A DNA Platform‐Assisted Cooperative Probe Addresses the Affinity–Selectivity Trade‐Off in Hybridization‐Based Nucleic Acid Detection at Room Temperature

Hybridization‐based nucleic acid detection remains central to molecular diagnostics but is often limited by slow kinetics, diminished sensitivity toward structured or double‐stranded targets and low selectivity in recognizing single‐nucleotide variation (SNV) at ambient temperatures. Here we introduce a DNA platform‐assisted cooperative probe (PCP) that enhances the performance of conventional molecular beacon (MB) probes through cooperative analyte binding mediated by a DNA scaffold. The PCP architecture spatially organizes two analyte‐binding arms (F and U) on a dsDNA platform for cooperative tight binding of nucleic acid analytes, which enables the use of a short platform‐detached strand m for highly selective SNV recognition. Using clinically relevant targets from Mycoplasma genitalium (23S rRNA fragment) and Neisseria gonorrhoeae (penA gene fragment), we demonstrate that PCP improves SNV selectivity from 10% to 14% for MB probe up to 99%, and lowers the limit of detection by up to an order of magnitude but does not significantly improve hybridization rate. The PCP sensor further enables room‐temperature detection of double‐stranded polymerase chain reaction (PCR) amplicons. These results establish DNA platform‐based probe organization as an advantageous strategy to resolve the affinity/specificity trade‐off in hybridization‐based diagnostics.