Nucleic Acids Res. 48, 8626–8644 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Hu, W., Sweet, T. J., Chamnongpol, S., Baker, K. E. & Coller, J. Co-translational mRNA decay in Saccharomyces cerevisiae. Nature 461, 225–229 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. van Dijk, E. et al. Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures. EMBO J. 21, 6915–6924 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  35. 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  PubMed  CAS  Google Scholar 

  36. Bohlen, J., Roiuk, M., Neff, M. & Teleman, A. A. PRRC2 proteins impact translation initiation by promoting leaky scanning. Nucleic Acids Res. 51, 3391–3409 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Garshott, D. M. et al. iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation. Cell Rep. 36, 109642 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Ostrowski, L. A., Hall, A. C. & Mekhail, K. Ataxin-2: from RNA control to human health and disease. Genes 8, 157 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Hildebrandt, A. et al. The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation. Genome Biol. 20, 216 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Pattabhi, S., Knoll, M. L., Gale, M. Jr & Loo, Y.-M. DHX15 is a coreceptor for RLR signaling that promotes antiviral defense against RNA virus infection. J. Interferon Cytokine Res. 39, 331–346 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Xing, J. et al. DHX15 is required to control RNA virus-induced intestinal inflammation. Cell Rep. 35, 109205 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Wang, Y. et al. Structural and functional insights into 5′-ppp RNA pattern recognition by the innate immune receptor RIG-I. Nat. Struct. Mol. Biol. 17, 781–787 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Wang, X. et al. Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361, eaat5691 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Zeng, H. et al. Spatially resolved single-cell translatomics at molecular resolution. Science 380, eadd3067 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. 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  Google Scholar 

  46. Lee, A. S., Kranzusch, P. J., Doudna, J. A. & Cate, J. H. D. eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation. Nature 536, 96–99 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Moerke, N. J. et al. Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G. Cell 128, 257–267 (2007).

    Article  PubMed  CAS  Google Scholar 

  48. Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell 74, 508–520 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Karikó, K. et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 1833–1840 (2008).

    Article  PubMed  Google Scholar 

  50. Cervantes, J. L., Weinerman, B., Basole, C. & Salazar, J. C. TLR8: the forgotten relative revindicated. Cell. Mol. Immunol. 9, 434–438 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Rehwinkel, J. & Gack, M. U. RIG-I-like receptors: their regulation and roles in RNA sensing. Nat. Rev. Immunol. 20, 537–551 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Chen, Y. G. et al. N6-methyladenosine modification controls circular RNA immunity. Mol. Cell 76, 96–109 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Lee, Y., Choe, J., Park, O. H. & Kim, Y. K. Molecular mechanisms driving mRNA degradation by m6A modification. Trends Genet. 36, 177–188 (2020).

    Article  PubMed  CAS  Google Scholar 

  54. Jain, S. et al. Modulation of translational decoding by m6A modification of mRNA. Nat. Commun. 14, 4784 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Ehret, F., Zhou, C. Y., Alexander, S. C., Zhang, D. & Devaraj, N. K. Site-specific covalent conjugation of modified mRNA by tRNA guanine transglycosylase. Mol. Pharm. 15, 737–742 (2018).

    Article  PubMed  CAS  Google Scholar 

  56. Meyer, K. D. et al. 5′ UTR m6A promotes cap-independent translation. Cell 163, 999–1010 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Kumar, P., Hellen, C. U. T. & Pestova, T. V. Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs. Genes Dev. 30, 1573–1588 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Hellen, C. U. & Sarnow, P. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 15, 1593–1612 (2001).

    Article  PubMed  CAS  Google Scholar 

  59. Brito Querido, J. et al. Structure of a human 48S translational initiation complex. Science 369, 1220–1227 (2020).

    Article  PubMed  CAS  Google Scholar 

  60. Querido, J. B. et al. The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A. Nat. Struct. Mol. Biol. 31, 455–464 (2024).

