Target-mediated assembly formation of multi-arm DNAzyme nanostructures for sensitive and accurate discrimination of single-nucleotide polymorphism in K-ras gene

Single-nucleotide polymorphism (SNP) is a typical way of genetic variation and commonly used as genetic marker to indicate various types of human diseases. We herein report a non-enzymatic and label-free sensing method for accurate and ultrasensitive differentiation of SNP in K-ras gene. The presence of target mutant K-ras gene (MtDNA) leads to the formation of “three-way junctions” (3WJ) structure with an exposure toehold site, which initiates the toehold strand displacement reaction (TSDR) to form multi-arm DNAzyme nanostructures. These DNAzymes further cyclically cleave the hairpin DNA substrates and release many G-rich fragments to interact with organic dye thioflavin T (ThT) and yield significantly enhanced fluorescence for sensitive discrimination of MtDNA. However, the presence of wild K-ras gene (WtDNA) trigger a base pairing frame shift on the 3WJ structure and result in the closure of the toehold site, thus neither TSDR nor DNAzyme-assistant cleavage reaction is initiated, and no obvious fluorescent signal is observed. Importantly, this strategy shows high sensitivity with a detection limit of 3.63 fM and exhibits high selectivity to discriminate MtDNA from a mixture with a concentration ratio of WtDNA to MtDNA as high as 5000:1. Moreover, this approach can be used to analyze target MtDNA in diluted serum samples, which demonstrates its potential application for future clinical diagnostics.