Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System,Nano Letters

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Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System
Nano Letters ( IF 9.6 ) Pub Date : 2018-03-22 00:00:00 , DOI: 10.1021/acs.nanolett.8b00526
Matthaeus Schwarz-Schilling ₁ , Aurore Dupin ₁ , Fabio Chizzolini ₂ , Swati Krishnan ₁ , Sheref S. Mansy ₂ , Friedrich C. Simmel _{1,

3}

Affiliation

Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.

中文翻译：

无细胞基因表达系统中多功能RNA蛋白质纳米结构的优化组装。

由RNA分子和蛋白质组成的分子复合物是有前途的多功能纳米结构，可用于生物细胞或人工细胞系统中的多种应用。在这项研究中，我们系统地解决了与使用无细胞基因表达系统表达和装配这种杂化结构有关的一些挑战。作为模型结构，我们研究了pRNA衍生的RNA支架，该支架被四个不同的适体功能化，其中三个与蛋白质，链霉亲和素和两个荧光蛋白结合，而一个与小分子染料孔雀石绿（MG）结合。我们利用RNA支架蛋白之间的MG荧光和Förster共振能量转移（FRET），评估了关键的组装参数，例如化学稳定性，结合效率，以及反应室内的资源共享效应。然后，我们优化单室无细胞基因表达系统内RNA蛋白纳米结构的同时表达和共装配。我们展示了乳液液滴内部的多组分纳米结构的表达和组装及其在链霉亲和素包被的基质上的适体介导的定位，以及细菌细胞内部的杂化结构的成功组装。

更新日期：2018-03-22

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