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基于 DNA 偶联金纳米粒子的比色法测定解旋酶活性:一种筛选潜在解旋酶抑制剂的新方法。

DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors.

机构信息

Nano Innovation Laboratory, Elettra-Sincrotrone Trieste S.C.p.A., Trieste, Italy.

Structural Biology Laboratory, Elettra-Sincrotrone Trieste S.C.p.A., Trieste, Italy.

出版信息

Sci Rep. 2017 Mar 13;7:44358. doi: 10.1038/srep44358.

Abstract

Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

摘要

解旋酶是普遍存在于所有生命形式中的必需酶。由于它们在核酸代谢中的核心作用,它们正成为抗病毒、抗菌和抗癌药物的重要靶点。开发简单、廉价、快速和强大的生化测定法来测量解旋酶活性,克服当前方法的局限性,是通过高通量筛选发现解旋酶抑制剂的前提。我们开发了一种利用与金纳米粒子(AuNP)结合的 DNA 的光学特性的方法,并满足了所需的标准。该方法用人源 RecQ4 解旋酶的催化结构域进行了测试,并与传统的 FRET 测定法进行了比较。基于 AuNP 的测定法产生了相似的结果,但比 FRET 更简单、更稳健且更便宜。因此,我们的基于纳米技术的平台显示出有潜力为现有的传统方法提供有用的替代方法,用于跟踪解旋酶活性,并筛选小分子文库作为潜在的解旋酶抑制剂。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fe17/5347027/008070645960/srep44358-f1.jpg

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