Two-Pronged Specialist (TPS) probe: an ultra-sensitive enzyme-driven universal system for dual-microRNA detection.

For multiplex mircoRNA (miRNA) profiling, the practical application of DNA logic gates is limited by insufficient sensitivity and operational complexity. Here, we developed an enzyme-powered Two-Pronged Specialist (TPS) probe method that utilizes a two-stage signal amplification cascade to simultaneously detect potential lung cancer biomarkers miR-21 and miR-221. This method combines an initial polymerase/nicking step, catalyzed by phi29 DNA polymerase and the nickase Nt.BstNBI, with a subsequent ligation-initiated rolling circle amplification (RCA) phase. This platform can perform programmable molecular calculations (AND/NAND/OR-AND/YES/OR/NOR/AND-INH) by using miRNAs and their templates as inputs, with different fluorescence signals (Cy3, ROX, Cy5) as outputs. Simultaneous quantitation of miR-21 and miR-221 showed a broad detection range (10 fM-100 pM) with the detection limits of 4.23 fM and 1.99 fM, respectively. The assay retained high specificity and reliability in serum and cell lysates. Furthermore, its modular template design allows for the detection of various nucleic acid targets through direct sequence replacement, demonstrating the versatility of the system in advanced molecular diagnostics and multi biomarker analysis based on its inherent dual detection capability. Using this probe to analyze miR-21 and miR-221 in lung cancer cells, we found that both miRNAs were significantly upregulated in lung cancer cell line A549 compared to human embryonic kidney cell line HEK-293T, and only miR-21 was upregulated in breast cancer cell line MCF-7. By optimizing experimental conditions, the optimal reaction conditions were determined as 1 nM S1/S2, 10 nM C1/C2, and 200 μM dNTPs for co-centration, 60 min for initial amplification, 6 h for T4 DNA polymerase, and 120 min for phi29 DNA polymerase.