    Article  Google Scholar 

  61. Kozak, M. Adherence to the first-AUG rule when a second AUG codon follows closely upon the first. Proc. Natl Acad. Sci. USA 92, 2662–2666 (1995).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Eschbach, J. W., Kelly, M. R., Haley, N. R., Abels, R. I. & Adamson, J. W. Treatment of the anemia of progressive renal failure with recombinant human erythropoietin. N. Engl. J. Med. 321, 158–163 (1989).

    Article  PubMed  CAS  Google Scholar 

  63. Karikó, K., Muramatsu, H., Keller, J. M. & Weissman, D. Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol. Ther. 20, 948–953 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Jang, D.-I. et al. The role of tumor necrosis factor alpha (TNF-α) in autoimmune disease and current TNF-α inhibitors in therapeutics. Int. J. Mol. Sci. 22, 2719 (2021).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Giannini, E. G., Testa, R. & Savarino, V. Liver enzyme alteration: a guide for clinicians. CMAJ 172, 367–379 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Liang, Q. et al. RBD trimer mRNA vaccine elicits broad and protective immune responses against SARS-CoV-2 variants. iScience 25, 104043 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Vogel, A. B. et al. BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature 592, 283–289 (2021).

    Article  PubMed  CAS  Google Scholar 

  68. Maher, K. et al. Mitigating autocorrelation during spatially resolved transcriptomics data analysis. Preprint at bioRxiv https://doi.org/10.1101/2023.06.30.547258 (2023).

  69. 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  PubMed  PubMed Central  CAS  Google Scholar 

  70. Riol-Blanco, L. et al. The chemokine receptor CCR7 activates in dendritic cells two signaling modules that independently regulate chemotaxis and migratory speed. J. Immunol. 174, 4070–4080 (2005).

    Article  PubMed  CAS  Google Scholar 

  71. Johnson, A. M. et al. Cancer cell-intrinsic expression of MHC class II regulates the immune microenvironment and response to anti-PD-1 therapy in lung adenocarcinoma. J. Immunol. 204, 2295–2307 (2020).

    Article  PubMed  CAS  Google Scholar 

  72. Kratky, W., Reis e Sousa, C., Oxenius, A. & Spörri, R. Direct activation of antigen-presenting cells is required for CD8+ T-cell priming and tumor vaccination. Proc. Natl Acad. Sci. USA 108, 17414–17419 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  73. Laczkó, D. et al. A single immunization with nucleoside-modified mRNA vaccines elicits strong cellular and humoral immune responses against SARS-CoV-2 in mice. Immunity 53, 724–732 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  74. Calabro, S. et al. Differential intrasplenic migration of dendritic cell subsets tailors adaptive immunity. Cell Rep. 16, 2472–2485 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  75. Dohnalkova, M. et al. Essential roles of RNA cap-proximal ribose methylation in mammalian embryonic development and fertility. Cell Rep. 42, 112786 (2023).

    Article  PubMed  CAS  Google Scholar 

  76. Haussmann, I. U. et al. CMTr cap-adjacent 2′-O-ribose mRNA methyltransferases are required for reward learning and mRNA localization to synapses. Nat. Commun. 13, 1209 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  77. Someya, T., Ando, A., Kimoto, M. & Hirao, I. Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry. Nucleic Acids Res. 43, 6665–6676 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  78. Pellestor, F. & Paulasova, P. The peptide nucleic acids (PNAs), powerful tools for molecular genetics and cytogenetics. Eur. J. Hum. Genet. 12, 694–700 (2004).

    Article  PubMed  CAS  Google Scholar 

  79. Corey, D. R. & Abrams, J. M. Morpholino antisense oligonucleotides: tools for investigating vertebrate development. Genome Biol. 2, reviews1015.1–reviews1015.3 (2001).

