Nanopore-Based Strategy for Single-Nucleotide Mutation Detection in Sickle Cell Disease Using Anthracycline Probes

Sickle cell disease (SCD) is an autosomal recessive genetic disease that is caused by a single nucleotide mutation from adenine to thymine in the β-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 α-hemolysin (α-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.