RCA-Supported Multipedal DNA Walker Integrated with TSDR for Simultaneous Detection of Single Nucleotide Polymorphisms in Circulating Tumor DNA

Peripheral blood circulating tumor DNA (ctDNA) is a crucial liquid biopsy biomarker that correlates overall systemic tumor burden with malignant progression. However, identifying multiple single nucleotide polymorphisms (SNPs) in ctDNA presents significant challenges. In this study, we developed a rolling circle amplification (RCA)-supported multipedal DNA walker integrated with toehold-mediated strand displacement (TSDR) to facilitate the detection of ctDNA SNPs. Initially, separate identification of ctDNAs is performed using TSDR procedures, along with the release of the triggers for RCA. Once the RCA processes are initiated, long single-stranded oligonucleotide products containing repeated DNAzyme sequences are generated. Finally, these RCA products function as multipedal legs that traverse a track in a quasi-mechanical manner, producing detectable fluorescence signals. The multipedal DNA walking mechanism exhibits a high walking efficiency and signal amplification capability, as the multipedal legs can interact with multiple signal probes simultaneously. Concurrent detection of the wild type (WT) and mutation type (MT) of multiple ctDNAs is achieved, and the detection limits are at the attomolar (aM) level, with a linear range of 10 aM to 5.0 μM. Furthermore, the proposed method demonstrates remarkable specificity for mismatched sequences and single-base mutant genes, due to the TSDR-assisted detection process. Additionally, the analysis of ctDNA SNPs in clinical serum samples reveals significant differences in ctDNA expression between the blood of cancer patients and that of healthy individuals. Therefore, the proposed method can be employed for the detection of clinical ctDNA SNPs and demonstrates significant potential for early diagnosis, treatment monitoring, and prognosis.