    Article  Google Scholar 

  80. Hewitt, S. L. et al. Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs. Sci. Transl. Med. 11, eaat9143 (2019).

    Article  PubMed  CAS  Google Scholar 

  81. Musunuru, K. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429–434 (2021).

    Article  PubMed  CAS  Google Scholar 

  82. Arevalo, C. P. et al. A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes. Science 378, 899–904 (2022).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  83. Foy, S. P. et al. Non-viral precision T cell receptor replacement for personalized cell therapy. Nature 615, 687–696 (2023).

    Article  PubMed  CAS  Google Scholar 

  84. Granit, V. et al. Safety and clinical activity of autologous RNA chimeric antigen receptor T-cell therapy in myasthenia gravis (MG-001): a prospective, multicentre, open-label, non-randomised phase 1b/2a study. Lancet Neurol. 22, 578–590 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  85. Cai, A. et al. Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation. Biochemistry 38, 8538–8547 (1999).

    Article  PubMed  CAS  Google Scholar 

  86. Flamme, M., McKenzie, L. K., Sarac, I. & Hollenstein, M. Chemical methods for the modification of RNA. Methods 161, 64–82 (2019).

    Article  PubMed  CAS  Google Scholar 

  87. Zhang, X. et al. Structural insights into FTO’s catalytic mechanism for the demethylation of multiple RNA substrates. Proc. Natl Acad. Sci. USA 116, 2919–2924 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  88. Shi, H. et al. Spatial atlas of the mouse central nervous system at molecular resolution. Nature 622, 552–561 (2023).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  89. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  PubMed  CAS  Google Scholar 

  90. Weigert, M., Schmidt, U., Haase, R., Sugawara, K. & Myers, G. Star-convex polyhedra for 3D object detection and segmentation in microscopy. In Proceedings of the IEEE/CVF Winter Conference on Applications of Computer Vision (eds Ross, A., Cox, D. & McCloskey, S.) 3666–3673 (IEEE, 2020).

  91. Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  92. Johnson, W. E., Li, C. & Rabinovic, A. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8, 118–127 (2007).

    Article  PubMed  Google Scholar 

  93. Edupuganti, R. R. et al. N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis. Nat. Struct. Mol. Biol. 24, 870–878 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  94. Alexander, S. C., Busby, K. N., Cole, C. M., Zhou, C. Y. & Devaraj, N. K. Site-specific covalent labeling of RNA by enzymatic transglycosylation. J. Am. Chem. Soc. 137, 12756–12759 (2015).

    Article  PubMed  CAS  Google Scholar 

  95. Meier, S., Güthe, S., Kiefhaber, T. & Grzesiek, S. Foldon, the natural trimerization domain of T4 fibritin, dissociates into a monomeric A-state form containing a stable β-hairpin: atomic details of trimer dissociation and local β-hairpin stability from residual dipolar couplings. J. Mol. Biol. 344, 1051–1069 (2004).

    Article  PubMed  CAS  Google Scholar 

  96. Chen, H. et al. Raw SILAC mass spectrometry data of chemical and topological design of multi-capped mRNA and capped circular RNA. Zenodo https://doi.org/10.5281/zenodo.12611469 (2024).

  97. Chen, H. et al. Lymph node STARmap/RIBOmap dataset of chemical and topological design of multi-capped mRNA and capped circular RNA. Zenodo https://doi.org/10.5281/zenodo.12518588 (2024).

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Acknowledgements

We would like to thank J. Ren (MIT and Broad Institute) and J. Tian (MIT and Broad Institute) for their help on the in situ sequencing experiments. We would also like to thank the flow cytometry core facility (Broad Institute) for providing access to their instruments. We would also like to thank M. Ford and others (MS Bioworks) for assistance with qMS. We would also like to thank other members of X.W.’s lab for helpful discussion throughout the project. X.W. acknowledges the support from the E. Scolnick Professorship, Ono Pharma Breakthrough Science Initiative Award, Merkin Institute Fellowship, Klarman Cell Observatory, Packard Fellowship, Sloan Research Fellowship and NIH DP2 New Innovator Award (1DP2GM146245). M.J.F. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) FR 4701/1-1. R.J.X. acknowledges support from NIH grants DK43351 and DK135492. F.Z. is supported by the Howard Hughes Medical Institute and NIH grant 2R01HG009761-05 and Broad Institute Programmable Therapeutics Gift Donors.

