Specific fluorescence detection strategy for single-stranded nucleic acids by dual-toehold branch migration

Small changes in nucleic acid sequences can produce large phenotypic differences and are closely associated with many serious diseases. Therefore, accurate detection of the nucleic acid targets is extremely necessary. Research focusing on improving the detection specificity of the single-stranded DNA (ssDNA) and RNA still needs to be developed. Herein, the research developed a specific fluorescence detection strategy for single-stranded nucleic acids through the innovative use of dual-toehold branch migration (DTBM). The ssDNA L₁ and miRNA let-7a were selected as single-stranded nucleic acid detection models, respectively. By extension with the KF polymerase, ssDNA L₁ was transformed into a double-stranded probe to trigger the DTBM, which ensures the detection specificity. For let-7a detection, a strand displacement amplification (SDA)-triggered DTBM strategy was further proposed. The SDA process formed the ssDNA L₂, which was also converted into a double-stranded probe by enzymes to trigger the subsequent DTBM. This specific fluorescence detection strategy showed high specificity for ssDNA L₁ and miRNA let-7a, and the average discrimination factors (DFs) were 24.49 and 30.59, respectively. And it displayed excellent analytical performance. Additionally, this strategy is user-friendly and does not require frequent temperature change steps or sample additions, which brings about great expectations regarding the practical application of ssDNA and miRNA detection.