化学
DNA
放大器
克拉斯
计算生物学
分子生物学
突变
生物化学
基因
聚合酶链反应
生物
作者
Chan Li,Hao Jia,Xiaoling Wei,Guohui Xue,Jianguo Xu,Ruize Cheng,Yinghao Cheng,Qiufeng Song,Zhifa Shen,Chang Xue
标识
DOI:10.1021/acs.analchem.3c05511
摘要
Accurate identification of single-nucleotide mutations in circulating tumor DNA (ctDNA) is critical for cancer surveillance and cell biology research. However, achieving precise and sensitive detection of ctDNAs in complex physiological environments remains challenging due to their low expression and interference from numerous homologous species. This study introduces single-nucleotide-specific lipidic nanoflares designed for the precise and visible detection of ctDNA via toehold-initiated self-priming DNA polymerization (TPP). This system can be assembled from only a single cholesterol-conjugated multifunctional molecular beacon (MMB) via hydrophobicity-mediated aggregation. This results in a compact, high-density, and nick-hidden arrangement of MMBs on the surface of lipidic micelles, thereby enhancing their biostability and localized concentrations. The assay commences with the binding of frequently mutated regions of ctDNA to the MMB toehold domain. This domain is the proximal holding point for initiating the TPP-based strand-displacement reaction, which is the key step in enabling the discrimination of single-base mutations. We successfully detected a single-base mutation in ctDNA (KRAS G12D) in its wild-type gene (KRAS WT), which is one of the most frequently mutated ctDNAs. Notably, coexisting homologous species did not interfere with signal transduction, and small differences in these variations can be visualized by fluorescence imaging. The limit of detection was as low as 10 amol, with the system functioning well in physiological media. In particular, this system allowed us to resolve genetic mutations in the KRAS gene in colorectal cancer, suggesting its high potential in clinical diagnosis and personalized medicine.
科研通智能强力驱动
Strongly Powered by AbleSci AI