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Author notes

  1. These authors contributed equally: Hongyu Chen, Dangliang Liu, Abhishek Aditham.

Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Hongyu Chen, Dangliang Liu, Jianting Guo, Jiahao Huang, Franklin Kostas & Xiao Wang

  2. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Hongyu Chen, Dangliang Liu, Abhishek Aditham, Jianting Guo, Jiahao Huang, Franklin Kostas, Kamal Maher, Mirco J. Friedrich, Ramnik J. Xavier, Feng Zhang & Xiao Wang

  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Abhishek Aditham, Mirco J. Friedrich & Feng Zhang

  4. Computational and Systems Biology Program, Massachusetts Institute of Technology, Cambridge, MA, USA

    Kamal Maher

  5. Howard Hughes Medical Institute, Cambridge, MA, USA

    Mirco J. Friedrich & Feng Zhang

  6. McGovern Institute for Brain Research at MIT, Cambridge, MA, USA

    Mirco J. Friedrich & Feng Zhang

  7. Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA

    Mirco J. Friedrich & Feng Zhang

  8. Center for Computational and Integrative Biology, Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA

    Ramnik J. Xavier

  9. Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Xiao Wang

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Conceptualization, X.W. Methodology and investigation, H.C. and D.L. Cloning, A.A. and J.G. Biochemical assays, H.C., D.L. and A.A. Data analysis, H.C., D.L., J.H. and K.M. Design and interpretation of vaccine tests, H.C., D.L., A.A., F.K., M.J.F., R.J.X., F.Z. and X.W. Supervision, X.W. Writing—original draft, H.C. and D.L. Writing—review and editing, all authors contributed to reviewing and editing the manuscript.

Corresponding author

Correspondence to Xiao Wang.

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Competing interests

X.W., H.C., D.L., A.A. and J.G. are inventors of patent applications related to this work. X.W. is a consultant, equity holder and scientific cofounder of Stellaromics and Convergence Bio. R.J.X. is a cofounder of Celsius Therapeutics and Jnana Therapeutics, scientific advisory board member at Nestle and board director at MoonLake Immunotherapeutics. F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, Proof Diagnostics, Aera Therapeutics and Moonwalk Biosciences. F.Z. is a scientific advisor for Octant. The remaining authors declare no competing interests.

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Nature Biotechnology thanks Ru-Yi Zhu 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 Synthesis and purification of fully capped synthetic oligonucleotides.

(a) Chemical capping and HPLC purification of solid-phase synthesized oligonucleotides. Crude chemical capping reactions with varying oligonucleotide counterions (triethylammonium or ammonium) were analyzed by analytical HPLC. Capped/uncapped oligos were fully resolved by using hexylammonium acetate as HPLC aqueous buffer. (b) Scalability of oligo capping. (c) Combination of m6A with sugar backbone modifications on the first 1 ~ 3 nucleotides. n = 3, biological replicates. Mean ± sem.

Source data

Extended Data Fig. 2 Synthesis and characterization of 5′/3′-modified dual-capped mRNA.

(a) Synthesis of dual-capped HiBiT mRNA. (b) Denaturing gel electrophoresis characterization of branched/linear HiBiT mRNAs (15% TBE-Urea). M, marker. Gel image is representative of two replicated experiments. (c) Relative HiBiT luminescence in HeLa cell normalized to chemically capped linear HiBiT mRNA. n = 3, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided t-test. (d) Synthesis workflow of 5′/3′-modified mRNA. 5′ triphosphorylated mRNA was synthesized by IVT, and ligated to exonuclease-resistant oligonucleotides containing 3′ blocking groups. The 5′ triphosphate was then hydrolyzed by RNA 5′ pyrophosphohydrolase (RppH) and ligated to capped oligonucleotides on the 5′ end. (e) The alkyne handle position does not significantly affect mRNA translation. n = 3, biological replicates. Mean ± sem. P values were calculated by ordinary one-way ANOVA. (f) Phosphorothioate modification on the branched oligo minimally further enhanced translation. (g) Representative RP-HPLC chromatogram of modified mRNA purification post LEGO and branched oligo recovery. (h ~ i) Gel electrophoresis characterization to confirm dual-capping using an RNase H assay (15% TBE) and no self-ligation (2% Agarose). M, marker. Gel images are representative of >20 assays performed under similar settings. (j) Effects of only tail stabilization on Fluc mRNA translation with a phosphorothioate_dideoxycytidine (PS_ddC) modified poly(A) tail or a phosphorothioate_2′-O-methoxyethyl_dideoxycytidine (PS_2MOE_ddC) modified poly(A) tail. n = 9, biological replicates. Mean ± sem.

