Invader assay-induced catalytic assembly of multi-DNAzyme junctions for sensitive detection of single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs) are main causes of differences in human phenotypes and drug resistance, especially the development of genetic diseases. Monitoring SNPs is of significant benefit to drug design and disease diagnosis. In this work, on the basis of tripartite DNAzyme junction formation induced by flap endonuclease 1 (FEN1) invader assay-triggered catalytic hairpin assembly (CHA), we describe an amplified and highly sensitive fluorescent strategy for detecting SNP of K-ras gene with substantial discrimination capability. The mutant DNA (MtDNA) of K-ras gene hybridizes with the sensing probe to inhibit the enzymatic activity of FEN1 to trigger subsequent CHA of three hairpins for the formation of tripartite DNAzyme junctions. And, the fluorescently quenched signal probes are efficiently and cyclically cleaved by the DNAzymes to restore largely magnified fluorescence for detecting MtDNA target sequence at 4.23 fM. Besides, the existence of low levels of MtDNA strands in diluted human serums and high concentrations of wild DNA solutions can be differentiated by such a method, showing its high potential for monitoring various SNPs for biological research and disease diagnosis.

Automated summary

This study describes an amplified and highly sensitive fluorescent strategy based on CHA for detecting SNPs in the K-ras gene, with substantial discrimination capability. This method has significant implications in drug design and disease diagnosis.