利用5′帽子亲和层析纯化IVT mRNA  

Purification of IVT mRNA by 5′cap affinity chromatography

作　　者：杜彬荷 林金钟 Binhe Du;Jinzhong Lin(State Key Laboratory of Genetic Engineering,Fudan University,Shanghai 200438,China;Center for mRNA Translational Research,Fudan University,Shanghai 200438,China)

机构地区：[1]复旦大学遗传工程国家重点实验室,上海200438 [2]复旦大学mRNA药物研发中心,上海200438

出　　处：《科学通报》2024年第33期4928-4938,共11页Chinese Science Bulletin

基　　金：国家自然科学基金(32371352);浙江大学上海高等研究院繁星科学基金(SN-ZJU-SIAS-009)资助。

摘　　要：mRNA作为指导蛋白质合成的活性分子,在翻译基础研究和mRNA药物研发中具有广阔的研究价值和应用前景.真核生物细胞质mRNA通常具有区别于其他类型RNA分子的5′端帽结构,在mRNA核转运、抵御核酸外切酶和介导翻译起始过程中发挥重要作用.无帽mRNA分子在机体内引起严重的免疫反应,因此mRNA的加帽率是评判其成药性的核心指标.虽然加帽mRNA可以通过基于T7 RNAP的共转录加帽技术和基于牛痘加帽酶的酶法加帽技术获得,但仍缺乏精确分选加帽mRNA的底盘技术.针对这一现象,本研究选取了覆盖真核生物、古菌、病毒在内的18种帽结合蛋白进行表达纯化测试,并表征了热稳定性、帽结构亲和性等参数.从中筛选获得3种代表性帽结合蛋白进行加帽RNA的亲和层析测试,实验结果表明,Hs IF4E K119A和Ct IF4E可以有效地富集加帽RNA,并且可以通过改变盐浓度的方法实现加帽RNA的洗脱.本研究开发的基于帽结构的亲和层析技术具有成本低、效率高、易于放大的特征,有望为分选加帽mRNA提供新的解决方案.Despite the remarkable strides in mRNA therapeutics,exemplified by the development of COVID-19 vaccines,their widespread application faces various challenges.These obstacles encompass concerns related to immunogenicity,inefficient in-vitro synthesis,and instability within preparations.A prominent factor contributing to these challenges is the presence of uncapped mRNA molecules,lacking the essential 5′cap and its 7-methylguanosine(m7 G)modification.This cap plays a pivotal role in enhancing stability,initiating translation,and evading the immune system.The absence of the cap leads to rapid degradation and potential immune activation.Current capping methods for in vitro transcribed mRNA(IVT mRNA)fall short of completely eliminating uncapped molecules.Both post-transcriptional capping and co-transcriptional approaches add the cap structure,but they cannot guarantee 100%efficiency.Due to the minor chemical distinction between capped and uncapped mRNA,there has been no established method for separating the two.Consequently,in the current pharmaceutical manufacturing of mRNA,uncapped RNA carries over from IVT reactions to the final drug products.This study bridges this critical gap by introducing a novel cap-affinity chromatography method for highly selective purification of IVT mRNA.Our approach harnesses the potent m7 G-binding capabilities of eukaryotic initiation factor 4E(eIF4E),a protein that plays a pivotal role in translation initiation by recognizing and binding the cap structure.We conducted a rigorous screening of 18 proteins,including eIF4E homologs from viruses,evaluating them for thermal stability,cap-binding affinity and specificity,and high-level recombinant expression.Three exceptional candidates emerged:(1)Ct.eIF4E from Chaetomium thermophilum,a thermophilic fungal eIF4E with excellent cap binding and thermal stability;(2)the La4E from Thermomyces lanuginosus,another thermophilic fungal eIF4E with strong cap affinity and stability;(3)human eIF4E mutant K119A,a modified human eIF4E exhibiting highly

关 键 词：帽结构 帽结合蛋白 亲和层析 mRNA纯化 mRNA药物 

分 类 号：Q75[生物学—分子生物学]