Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA-protein correlations at the level of single cells.
Characterizing exogenous mRNA delivery, trafficking, cytoplasmic release and RNA-protein correlations at the level of single cells.
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DOI：
10.1093/nar/gkx290
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发表时间：
2017-07-07
影响因子：
14.9
通讯作者：
Santangelo PJ
中科院分区：
文献类型：
--
作者：
Kirschman JL;Bhosle S;Vanover D;Blanchard EL;Loomis KH;Zurla C;Murray K;Lam BC;Santangelo PJ
使用合成信使核糖核酸（mRNA）来表达特定蛋白质是一种非常有前途的治疗和疫苗方法，可以避免与病毒或基于DNA的系统相关的许多安全问题。然而，为了优化mRNA设计和递送，需要技术进步来研究mRNA在单细胞和组织水平上摄取和定位的基本机制。在这里，我们提出了一个单一的RNA敏感的荧光标记方法，使我们能够标记和可视化合成的mRNA，而不会显着影响功能。这种方法使单细胞表征的mRNA摄取和释放动力学从内吞隔室，mRNA/蛋白质的相关性的测量，并激发了mRNA诱导的细胞应激的研究，所有重要的机制影响蛋白质的生产。此外，我们证明了这种方法可以促进体内和离体组织切片中mRNA定位的近红外成像，这将促进临床前模型中的mRNA运输研究。总体而言，我们证明了研究优化递送和治疗策略所需的基本机制的能力，以设计下一代新型mRNA治疗剂和疫苗。
The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will facilitate mRNA trafficking studies in pre-clinical models. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines.