(638h) Theoretical and Experimental Investigation of the Impact of Surfaces On Oligonucleotide Melting Temperature: The Effects of Spacer Length and Probe Density

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, semi-empirical models have been developed to look at how these influences, namely electrostatic and entropic blocking, are affected by various system design parameters. Free energy penalty imposed by electrostatic blocking was calculated through the utilization of Electrical Double Layer theory, zeta potential measurements and Surface Partition Model. Entropic blocking free energy penalty term was modeled using a Polymer Physics approach. To further support these findings, various on-surface melting temperature experiments were carried out by utilizing a specially designed integrated real-time microfluidic system. Two design parameters, spacer length and probe density, were examined. Theoretical and experimental results were found to be in agreement with each other and the published trends reached via simulations. At the experimental conditions tested, probe density seems to have more influence in decreasing duplex stability when compared to spacer length. These findings can be used as guidelines in the design of experiments and optimization of hybridization conditions, when spacer length and probe density are considered as design parameters.