摘要
为了在活细胞RNA成像中提供更加精准的工具,本文旨在基于broccoli的RNA荧光标记创建荧光强度高、稳定性好的绿色荧光适体.采用理性设计对broccoli进行优化,并通过测定光谱特征和理化性质对broccoli变体进行多轮筛选.结果发现基于对RNA荧光适配体broccoli的三轮突变筛选,获得了荧光强度提升3.3倍的broccoli四位点突变体(命名为Bro);再利用设计的3WJ支架,组建了3WJ-n×Bro RNA荧光适配体家族;采用嵌入串联重复Bro的方式可以显著提升适体的荧光强度,其中3WJ-4×Bro的荧光强度超过3WJ-Bro的2.7倍,且表现出更低的盐离子依赖性和更高的热稳定性、光稳定性和监测灵敏度.结果表明本文设计出的3WJ-n×Bro家族并从中筛选出优异的核酸适配体3WJ-4×Bro,可为在RNA水平上实现活细胞RNA成像提供可视化工具.
In order to provide more precise tools for RNA imaging in living cells,this paper aims to create green fluorescent aptamers with high fluorescence intensity and good stability based on broccoli's RNA fluorescent labeling.The broccoli was optimized by rational design,and its variants were screened for multiple rounds by measuring spectral characteristics and physicochemical properties.The results showed that based on three rounds of mutation screening of the RNA fluorescent aptamer broccoli,a broccoli four-site mutant(named Bro)with a fluorescence intensity increase of 3.3 times was obtained;The 3WJ-n×Bro RNA fluorescent aptamer family was established by using the designed 3WJ scaffold.The fluorescence intensity on the aptamer can be significantly improved by embedding tandem repeat Bro,with the fluorescence intensity of 3WJ-4×Bro is 2.7 times higher than that of 3WJ-Bro;It also shows lower salt ion dependence and higher thermal stability,light stability and monitoring sensitivity.In this paper,we designed the 3WJ-n×Bro family and screened out the excellent aptamer 3WJ-4×Bro,which provided a visualization tool for live cell RNA imaging at the RNA level.
作者
何祖洋
白九元
赵云
HE Zu-Yang;BAI Jiu-Yuan;ZHAO Yun(Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,College of Life Sciences,Sichuan University,Chengdu 610065,China)
出处
《四川大学学报（自然科学版）》
CAS
CSCD
北大核心
2024年第4期310-319,共10页
Journal of Sichuan University(Natural Science Edition)
基金
高校基本科研业务费-专职博士后研发基金(2022SCU12100)。
