Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), 2 Fusionopolis Way, Innovis #08-03, 138634, Singapore; Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore, 117574, Singapore.
p53 Laboratory (A*STAR), 8A Biomedical Grove Immunos, 138648, Singapore.
Colloids Surf B Biointerfaces. 2018 Sep 1;169:214-221. doi: 10.1016/j.colsurfb.2018.05.007. Epub 2018 May 5.
The tumor suppressor protein p53 plays a central role in preventing cancer through interaction with DNA response elements (REs) to regulate target gene expression in cells. Due to its significance in cancer biology, relentless efforts have been directed toward understanding p53-DNA interactions for the development of cancer therapeutics and diagnostics. In this paper, we report a rapid, label-free and versatile colorimetric assay to detect wildtype p53 DNA-binding function in complex solutions. The assay design is based on a concept that alters interparticle-distances between RE-AuNPs from a crosslinking effect induced through tetramerization of wildtype p53 protein (p53-WT) upon binding to canonical DNA motifs modified on gold nanoparticles (RE-AuNPs). This leads to a visible solution color change from red to blue, which is quantifiable by the UV- visible absorption spectra with a detection limit of 5 nM. Contrastingly, no color change was observed for the binding-deficient p53 mutants and non-specific proteins due to their inability to crosslink RE-AuNPs. Based on this sensing principle, we further demonstrate its utility for fast detection of drug-induced DNA binding function to cancer-associated Y220C mutant p53 protein using well-established reactivating compounds. By exploiting the dominant-negative property of mutant p53 over p53-WT and interactions with RE-AuNPs, this assay is configurable to detect low numbers of mutant p53 expressing cells in miniscule sample fractions obtained from typical core needle biopsy-sized tissues without signal attrition, alluding to the potential for biopsy sampling in cancer diagnostics or for defining cancer margins. This nanogold enabled colorimetric assay provides a facile yet robust method for studying important parameters influencing p53-DNA interactions with great promises for clinically pertinent applications.
抑癌蛋白 p53 通过与 DNA 反应元件 (RE) 相互作用，在细胞中调节靶基因表达，从而在预防癌症中发挥核心作用。由于其在癌症生物学中的重要性，人们一直在努力理解 p53-DNA 相互作用，以期开发癌症治疗和诊断方法。在本文中，我们报告了一种快速、无标记且多功能的比色测定法，用于检测复杂溶液中野生型 p53 DNA 结合功能。该测定法的设计基于这样一种概念，即通过野生型 p53 蛋白（p53-WT）四聚化诱导的交联作用改变 RE-AuNPs 之间的粒子间距离，从而改变结合到金纳米粒子 (RE-AuNPs) 上的经典 DNA 基序修饰的 RE-AuNPs 之间的粒子间距离。这导致溶液颜色从红色变为蓝色，可通过紫外-可见吸收光谱进行定量，检测限为 5 nM。相比之下，由于结合缺陷的 p53 突变体和非特异性蛋白无法交联 RE-AuNPs，因此它们的结合没有观察到颜色变化。基于这一传感原理，我们进一步证明了它在使用已建立的再激活化合物快速检测与癌症相关的 Y220C 突变 p53 蛋白诱导的 DNA 结合功能方面的效用。利用突变 p53 对 p53-WT 的显性负性和与 RE-AuNPs 的相互作用，该测定法可配置用于检测从小体积组织芯活检大小的组织中获得的微小样本分数中低数量表达突变 p53 的细胞，而不会导致信号衰减，这暗示了在癌症诊断中进行活检采样的潜力，或用于定义癌症边界。这种基于纳米金的比色测定法为研究影响 p53-DNA 相互作用的重要参数提供了一种简便而强大的方法，为临床相关应用带来了巨大的希望。
