Detection of single nucleotide polymorphisms based on triple-helix molecular switch combined with invader assay

Single-nucleotide polymorphisms (SNPs) can be relevant to disease susceptibility, disease pathogenesis, and efficacy of specific drugs. Monitoring SNPs plays a significant role in disease susceptibility and individual response to treatment, wherein optimal care can be provided to individuals based on their unique genetic and disease profiles. In this work, we have formulated a rapid and sensitive fluorescent biosensing platform based on a target-activated triple-helix molecular switch (THMS)-conformation-change-induced invader assay strategy for the detection of single nucleotide polymorphisms in the apolipoprotein E (APOE). The mutant DNA (MtDNA) of APOE gene hybridized with the THMS to form the three-dimensional invader structure over the SNP site. Subsequently, the specific structure would be recognized and cleaved by FEN1, leading to the recovery of FAM quenched by BHQ 1. Based on the high binding affinity of THMS to the target, our strategy could detect the MtDNA of AOPE gene without amplification at 0.27 nM. Furthermore, we achieved genotyping based on the difference in fluorescence signals and successfully applied this strategy to detect MtDNA of APOE gene in clinical samples, showing the great potential for monitoring various SNPs for biological research and disease diagnosis.