A gold-nanoparticle-based real-time colorimetric screening method for endonuclease activity and inhibition
A gold-nanoparticle-based real-time colorimetric screening method for endonuclease activity and inhibition
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DOI：
10.1002/anie.200605249
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发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Mirkin, Chad A.
中科院分区：
文献类型：
--
作者：
Xu, Xiaoyang;Han, Min Su;Mirkin, Chad A.
核酸作为遗传信息的载体，大多数生物体含有一种称为核酸内切酶的酶，[1]它可以水解核酸骨架中的磷酸二酯键。这些核酸酶对于涉及核酸的复制、修复和重组的许多过程是重要的。核酸内切酶如DNA旋转酶和病毒整合酶在DNA的拓扑结构改变和前病毒DNA插入宿主染色体DNA等生物过程中起着关键作用。[2]因此，抑制核酸内切酶的分子被认为是各种抗微生物和抗病毒药物的候选物。因此，用于高通量筛选不同核酸酶抑制剂的方法已成为药物开发过程的核心部分。[3]最广泛使用的测定包括基于使用显色或荧光底物产生分光光度信号的测定。[4]然而，在许多情况下，期望测量酶与明确定义的感兴趣的核酸底物之间的反应，而不是该底物的荧光或显色衍生物。历史上，除了最近的基于荧光的方法之外，已经通过粘度法、放射性标记和凝胶电泳来筛选核酸内切酶活性。[5]这些方案中的大多数是耗时的，并且不提供真实的时间内的核酸内切酶活性的量度。在这些方法中，只有荧光被明显地用于高通量筛选，并且基于荧光的方法最近刚刚被实施。[6]在此，我们报告了一种操作简单的比色内切酶抑制试验，它能够实时监测内切酶活性和同时测定内切酶抑制剂（例如，DNA结合分子）的效率。[7]该新方法依赖于DNA功能化的金纳米粒子（DNA-AuNPs）与DNA双链体互连的聚合物聚集体。[8]DNA-AuNP以前已用于检测DNA、蛋白质、金属离子和DNA结合分子。[9]别人
Nucleic acids act as the carriers of genetic information, and most organisms contain a type of enzymes called endonucleases,[1] which can hydrolyze the phosphodiester linkages in the nucleic acid backbone. These nucleases are important for many processes involving the replication, repair, and recombination of nucleic acids. Endonucleases such as DNA gyrase and virus integrase play key roles in biological process such as the topological altering of DNA and the insertion of proviral DNA into host chromosomal DNA.[2] Molecules that inhibit endonucleases are therefore considered candidates for a variety of antimicrobial and antiviral drugs. As such, methods for the high-throughput screening of different nuclease inhibitors have become a central part of the drug-development process.[3]The most widely used assays include those based on the use of chromogenic or fluorogenic substrates to produce a spectrophotometric signal.[4] In many cases, however, it is desirable to measure the reaction between an enzyme and a well-defined nucleic acid substrate of interest, as opposed to a fluorogenic or chromogenic derivative of that substrate. Historically, endonuclease activity has been screened by viscometry, radioactive labeling, and gel electrophoresis, in addition to the more recent fluorescence-based approaches.[5] Most of these protocols are time consuming and do not provide a measure of endonuclease activity in real time. Of these methods, only fluorescence is appreciably used for highthroughput screening, and the fluorescence-based approach has just recently been implemented.[6] Herein, we report an operationally simple colorimetric endonuclease-inhibition assay, which enables the real-time monitoring of endonuclease activity and the simultaneous determination of the efficiencies of endonuclease inhibitors (eg, DNA-binding molecules).[7] The new method relies on polymeric aggregates of DNA-functionalized gold nanoparticles (DNA-AuNPs) with DNA-duplex interconnects.[8] DNA-AuNPs have been used previously to detect DNA, proteins, metal ions, and DNA-binding molecules.[9] Others