Kinetic inversion in hybridization-based biosensors with multi-step reaction mechanisms

Abstract

DNA plays a major role in many biological processes and technological applicaFons. The recent increased interest in minimally invasive techniques in medicine shows the need to improve mutant detecFon in an excess of single nucleoFde variant background. We explore new detecFon and quanFficaFon concepts by improving our understanding of the thermodynamic and kineFc properFes of DNA. Typically, hybridizaFon-based biosensors make use of the difference in binding free energy between the matching target and mismatched target. In a single-step reacFon, i.e. the formaFon of a probe + target duplex, this leads to opFmal discriminaFon at equilibrium. Earlier work on a mulF-stranded sensor a1ributed the improved selecFvity to the sensor operaFng under non-equilibrium condiFons. In contrast to single-step reacFons, it was also observed that matching target equilibrated faster compared to mismatched target, a novel phenomenon dubbed ‘kineFc inversion.' It is our understanding that this behavior is unique to sensors with mulF-step reacFon mechanisms. In such a system, the opFmal Fme scale to discriminate between matching and mismatched target is not at equilibrium but at a specified Fme during the reacFon kineFcs (see figure). We studied the presence and properFes of kineFc inversion in a mulF-stranded probe with a two-step reacFon mechanism. A theoreFcal analysis shows that it is sensiFvely dependent on the reacFon rates and the thermodynamics of the interacFng strands. SelecFve binding should occur in the first of the two steps, and the second step should be indiscriminate. In addiFon to this, the first step should be noFceably faster compared to the second step. Experimental data is currently being collected using a mulF-stranded probe designed to operate around room temperature. KineFc inversion could increase the biosensor performance by extending the dynamic range and shortening the readout Fme.