基于DNA敏感的Ter（III）探针的蛋白质乙酰化相关酶的连续活性检测的时间分辨发光生物传感器。,Analytical Chemistry

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基于DNA敏感的Ter（III）探针的蛋白质乙酰化相关酶的连续活性检测的时间分辨发光生物传感器。
Analytical Chemistry ( IF 6.7 ) Pub Date : 2015-09-01 00:00:00 , DOI: 10.1021/acs.analchem.5b01338
Yitao Han ₁ , Hao Li ₁ , Yufang Hu ₁ , Pei Li ₁ , Huixia Wang ₁ , Zhou Nie ₁ , Shouzhuo Yao ₁

Affiliation

组蛋白的蛋白质乙酰化是表观遗传基因调控中必不可少的翻译后修饰（PTM）机制，其状态由组蛋白乙酰转移酶（HATs）和组蛋白脱乙酰基酶（HDACs）可逆地控制。在这里，我们已经开发了一种灵敏的，无标记的时间分辨发光（TRL）生物传感器，用于分别基于乙酰化介导的肽/ DNA相互作用和Tb ³⁺ / DNA发光探针分别连续检测HAT和HDAC的酶活性。使用富含鸟嘌呤（G）的DNA敏感的Tb ³⁺发光作为输出信号，聚阳离子底物肽以高亲和力与DNA相互作用，随后取代Tb ³⁺，消除发光信号。HAT催化的乙酰化作用显着降低了肽的正电荷，并减少了肽/ DNA的相互作用，从而通过恢复DNA敏化的Tb ³⁺发光产生了检测信号。使用该TRL传感器，可以在0.2到100 nM的宽线性范围内和0.05 nM的低检测限范围内灵敏地检测到HAT（p300）。所提出的传感器还被用来连续地实时监测HAT的活动。此外，TRL生物传感器已成功应用于评估两种特异性抑制剂（漆树酸和C464）和令人满意的Z抑制HAT的能力获得了高于0.73的'因子。此外，该传感器对于以0.5到500 nM的线性范围和0.5 nM的检出限连续监测HDAC（Sirt1）催化的脱乙酰作用是可行的。拟议中的传感器是一种方便，灵敏且可混合阅读的测定方法，为蛋白质乙酰化靶向表观遗传学研究和药物发现提供了一个有希望的平台。

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Time-Resolved Luminescence Biosensor for Continuous Activity Detection of Protein Acetylation-Related Enzymes Based on DNA-Sensitized Terbium(III) Probes

Protein acetylation of histone is an essential post-translational modification (PTM) mechanism in epigenetic gene regulation, and its status is reversibly controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Herein, we have developed a sensitive and label-free time-resolved luminescence (TRL) biosensor for continuous detection of enzymatic activity of HATs and HDACs, respectively, based on acetylation-mediated peptide/DNA interaction and Tb³⁺/DNA luminescent probes. Using guanine (G)-rich DNA-sensitized Tb³⁺ luminescence as the output signal, the polycationic substrate peptides interact with DNA with high affinity and subsequently replace Tb³⁺, eliminating the luminescent signal. HAT-catalyzed acetylation remarkably reduces the positive charge of the peptides and diminishes the peptide/DNA interaction, resulting in the signal on detection via recovery of DNA-sensitized Tb³⁺ luminescence. With this TRL sensor, HAT (p300) can be sensitively detected with a wide linear range from 0.2 to 100 nM and a low detection limit of 0.05 nM. The proposed sensor was further used to continuously monitor the HAT activity in real time. Additionally, the TRL biosensor was successfully applied to evaluating HAT inhibition by two specific inhibitors, anacardic acid and C464, and satisfactory Z′-factors above 0.73 were obtained. Moreover, this sensor is feasible to continuously monitor the HDAC (Sirt1)-catalyzed deacetylation with a linear range from 0.5 to 500 nM and a detection limit of 0.5 nM. The proposed sensor is a convenient, sensitive, and mix-and-read assay, presenting a promising platform for protein acetylation-targeted epigenetic research and drug discovery.

更新日期：2015-09-01

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