Complex, multi-target DNA hybridization: applications to molecular diagnostics and nucleic acid amplification techniques

Abstract

Nucleic acid (NA) hybridization underlies many different DNA and RNA detection assays. While polymerase chain reaction (PCR) remains the gold standard, its susceptibility to contamination and reliance on thermocycling have motivated the development of alternative isothermal amplification strategies. Although these isothermal methods simplify instrumentation, they remain limited by non-specific amplification and inhibition. As a result, emerging sensor designs have sought to integrate allosteric-like interactions (e.g. DNA switches, multi-site receptors, and coaxial base-stacking) to improve tunability and specificity. Allostery is generally defined as the energetic coupling between two binding sites. However, despite well-established thermodynamic parameters for canonical base pairing, the thermodynamics and kinetics of complex, multi-site DNA receptors remain less understood. In this work, we investigated the thermodynamics and kinetics of looped DNA receptors containing one or more target-binding sites. We developed a mathematical model that predicts fluorescence outputs for receptors bound to one or two targets, uniquely incorporating target dimerization and non-constant target concentrations. Our model provides mechanistic insights not captured by classic Hill-type models and enables quantification of how receptor sequence and binding-site number influence the sensitivity and dynamic range. Using these insights, we provided design recommendations for allosteric-like DNA receptors and established a framework for predicting signal outputs from DNA structure. To further characterize multi-site DNA hybridization, we measured the binding kinetics of related receptors using Surface Plasmon Resonance, an optical technique to study the real-time interactions of unlabeled molecules. We developed a protocol for small DNA binding pairs despite challenges posed by instrument sensitivity and small target size. We applied insights from multi-site hybridization to improve micro- RNA transduction strategies for Ultrasensitive DNA Amplification Reaction (UDAR), an isothermal amplification reaction. We evaluated a ligation-based, split-transduction scheme, identifying key limitations arising from enzyme behavior and unpredicted NA interactions. Finally, we investigated the relevance of micro-RNA biomarkers by profiling micro-RNA dysregulation in a neurodegenerative model based on okadaic acid neurotoxicity. This research advances the mechanistic understanding of allosteric-like interactions in NA hybridization reactions. The results demonstrate how complex kinetic and thermodynamic designs can be leveraged to create more tunable and robust molecular diagnostics for DNA and RNA disease biomarkers.