iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications.
iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications.
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DOI：
10.1093/nar/gkw083
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发表时间：
2016-04-07
影响因子：
14.9
通讯作者：
Ryckelynck M
中科院分区：
文献类型：
--
作者：
Autour A;Westhof E;Ryckelynck M
使用随机诱变和高通量筛选的微流体辅助体外区室化，我们报告了一个数量级倍更亮的突变体的照明RNA适体菠菜的分离，远低于盐敏感性和具有比母体分子高得多的热稳定性。进一步工程化得到了iSpinach，其折叠和荧光性质超过了所有目前已知的基于荧光辅因子3，5-二氟-4-羟基亚苄基咪唑啉酮（DFHBI）的适体。我们说明了潜在的iSpinach在一个新的敏感和高通量兼容的荧光检测，测量共转录的催化常数（KCAT）的模型核酶。
Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme.