Li Zhen, Liu Xingtong, Li Linna, Hui Yandi, Liu Lili, Yao Fujun, Kang Xiaofeng, Guo Yanli
Key Laboratory of Synthetic and Natural Functional Molecular Chemistry, College of Chemistry & Materials Science, Northwest University, Xi'an 710127, P R China.
Anal Chem. 2025 Aug 5;97(30):16432-16438. doi: 10.1021/acs.analchem.5c02351. Epub 2025 Jul 22.
Sickle cell disease (SCD) is an autosomal recessive genetic disease that is caused by a single nucleotide mutation from adenine to thymine in the -bead protein gene (HBB gene). Currently, there is no completely effective cure for SCD, and management is primarily to prevent. Therefore, timely detection of the genotype is crucial for subsequent diagnosis. In this study, we developed a rapid and straightforward method utilizing the high spatiotemporal resolution of the -hemolysin (-HL) nanopore to detect wild-type (HBBW) and mutant (HBBM) HBB gene. This approach uses the anthracycline drug doxorubicin (DOX) as a probe and leverages the distinct characteristic signals generated from its interactions with HBBW and HBBM. When DOX interacts with HBBW, it forms a double-stranded structure of the DNA-DOX complexes, whereas it induces the formation of hairpin structures when interacting with HBBM. These two structures exhibit significant differences in dwell time and amplitude, enabling clear and efficient identification. By mixing different ratios of HBBW and HBBM, we were able to accurately identify genotypes based on the peak area occupied by the characteristic HBBM signals. This method has been successfully applied for detection of HBBM in simulated artificial amniotic fluid (AAF), demonstrating its practicality and reliability. Our approach enables early diagnosis of SCD at the single-molecule level, providing a new avenue for rapid detection of single-nucleotide mutations in gene sequences using DNA-drug interactions.
镰状细胞病（SCD）是一种常染色体隐性遗传病，由β珠蛋白基因（HBB基因）中的单个核苷酸从腺嘌呤突变为胸腺嘧啶引起。目前，SCD尚无完全有效的治愈方法，治疗主要以预防为主。因此，及时检测基因型对后续诊断至关重要。在本研究中，我们开发了一种快速且直接的方法，利用α-溶血素（α-HL）纳米孔的高时空分辨率来检测野生型（HBBW）和突变型（HBBM）HBB基因。该方法使用蒽环类药物阿霉素（DOX）作为探针，并利用其与HBBW和HBBM相互作用产生的独特特征信号。当DOX与HBBW相互作用时，会形成DNA-DOX复合物的双链结构，而与HBBM相互作用时会诱导发夹结构的形成。这两种结构在驻留时间和幅度上表现出显著差异，从而能够清晰有效地进行识别。通过混合不同比例的HBBW和HBBM，我们能够根据特征性HBBM信号所占的峰面积准确识别基因型。该方法已成功应用于模拟人工羊水（AAF）中HBBM的检测，证明了其实用性和可靠性。我们的方法能够在单分子水平上对SCD进行早期诊断，为利用DNA-药物相互作用快速检测基因序列中的单核苷酸突变提供了一条新途径。
