Fast complementation of split fluorescent protein triggered by DNA hybridization
Fast complementation of split fluorescent protein triggered by DNA hybridization
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DOI：
10.1073/pnas.0511078103
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发表时间：
2006-02-14
影响因子：
11.1
通讯作者：
Broude, NE
中科院分区：
文献类型：
--
作者：
Demidov, VV;Dokholyan, NV;Broude, NE
荧光蛋白已被证明是优秀的报告分子和生化传感器，具有广泛的应用。在分裂形式下，它们不发荧光，但可以通过重新组装分裂多肽的补充蛋白质-蛋白质或蛋白质-核酸相互作用来恢复它们的荧光。然而，在之前的研究中，需要数小时才能恢复分裂荧光蛋白的荧光，因为蛋白质发色团的形成与重新组装同时缓慢地发生。在这里，我们提供的证据表明，荧光发色团可以在增强型绿色荧光蛋白（EGFP）的分离的 N 末端片段内自催化形成。我们表明，核酸互补相互作用可以驱动分裂蛋白荧光的恢复。在我们的测定中，当拆分 EGFP 的互补寡核苷酸连接片段组合时，荧光产生速度很快（几分钟内）。我们的 EGFP 系统对 DNA 杂交快速响应的能力应该有助于检测体外和活细胞中许多其他类型的成对相互作用的动力学。
Fluorescent proteins have proven to be excellent reporters and biochemical sensors with a wide range of applications. In a split form, they are not fluorescent, but their fluorescence can be restored by supplementary protein-protein or protein-nucleic acid interactions that reassemble the split polypeptides. However, in prior studies, it took hours to restore the fluorescence of a split fluorescent protein because the formation of the protein chromophore slowly occurred de novo concurrently with reassembly. Here we provide evidence that a fluorogenic chromophore can self-catalytically form within an isolated N-terminal fragment of the enhanced green fluorescent protein (EGFP). We show that restoration of the split protein fluorescence can be driven by nucleic acid complementary interactions. In our assay, fluorescence development is fast (within a few minutes) when complementary oligonucleotide-linked fragments of the split EGFP are combined. The ability of our EGFP system to respond quickly to DNA hybridization should be useful for detecting the kinetics of many other types of pairwise interactions both in vitro and in living cells.