多克隆mRNA和带帽环状RNA的化学和拓扑设计以增强翻译-动脉网

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

Nature 等信源发布 2024-09-23 18:15

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AbstractProtein 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.

摘要基于mRNA的蛋白质和疫苗疗法将受益于翻译能力的提高。在这里，我们报告了一种增强翻译的方法，称为连接启用的mRNA-寡核苷酸组装（LEGO）。我们系统地筛选了不同的化学拓扑基序，发现分支的mRNA帽有效地启动了线性或环状mRNA的翻译，而没有内部核糖体进入位点。

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.

两种类型的化学修饰，即帽上的锁核酸（LNA）N7甲基鸟苷修饰和5'非翻译区上的LNA+×2'O-甲基，可增强RNA-真核翻译起始因子（eIF4E-eIF4G）的结合和RNA在体外的稳定性。通过双封端mRNA和封端环状RNA的多维化学拓扑工程，我们在体内将mRNA蛋白产量提高了10倍，导致在严重急性呼吸综合征冠状病毒2疫苗中初次和加强剂量后抗体产量分别提高了17倍和3.7倍。

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..

乐高平台为基础研究和治疗应用提供了超越经典线性和环状RNA的非自然RNA结构和拓扑设计的可能性。。

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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.

图1:mRNA帽和5'UTR修饰的多维化学筛选。图2：双帽mRNA的化学拓扑设计和工程。图3：mRNA翻译中5'化学和拓扑修饰的机理表征。图4:m1Ψ修饰的QRNA的合成和cap近端翻译诱导的机制探测。图。

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..

5：化学拓扑优化的mRNA在体内表现出增强的蛋白质表达。图6：化学修饰的双帽mRNA增强了SARS-CoV-2 mRNA疫苗在小鼠中的效力。。

Data availability

数据可用性

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.

补充表1提供了本研究中使用的所有RNA构建体的序列和修饰信息。本研究中使用的质粒被保存到Addgene。用于SILAC下拉实验的原始MS数据可在Zenodo96上获得。SARS-CoV-2疫苗淋巴结星图和RIBOmap数据集可在Zenodo97上获得。

Source data are provided with this paper..

本文提供了源数据。。

Code availability

代码可用性

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

GitHub上提供了用于数据分析的代码(https://github.com/wanglab-broad/Multicap-analysis)。

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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).Chen, H. et al. Lymph node STARmap/RIBOmap dataset of chemical and topological design of multi-capped mRNA and capped circular RNA.

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Zenodo https://doi.org/10.5281/zenodo.12518588 (2024).Download referencesAcknowledgementsWe 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.

泽诺多https://doi.org/10.5281/zenodo.12518588（2024年）。下载参考文献致谢我们要感谢J.Ren（麻省理工学院和布罗德研究所）和J.Tian（麻省理工学院和布罗德研究所）在原位测序实验方面的帮助。我们还要感谢流式细胞仪核心设施（Broad Institute）提供对其仪器的访问。

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).

我们还要感谢M.Ford和其他人（Bioworks女士）对qMS的帮助。我们还要感谢X.W.实验室的其他成员在整个项目中进行了有益的讨论。十、 W.感谢E.Scolnick教授、Ono Pharma突破性科学倡议奖、Merkin研究所奖学金、Klarman细胞天文台、Packard奖学金、Sloan研究奖学金和NIH DP2新创新者奖（1DP2GM146245）的支持。

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.Author informationAuthor notesThese authors contributed equally: Hongyu Chen, Dangliang Liu, Abhishek Aditham.Authors and AffiliationsDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USAHongyu Chen, Dangliang Liu, Jianting Guo, Jiahao Huang, Franklin Kostas & Xiao WangBroad Institute of MIT and Harvard, Cambr.

M、 J.F.由Deutsche Forschungsgemeinschaft（DFG，德国研究基金会）FR 4701/1-1资助。R、 J.X.感谢NIH拨款DK43351和DK135492的支持。F、 Z.得到了霍华德·休斯医学研究所和NIH grant 2R01HG009761-05以及Broad Institute Programmable Therapeutics礼品捐赠者的支持。作者信息作者注意到这些作者做出了同样的贡献：陈宏宇，刘当良，阿披实·阿迪瑟姆。作者和附属机构麻省理工学院化学系，剑桥，马萨诸塞州，美国陈宏宇，刘当良，郭建庭，黄家豪，麻省理工学院和哈佛大学Franklin Kostas＆Xiao WangBroad研究所，坎布尔。

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PubMed Google ScholarContributionsConceptualization, 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 authorCorrespondence to.

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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.

十、 W.，H.C.，D.L.，A.A.和J.G.是与这项工作相关的专利申请的发明人。X.W.是Stellaromics和Convergence Bio的顾问、股权持有人和科学联合创始人。R.J.X.是Celsius Therapeutics和Jnana Therapeutics的联合创始人，雀巢的科学顾问委员会成员和MoonLake Immunotherapeutics的董事。

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..

F、 Z.是Editas Medicine、Beam Therapeutics、成对植物、Arbor Biotechnologies、Proof Diagnostics、Aera Therapeutics和Moonwalk Biosciences的科学顾问和联合创始人。F、 Z.是Octant的科学顾问。其余作者声明没有利益冲突。。

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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.

Nature Biotechnology感谢朱汝毅和另一位匿名审稿人为这项工作的同行评审做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Synthesis and purification of fully capped synthetic oligonucleotides.(a) Chemical capping and HPLC purification of solid-phase synthesized oligonucleotides.

Additional informationPublisher的注释Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。扩展数据扩展数据图1完全封端的合成寡核苷酸的合成和纯化。（a）固相合成寡核苷酸的化学封端和HPLC纯化。

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.

通过分析HPLC分析了具有不同寡核苷酸抗衡离子（三乙基铵或铵）的粗化学封端反应。通过使用乙酸己基铵作为HPLC水性缓冲液，完全解析了加帽/未加帽的寡核苷酸。（b）寡核苷酸加帽的可扩展性。（c） m6A与前1〜3个核苷酸上的糖骨架修饰的组合。

n = 3, biological replicates. Mean ± sem.Source dataExtended 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.

n=3，生物学重复。平均sem.Source数据扩展数据图2 5'/3'修饰的双帽mRNA的合成和表征。（a）双帽HiBiT mRNA的合成。（b）支链/线性HiBiT mRNA的变性凝胶电泳表征（15%TBE尿素）。M、标记。凝胶图像是两个重复实验的代表。

(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.

（c） HeLa细胞中的相对HiBiT发光归一化为化学封端的线性HiBiT mRNA。n=3，生物学重复。通过不成对的双侧t检验计算平均sem.P值。（d） 5'/3'修饰mRNA的合成流程。通过IVT合成5'三磷酸化mRNA，并连接到含有3'阻断基团的核酸外切酶抗性寡核苷酸上。

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.

然后用RNA 5'焦磷酸水解酶（RppH）水解5'三磷酸，并在5'端连接到带帽的寡核苷酸上。（e）炔烃手柄位置不会显着影响mRNA翻译。n=3，生物学重复。通过普通单因素方差分析计算平均sem.P值。

(f) Phosphorothioate modification on the branched oligo minima.

（f）分支寡核苷酸的硫代磷酸酯修饰。

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