Abstract
<jats:p>Nucleic acids are powerful recognition elements because base pairing enables straightforward probe design, yet selective target recognition under physiological conditions remains challenging when specificity cannot rely on melting-based discrimination. Here we introduce a programmable multi-domain DNA probe that converts target binding into a cooperative self-assembly process. The recognition sequence is split into two short domains carried on separate strands, each coupled to a programmable stem domain through a flexible linker. Target hybridization increases the effective local concentration of the stems and drives their cooperative assembly into a stable three-way junction. This architecture enables independent control over probe performance: stem length tunes thermodynamic stability and apparent affinity, linker length regulates signal transduction without significantly affecting recognition, and cooperative assembly introduces an activation threshold that sharpens discrimination against mismatched and truncated targets. We demonstrate high sequence specificity with model DNA targets, extend the approach to disease-relevant miRNAs, and show that distinct probes operate orthogonally in a multiplexed format. Finally, we translate the same mechanism to a signal-ON electrochemical platform that supports point-of-care formats and operates directly in whole blood. Multi-domain probe design thus provides a modular, amplification-free strategy for programmable, high-specificity nucleic acid detection.</jats:p>