Source data

Extended Data Fig. 3 Biochemical evaluation of chemically and topologically modified oligonucleotides on mRNA translation.

(a ~ f) EMSA of modified capped oligonucleotides (0.1 nM) with eIF4E (4 ~ 20% gradient native TBE gel). (g) EMSA of modified oligo with 1.3 µM recombinant eIF4E and 200 nM recombinant eIF4G, 3 replicated experiments (4 ~ 20% gradient native TBE gel). (h ~ i) Denaturing gel electrophoresis analysis of chemically modified capped oligos by hDcp2 at various time points (15% TBE-Urea). (j) EMSA of dual-capped oligonucleotides (0.1 nM) with eIF4E (4 ~ 20% gradient native TBE gel). (k) Competitive EMSA of dual-m7G-capped vs mono-m7G-capped oligonucleotides (0.1 nM) with eIF4E (4 ~ 20% gradient native TBE gel). (l) Evaluation of immune toxicity in HeLa cells of chemically and topologically modified mRNAs by qPCR detection of key molecules involved in RIG-1 signaling pathways, normalized to GAPDH level and then to lipofectamine transfection control. n = 3, biological replicates. Mean ± sem. P values were calculated by ordinary one-way ANOVA.

Source data

Extended Data Fig. 4 Probing the involvement of eIF3D-dependent translation initiation by dual-capped mRNA.

(a) Probing involvement of eIF3D-dependent translation initiation using eIF3D knockdown or eIF4E/eIF4G interaction inhibitor. (b ~ d) Expression of mono-capped RLuc and dual-capped FLuc mRNA co-transfected in wild-type or eIF3D-knockdown HeLa cells by siRNA. n = 3, biological replicates. Mean ± sem. P values were calculated by two-sided unpaired t-test. (e ~ g) In vitro translation of mono-capped FLuc and dual-capped NLuc with or without 4EGI-1 in rabbit reticulocyte lysates (RRL). n = 3, biological replicates. Mean ± sem. P values were calculated by two-sided unpaired t-test.

Source data

Extended Data Fig. 5 Synthesis and characterization of QRNA.

(a) Synthesis of a minimal QRNA encoding a HiBiT tag. (b) Relative HiBiT luminescence in HeLa cell normalized to HiBiT circRNA. n = 3, biological replicates. Mean ± sem. P values were calculated by two-sided unpaired t-test. (c) Synthetic workflow of QRNA encoding the full length Nano luciferase (NLuc) protein. A tRNA hairpin was placed in the 5′ untranslated region. The synthetic mRNA (without m1ψ) was circularized by intron mediated back splicing and labeled with a tRNA-guanine transglycosylase and a azide-labeled pre-queuosine 1 (preQ1) analogue. A synthetic oligonucleotide with a 5′ Bn7G cap and 3′ alkyne was conjugated to the azide-circRNA, and the QRNA/circRNA were separated on HPLC by the hydrophobicity of the benzyl group. (d) Agarose gel electrophoresis characterization of NLuc circRNAs (with or without an IRES) by intron-mediated backsplicing and then RNase R treatment. M, marker. 2% Agarose gel. (e) Expression of circRNA/QRNA without m1ψ in HeLa cells. (f) LEGO enables site-specific chemical and topological modification of circRNA in a m1Ψ-compatible manner. mRNA is synthesized by IVT with 100% m1Ψ replacement and digested with calf-intestinal alkaline phosphatase (CIAP) to generate 5′/3′-hydroxylated mRNA. A synthetic 5′/3′-phosphorylated oligo (with/without a branched cap or other chemical modifications) was ligated to the mRNA first by T4 RNA ligase and then circularized by RtcB ligase. (g) Exemplary HPLC trace for separation of circular products from linear precursor post LEGO workflow. (h) Confirmation of circular topology and branch capping for QRNA products by double RNase H assay. RNase H with a single primer leads to linearization rather than fragmentation, confirming the circular topology. RNase H with two primers flanking both the downstream and upstream sites of the branching point generated the fragment, where branch capping leads to further band shift in gel electrophoresis due to topological patterns. Gel image is representative of >4 assays with similar settings.

