A DNA Platform-Assisted Cooperative Probe Addresses the Affinity-Selectivity Trade-Off in Hybridization-Based Nucleic...

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.