RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation.
RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation.
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通过双分子荧光互补检测活细胞中按确定序列形成的 RNA G 四链体
DOI：
10.1039/c5sc03946k
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发表时间：
2016-07-01
期刊：
影响因子：
8.4
通讯作者：
Tan Z
中科院分区：
文献类型：
--
作者：
Liu HH;Zheng KW;He YD;Chen Q;Hao YH;Tan Z
RNA G-四链体的形成在活的细菌细胞中的一个确定的序列中被检测的双分子荧光互补与序列和结构特异性。G-四链体参与许多重要的细胞过程，并且具有形成G-四链体的潜力的序列广泛存在于DNA和RNA中。然而，很难知道感兴趣的序列是否在活细胞中天然形成G-四链体。在这里，我们报告的G-四链体在定义的RNA序列在活细胞中的自然细胞内环境中的检测。RNA转录物中的G-四链体形成序列在适体附近被标记。这两种结构分别被两种探针蛋白识别，每种探针蛋白与增强型绿色荧光蛋白（eGFP）的分裂的一半融合。这两种蛋白质与RNA的同时结合使eGFP的两半接近，允许它们重建成功能性eGFP，其发射荧光以发出RNA中G-四链体形成的信号。我们表明，一个G-四链体可以在RNA中形成，并可以在体外和体内条件下检测序列和结构特异性。因此，这些结果为活细胞中RNA G-四链体的形成提供了直接证据，并且该方法提供了一种有用的工具来验证在天然细胞条件下特定序列中的G-四链体形成。
RNA G-quadruplex formation in a defined sequence in living bacterial cells is detected by bimolecular fluorescence complementation with sequence and structure specificity. G-quadruplexes are implicated in many essential cellular processes and sequences with potential to form a G-quadruplex are widely present in DNA and RNA. However, it is difficult to know whether a sequence of interest naturally forms a G-quadruplex in living cells. Here we report the detection of a G-quadruplex in defined RNA sequences in living cells in a natural intracellular environment. A G-quadruplex forming sequence in a RNA transcript is tagged at proximity with an aptamer. The two structures are recognized respectively by two probe proteins each of which is fused with a split half of enhanced green fluorescent protein (eGFP). Simultaneous binding of the two proteins to RNA brings the two halves of eGFP into proximity, permitting them to reconstitute into a functional eGFP that emits fluorescence to signal the formation of a G-quadruplex in RNA. We show that a G-quadruplex can form in RNA and can be detected with sequence and structure specificity under both in vitro and in vivo conditions. The results, therefore, provide direct evidence for the formation of RNA G-quadruplexes in live cells and the method provides a useful tool to validate G-quadruplex formation in a specific sequence under a natural cellular condition.
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影响因子：
46.9
作者：
Chambers, Vicki S.;Marsico, Giovanni;Balasubramanian, Shankar
通讯作者：
Balasubramanian, Shankar
影响因子：
14.9
作者：
Bugaut A;Balasubramanian S
通讯作者：
Balasubramanian S
影响因子：
14.9
作者：
Lattmann S;Giri B;Vaughn JP;Akman SA;Nagamine Y
通讯作者：
Nagamine Y
DOI：
10.1073/pnas.1422605112
发表时间：
2015-08-04
影响因子：
11.1
作者：
Heddi, Brahim;Cheong, Vee Vee;Anh Tuan Phan
通讯作者：
Anh Tuan Phan
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III