Peptide-mediated energy transfer between an anionic water-soluble conjugated polymer and Texas red labeled DNA for protease and nuclease activity study.
Peptide-mediated energy transfer between an anionic water-soluble conjugated polymer and Texas red labeled DNA for protease and nuclease activity study.
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DOI：
10.1021/ac802488m
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发表时间：
2009-04
影响因子：
7.4
通讯作者：
Yong Zhang;Yanyan Wang;B. Liu
中科院分区：
文献类型：
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作者：
Yong Zhang;Yanyan Wang;B. Liu
我们首次报告肽可以作为一种介质，使阴离子缀合聚合物和染料标记的DNA紧密接触以进行能量转移。利用荧光素（FI）标记的肽介导的聚（9，9-双（4 '-磺酸丁基）芴-alt-1，4-亚苯基）钠盐（PFP-SO（3）Na）与德克萨斯红（TR）标记的单链DNA（ssDNA）之间的能量转移，我们建立了一种以肽/DNA复合物为底物的蛋白酶和核酸酶活性的均相检测方法。作为概念验证的酶分别是胰蛋白酶（蛋白酶）和S1（核酸酶）。在无酶存在下，从PFP-SO（3）Na到Fl和TR以及从Fl到TR发生了多步荧光能量转移，TR发射在溶液荧光中占主导地位。在胰蛋白酶的存在下，肽被切割成片段;小肽片段和聚合物之间相对弱的静电相互作用不能使TR-ssDNA和聚合物紧密靠近以进行能量转移。溶液的TR发射明显减弱，PFP-SO（3）Na发射增强，溶液荧光呈现蓝色。当用S1核酸酶切割TR-ssDNA时，TR发射明显降低，F1发射明显增强，溶液荧光呈绿色。开发的检测方法是检测具有DNA或蛋白质切割活性的化学和生物分子的理想方法。
We report for the first time that peptide could serve as a medium to bring an anionic conjugated polymer and a dye-labeled DNA into close proximity for energy transfer. By taking advantage of the fluorescein (Fl)-labeled peptide-mediated energy transfer between poly(9,9-bis(4'-sulfonatobutyl)fluorene-alt-1,4-phenylene) sodium salt (PFP-SO(3)Na) and Texas red (TR)-labeled single-stranded DNA (ssDNA), we develop a homogeneous assay for detection and monitoring of protease and nuclease activity in one solution using peptide/DNA complexes as the substrate. The enzymes as a proof of concept are trypsin (protease) and S1 (nuclease), respectively. In the absence of enzyme, multistep fluorescence energy transfer occurs from PFP-SO(3)Na to Fl and TR and from Fl to TR, and the TR emission dominates the solution fluorescence. In the presence of trypsin, the peptide is cleaved into fragments; the relatively weak electrostatic interaction between the small peptide fragments and the polymer fails to bring the TR-ssDNA and the polymer into close proximity for energy transfer. There is a significant decrease in TR emission and an increase in PFP-SO(3)Na emission, and the solution fluorescence appears blue. When S1 nuclease is used to cleave TR-ssDNA, a significant decrease in TR emission and an obvious increase in Fl emission are found, and the solution fluorescence appears green. The developed assay is ideal for the detection of chemical and biological molecules with DNA or protein cleaving activities.