(321c) Theoretical and Experimental Investigation of the Impact of Surfaces on Oligonucleotide Melting Temperature

The most commonly used method to predict the melting temperature of an oligonucleotide duplex in solution is the Nearest Neighbor method, which relies on the sequential arrangement of different dinucleotides based on the stabilization effect of the base-stacking interactions between them. It is also the choice of the researchers designing oligonucleotide sequences or probes on the surface for microarray applications. However, the absence of the inclusion of the effects of the surface on oligonucleotide stability and melting temperature leads to an unrealistic representation of the oligonucleotide duplex formation and denaturation in close proximity to the microarray substrate. In this part of the study, a semi-empirical model has been developed to look at how these influences, namely electrostatic and entropic blocking, are affected by various system design parameters, probe length, probe density and spacer length, with respect to different surface coverages and salt concentrations. To further support these findings, various on-surface melting temperature experiments with different sequences are carried out by utilizing a specially designed integrated real-time microfluidic system. The theoretical and experimental results are found to be in agreement with each other and the published trends reached via simulations. A Gibbs Free Energy model incorporating these findings within a Nearest-Neighbor approach is developed, and its performance in realistically representing the thermodynamics on the surface is discussed.