Direct Fluorescence Anisotropy Detection of miRNA Based on Duplex-Specific Nuclease Signal Amplification

The dysregulation of microRNAs (miRNAs) is associated with various diseases, including cancer, so miRNAs are considered a potential biomarker candidate for disease diagnosis and therapy. However, the direct, rapid, sensitive, and specific detection of miRNAs remains quite challenging due to their short length, sequence homology, and low abundance. Herein, we propose a simple and homogeneous fluorescence anisotropy (FA) strategy for the direct and rapid (∼35 min) quantification of miRNA-21 based on duplex-specific nuclease (DSN)-assisted signal amplification. In the presence of target miRNA-21, the complementary single-stranded DNA (ssDNA) probes labeled with a single fluorophore, tetramethylrhodamine (TMR), are specifically hydrolyzed into small fragments by endonuclease DSN upon formation of the DNA/RNA hybrid, which leads to a reduction in FA due to the decrease in molecular size. However, the target miRNA remains intact during the enzymatic digestion process and is released in solution for the next round of binding, hydrolysis, and release for recycling. It is observed that the ssDNA probe labeled with TMR at the 5'-end, in which the fluorophore is nine nucleotides away from the nearest dG base to eliminate/reduce photoinduced electron transfer interaction between TMR and the dG base, exhibits the maximum FA change in response to the target miRNA-21. The change in FA enables the sensitive detection of miRNA-21 ranging from 0.050 to 2.0 nM, with a detection limit of 40 pM. In addition, this amplification strategy exhibits high selectivity and can even discriminate single-base mutations between miRNA family members. We further applied this method to detect miRNA-21 in the extract of various cancer cell lines. Therefore, this method holds great potential for miRNA analysis in tissues or cells, providing valuable information for biomedical research, clinical diagnostics, and therapeutic applications.