Systematic reconstruction of binding and stability landscapes of the fluorogenic aptamer spinach.
Systematic reconstruction of binding and stability landscapes of the fluorogenic aptamer spinach.
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DOI：
10.1093/nar/gkv944
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发表时间：
2015-10-30
影响因子：
14.9
通讯作者：
Meier M
中科院分区：
文献类型：
--
作者：
Ketterer S;Fuchs D;Weber W;Meier M
基于由3，5-二氟-4-羟基苯亚甲基咪唑啉酮（DFHBI）衍生物和名为Spinach的RNA适体形成的复合物的荧光RNA被用于工程化新一代用于生物分析的体外和体内传感器。随着RNA/小分子复合物的解析晶体结构，工程图变得可用，但缺少关于分子热力学特征的综合信息。在这里，我们重建了DFHBI和第一代菠菜的截短序列之间的完整热力学结合和稳定性景观。为此，我们建立了一个系统的筛选程序，单点和双点突变的微流控大规模集成芯片平台上的87-nt长的RNA。具有单碱基分辨率的热力学曲线用于通过定向而非进化方法来设计改进的荧光菠菜代。
Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new generation of in vitro and in vivo sensors for bioanalytics. With the resolved crystal structure of the RNA/small molecule complex, the engineering map becomes available, but comprehensive information regarding the thermodynamic profile of the molecule is missing. Here, we reconstructed the full thermodynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a systematic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermodynamic profile with single base resolution was used to engineer an improved fluorogenic spinach generation via a directed rather than evolutional approach.