Designing and using RNA scaffolds to assemble proteins in vivo
Designing and using RNA scaffolds to assemble proteins in vivo
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DOI：
10.1038/nprot.2012.102
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发表时间：
2012-10-01
期刊：
影响因子：
14.8
通讯作者：
Lindner, Ariel B.
中科院分区：
文献类型：
--
作者：
Delebecque, Camille J.;Silver, Pamela A.;Lindner, Ariel B.
RNA支架是合成的非编码RNA分子，具有工程化的3D折叠，用于在体内空间组织蛋白质。在这里，我们提供了一个协议，设计，表达和表征RNA支架及其同源蛋白在1个月内。这里描述的RNA支架设计是基于携带RNA适体作为蛋白质对接位点的单体或多聚体单元。将支架和蛋白质克隆到诱导型质粒中并表达以形成功能组装体。RNA支架在许多领域中找到应用，其中生物分子的体内组织是感兴趣的。与DNA或蛋白质支架策略相比，RNA支架通过编程调节多个蛋白质化学计量和数量以及蛋白质的相对距离和空间取向提供了扩展的灵活性。对于合成生物学，RNA支架提供了一个新的平台，可用于增加连续代谢途径的产量。
RNA scaffolds are synthetic noncoding RNA molecules with engineered 3D folding harnessed to spatially organize proteins in vivo. Here we provide a protocol to design, express and characterize RNA scaffolds and their cognate proteins within 1 month. The RNA scaffold designs described here are based on either monomeric or multimeric units harboring RNA aptamers as protein docking sites. The scaffolds and proteins are cloned into inducible plasmids and expressed to form functional assemblies. RNA scaffolds find applications in many fields in which in vivo organization of biomolecules is of interest. RNA scaffolds provide extended flexibility compared with DNA or protein scaffolding strategies through programmed modulation of multiple protein stoichiometry and numbers, as well as the proteins' relative distances and spatial orientations. For synthetic biology, RNA scaffolds provide a new platform that can be used to increase yields of sequential metabolic pathways.