Thermostable DNA immobilization and temperature effects on surface hybridization.

Monolayer films of nucleic acids on solid supports are encountered in a range of diagnostic and bioanalytical applications. These applications often rely on elevated temperatures to improve performance; moreover, studies at elevated temperatures can provide fundamental information on layer organization and functionality. To support such applications, this study compares thermostability of oligonucleotide monolayers immobilized to gold by first coating the gold with a nanometer-thick film (an "anchor layer") of a polymercaptosiloxane, to which DNA oligonucleotides are subsequently tethered through maleimide-thiol conjugation, with thermostability of monolayers formed via widely used attachment through a terminal thiol moiety on the DNA. The temperature range covered is from 25 to 90 °C. After confirming stability of immobilization and, more importantly, retention of hybridization activity even under the harshest conditions investigated, these thermostable films are used to demonstrate measurements of (1) reversible surface melting transitions and (2) temperature dependence of competitive hybridization, when fully matched and mismatched sequences compete for binding to immobilized DNA oligonucleotides. The competitive hybridization experiments reveal a pronounced impact of temperature on rates of approach to equilibrium, with kinetic freezing into nonequilibrium states close to room temperature and rapid approach to equilibrium at elevated temperatures. Modeling of competitive surface hybridization equilibria using thermodynamic parameters derived from surface melting transitions of the individual sequences is also discussed.