Source data

Extended Data Fig. 6 Evaluation of the ribosome landing site induced by the branched cap.

Designs and evaluations of two-AUG HiBiT reporters (S21 ~ 26) and evaluation of translation activity in RRL. All constructs contained 5′-exonuclease resistant modifications, a fixed branching site at the +4 base, a fixed out-of-frame start codon 46 nt downstream the branching site, a 30-A polyA tail, and an in-frame start codon encoding the HiBiT tag 26/29/32/35/38 nt downstream the branching site. 200 ng of the mRNA was translated in the rabbit reticulocyte lysate (RRL) for 1 hour before assayed for HiBiT activity. n = 3, independent experiments. Mean ± sem. P values were calculated by unpaired two-sided t-test with Welch’s correction.

Source data

Extended Data Fig. 7 Dual-capped hEPO mRNA enhances generation of reticulocytes in mouse plasma.

(a) Flow cytometry gating strategy for reticulocytes counting from peripheral mouse plasma. (b) Representative reticulocyte counting for each condition using flow cytometry on Day 6.

Source data

Extended Data Fig. 8 Spatial transcriptomics analysis of mouse lymph nodes after booster injection.

(a) The preprocessed lymph node samples were clustered by regions with SPIN to identify T/B/margin/capsule regions, and subsequently clustered by k-means. (b) Dot plot quantification of cell type specific gene expression for major cell types present in the lymph nodes. (c) Quantification of antigen presenting B cell/dendritic cells(DC)/macrophages(Mφ.) percentage in specified lymph node region (that is APC (antigen presenting cells) counts in region / total counts of specific cell types). n = 4. Mean ± s.e.m. P values were calculated by ordinary one-way ANOVA.

Source data

Extended Data Fig. 9 Quantification of SARS-CoV-2-RBD-specific T cells in mice after vaccination.

(a) Gating strategy for single and viable T cells in splenocytes. CD4+ or CD8+ T-effector memory (Tem) cells (CD44+CD62L−) were further analyzed to detect the expression of cytokines stimulated by corresponding RBD peptide pools (b ~ c) Representative flow plots for specific CD4+ T cell response (b) or CD8+ T cell response (c). (d ~ h) Measurement of the level of IL-2 (d), IL-4 (e), IL-13 (f), TNF (g) or IFN-γ (h) in the supernatants of peptide pool-stimulated splenocytes with ELISA. n = 4, biological replicates. Mean ± sem. P values were calculated by unpaired two-sided t-test for comparison within each treatment conditions (DMSO vs S-peptide). P values were calculated by one-way ANOVA with Šídák’s multiple comparisons test for comparison between constructs under S-peptide treatment conditions.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

Compiled sequences of all constructs and probes used in this study.

Source data

Source Data Figs. 1–6 and Extended Data Figs. 1–9

Compiled statistical source data for all figures presented.

Source Data Fig. 2d and Extended Data Figs. 2b,h,i, 3a–k and 5g,h

Full-length gel scans for gel images that were vertically clipped in the main figures.

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

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