Thermodynamic and modeling insights of DNA molecular beacons with dual target binding to design for tunable fluorescent outputs

Molecular beacons (MBs) offer a versatile platform for detecting specific nucleic acid targets based on the relative proximity of fluorophore and quencher molecules. Conventional MBs contain a single target-binding site in the loop, and hybridization has been modeled based on three specific phases: closed hairpin, random coil, and target-bound. The MB-target interactions can be tuned by stem length and target site location, while multi-site MBs introduce allosteric effects that modulate affinity and produce steeper, switch-like responses. Despite available thermodynamic models, current in silico methods do not adequately predict complex sensor outputs, especially for multi-site interactions. We present a new mathematical framework for calculating the thermodynamic parameters of MBs with two target binding sites. Our model additionally accounts for non-saturating target concentrations and target dimerization, extending its application to more complex reactions. We used three novel MB designs to validate the model, comparing single- and dual-target interactions and assessing temperature-dependent effects. Our approach captures both occupancy and temperature-dependent complex behavior of multi-site and single-site MBs, revealing how a second binding event can enhance affinity and tune the dynamic range. Full derivations and design recommendations are provided, offering a predictive tool to guide the rational design of multi-site MBs with controlled thermodynamics and fluorescent responses.