Reassembly of a bioluminescent protein Renilla luciferase directed through DNA hybridization.
Reassembly of a bioluminescent protein Renilla luciferase directed through DNA hybridization.
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通过 DNA 杂交引导生物发光蛋白海肾荧光素酶的重新组装。
DOI：
10.1021/bc8003099
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发表时间：
2009
影响因子：
4.7
通讯作者：
Deo,SapnaK
中科院分区：
文献类型：
--
作者：
Cissell,KyleA;Rahimi,Yasmeen;Shrestha,Suresh;Deo,SapnaK
分离报告蛋白的重组，也被称为蛋白质互补，用于检测蛋白质−蛋白质或蛋白质−核酸的相互作用。在这个策略中，一个报告蛋白被分成两个不活跃的多肽，相互作用/结合的伙伴被融合到一起。融合的伙伴之间的相互作用导致了重新组装的、活跃的记者的形成。在这篇通讯中，我们提出了一种基于生物发光报告基因Renillaluciferase(Rluc)的重组的概念验证，该重组是由DNA杂交驱动的。虽然，通过蛋白质相互作用重组Rluc已经被证明，但通过DNA杂交来重组Rluc还没有被证明，这是本工作的新奇之处。众所周知，由于没有任何背景信号，生物发光检测具有显著的优势。在我们的研究中，两个设计合理的Rluc片段被连接到互补的寡核苷酸探针上。两个探针与融合的Rluc片段杂交导致片段的重新组装，产生活性Rluc，可通过加入coelenterazine时发出的光强度来测量。我们的研究还表明，Rluc的重组可以被竞争与杂交探针−Rluc片段复合体结合的寡核苷酸探针所抑制，这表明了一种潜在的定量检测靶核酸的策略。我们能够利用毫微摩尔量的探针−片段蛋白结合物实现Rluc与寡核苷酸探针的重组。这一浓度比以绿色荧光蛋白(GFP)为报道的浓度低约4个数量级。在细胞基质中进行的DNA驱动的Rluc重组研究没有显示任何来自基质的干扰。
Reassembly of split reporter proteins, also referred to as protein complementation, is utilized in the detection of protein−protein or protein−nucleic acid interactions. In this strategy, a reporter protein is fragmented into two inactive polypeptides to which interacting/binding partners are fused. The interaction between fused partners leads to the formation of a reassembled, active reporter. In this Communication, we have presented a proof-of-concept for the detection of a target nucleic acid sequence based on the reassembly of the bioluminescent reporterRenillaluciferase (Rluc), which is driven by DNA hybridization. Although, reassembly of Rluc though protein interactions has been demonstrated by others, the Rluc reassembly through DNA hybridization has not been shown yet, which is the novelty of this work. It is well established that bioluminescence detection offers significant advantages due to the absence of any background signal. In our study, two rationally designed fragments of Rluc were conjugated to complementary oligonucleotide probes. Hybridization of the two probes with fused Rluc fragments resulted in the reassembly of the fragments, generating active Rluc, measurable by the intensity of light given off upon addition of coelenterazine. Our study also shows that the reassembly of Rluc can be inhibited by an oligonucleotide probe that competes to bind to the hybridized probe−Rluc fragment complex, indicating a potential strategy for the quantitative detection of target nucleic acid. We were able to achieve the reassembly of Rluc fused to oligonucleotide probes using femtomole amounts of the probe−fragment protein conjugate. This concentration is approximately 4 orders of magnitude less than that reported using green fluorescent protein (GFP) as the reporter. A DNA-driven Rluc reassembly study performed in a cellular matrix did not show any interference from the matrix.