One-pot Ligation of Multiple MRNA Fragments on DsDNA Splint Advancing Regional Modification and Translation

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Date 2025 Jan 8

PMID 39778864

Authors

Yunfan Xu

Shuopeng Qi

Gongrui Zhang

Dan Liu

Dejin Xu

Tong Qin

Qin Cheng

Han Kang

Bei Hu

Zhen Huang

Affiliations

Soon will be listed here.

Abstract

Region-specific RNA modifications are crucial for advancing RNA research and therapeutics, including messenger RNA (mRNA)-based vaccines and immunotherapy. However, the predominant method, synthesizing regionally modified mRNAs with short single-stranded DNA (ssDNA) splints, encounters challenges in ligating long mRNA fragments due to the formation of RNA self-folded complex structures. To address this issue, we developed an efficient strategy using an easily obtained long double-stranded DNA (dsDNA) as a ligation splint after in situ denaturing, while parts of this dsDNA are the templates for transcribing mRNA fragments. We observed that the denatured dsDNA formed a long hybrid duplex with these mRNA fragments, overcoming their structures. Further, our novel strategy remarkably facilitated the ligation of long mRNA fragments (especially structured ones), offering ligation efficiency up to 106-fold higher than the ssDNA method. Using this one-pot strategy, we conveniently synthesized the mRNAs with N1-methylpseudouridine (m1ψ) and 5-methylcytidine (m5C) modifications in specific regions. We have found that compared with the fully modified mRNAs, the 3'UTR m1ψ modifications alone increased the translation efficiency, and the combined modifications of the m1ψ-3'UTR and m5C-5'UTR/CDS exhibited higher translation efficiency and lower immunogenicity in general. Our study presents a broadly applicable strategy for producing regionally modified mRNAs, advancing the potential of mRNA therapeutics.

References

Muller S, Appel B . In vitro circularization of RNA. RNA Biol. 2016; 14(8):1018-1027. PMC: 5680678. DOI: 10.1080/15476286.2016.1239009. View

Chen H, Cheng K, Liu X, An R, Komiyama M, Liang X . Preferential production of RNA rings by T4 RNA ligase 2 without any splint through rational design of precursor strand. Nucleic Acids Res. 2020; 48(9):e54. PMC: 7229815. DOI: 10.1093/nar/gkaa181. View

Wang X, Liu C, Rcheulishvili N, Papukashvili D, Xie F, Zhao J . Strong immune responses and protection of PcrV and OprF-I mRNA vaccine candidates against Pseudomonas aeruginosa. NPJ Vaccines. 2023; 8(1):76. PMC: 10209580. DOI: 10.1038/s41541-023-00672-4. View

Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M . Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 2015; 208:44-53. DOI: 10.1016/j.jbiotec.2015.04.024. View

Wang P, Ko S, Tian C, Hao C, Mao C . RNA-DNA hybrid origami: folding of a long RNA single strand into complex nanostructures using short DNA helper strands. Chem Commun (Camb). 2013; 49(48):5462-4. DOI: 10.1039/c3cc41707g. View

Nakamoto K, Abe N, Tsuji G, Kimura Y, Tomoike F, Shimizu Y . Chemically synthesized circular RNAs with phosphoramidate linkages enable rolling circle translation. Chem Commun (Camb). 2020; 56(46):6217-6220. DOI: 10.1039/d0cc02140g. View

Kariko 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. 2005; 23(2):165-75. DOI: 10.1016/j.immuni.2005.06.008. View

Yeh A, Norn C, Kipnis Y, Tischer D, Pellock S, Evans D . De novo design of luciferases using deep learning. Nature. 2023; 614(7949):774-780. PMC: 9946828. DOI: 10.1038/s41586-023-05696-3. View

Creech C, Anderson E, Berthaud V, Yildirim I, Atz A, Melendez Baez I . Evaluation of mRNA-1273 Covid-19 Vaccine in Children 6 to 11 Years of Age. N Engl J Med. 2022; 386(21):2011-2023. PMC: 9127699. DOI: 10.1056/NEJMoa2203315. View

10.

Davanloo P, Rosenberg A, Dunn J, Studier F . Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984; 81(7):2035-9. PMC: 345431. DOI: 10.1073/pnas.81.7.2035. View

11.

Abe N, Hiroshima M, Maruyama H, Nakashima Y, Nakano Y, Matsuda A . Rolling circle amplification in a prokaryotic translation system using small circular RNA. Angew Chem Int Ed Engl. 2013; 52(27):7004-8. DOI: 10.1002/anie.201302044. View

12.

Edupuganti R, Geiger S, Lindeboom R, Shi H, Hsu P, Lu Z . N-methyladenosine (mA) recruits and repels proteins to regulate mRNA homeostasis. Nat Struct Mol Biol. 2017; 24(10):870-878. PMC: 5725193. DOI: 10.1038/nsmb.3462. View

13.

Watson O, Barnsley G, Toor J, Hogan A, Winskill P, Ghani A . Global impact of the first year of COVID-19 vaccination: a mathematical modelling study. Lancet Infect Dis. 2022; 22(9):1293-1302. PMC: 9225255. DOI: 10.1016/S1473-3099(22)00320-6. View

14.

Hughes R, Ellington A . Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harb Perspect Biol. 2017; 9(1). PMC: 5204324. DOI: 10.1101/cshperspect.a023812. View

15.

Weber J, Carlino M, Khattak A, Meniawy T, Ansstas G, Taylor M . Individualised neoantigen therapy mRNA-4157 (V940) plus pembrolizumab versus pembrolizumab monotherapy in resected melanoma (KEYNOTE-942): a randomised, phase 2b study. Lancet. 2024; 403(10427):632-644. DOI: 10.1016/S0140-6736(23)02268-7. View

16.

Praetorius F, Kick B, Behler K, Honemann M, Weuster-Botz D, Dietz H . Biotechnological mass production of DNA origami. Nature. 2017; 552(7683):84-87. DOI: 10.1038/nature24650. View

17.

Mauger D, Cabral B, Presnyak V, Su S, Reid D, Goodman B . mRNA structure regulates protein expression through changes in functional half-life. Proc Natl Acad Sci U S A. 2019; 116(48):24075-24083. PMC: 6883848. DOI: 10.1073/pnas.1908052116. View

18.

Martin C, Coleman J . T7 RNA polymerase does not interact with the 5'-phosphate of the initiating nucleotide. Biochemistry. 1989; 28(7):2760-2. DOI: 10.1021/bi00433a002. View

19.

Thomas S, Moreira Jr E, Kitchin N, Absalon J, Gurtman A, Lockhart S . Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine through 6 Months. N Engl J Med. 2021; 385(19):1761-1773. PMC: 8461570. DOI: 10.1056/NEJMoa2110345. View

20.

Nance K, Meier J . Modifications in an Emergency: The Role of N1-Methylpseudouridine in COVID-19 Vaccines. ACS Cent Sci. 2021; 7(5):748-756. PMC: 8043204. DOI: 10.1021/acscentsci.1c00197. View