Xu Yunfan, Qi Shuopeng, Zhang Gongrui, Liu Dan, Xu Dejin, Qin Tong, Cheng Qin, Kang Han, Hu Bei, Huang Zhen
Key Laboratory of Bio-resource and Eco-environment of Ministry of Education, The College of Life Sciences, Sichuan University, 24 South Section 1, 1st Ring Road, Chengdu, Sichuan 610064, P.R. China.
SeNA Research Institute, School of Life Sciences, Hubei University, 368 Youyi Avenue, Wuhan, Hubei 430062, P.R. China.
Nucleic Acids Res. 2025 Jan 7;53(1). doi: 10.1093/nar/gkae1280.
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.
区域特异性RNA修饰对于推进RNA研究和治疗至关重要，包括基于信使核糖核酸（mRNA）的疫苗和免疫疗法。然而，目前的主要方法是用短单链DNA（ssDNA）夹板合成区域修饰的mRNA，由于RNA自折叠复杂结构的形成，在连接长mRNA片段时遇到了挑战。为了解决这个问题，我们开发了一种高效策略，在原位变性后使用容易获得的长双链DNA（dsDNA）作为连接夹板，而该dsDNA的部分区域是转录mRNA片段的模板。我们观察到变性后的dsDNA与这些mRNA片段形成了长杂交双链体，克服了它们的结构。此外，我们的新策略显著促进了长mRNA片段（特别是结构化片段）的连接，连接效率比ssDNA方法高出106倍。使用这种一锅法策略，我们方便地合成了在特定区域具有N1-甲基假尿苷（m1ψ）和5-甲基胞苷（m5C）修饰的mRNA。我们发现，与完全修饰的mRNA相比，仅3'非翻译区（3'UTR）的m1ψ修饰提高了翻译效率，而m1ψ-3'UTR和m5C-5'非翻译区/编码区（5'UTR/CDS）的联合修饰总体上表现出更高的翻译效率和更低的免疫原性。我们的研究提出了一种广泛适用的策略来生产区域修饰的mRNA，提升了mRNA治疗的潜力。
