Thermostable DNA Immobilization and Temperature Effects on Surface Hybridization

Abstract

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.

Temperature Gradient Approach for Rapidly Assessing Sensor Binding Kinetics and Thermodynamics

“…These observations are consistent with other reports comparing DNA hybridization on gold surfaces to hybridization in solution. 4,7,11 To demonstrate the practical advantages of using the gradient to differentiate types of DNA hybrids, we performed a set of hybridization experiments using variables that have known influences on hybrid stability in solution. Figure 6 shows temperature profiles of hybridization activity for P15 probes with P15′ targets in different salt concentration hybridization buffers, with targets that form shorter length hybrids (P12′-T3 and P10′-T5), and with a target that results in hybrids with an internal nucleotide mismatch (P15′-MM).…”

“…This quantitative knowledge will provide information needed to refine predictive models, allowing accurate modeling of kinetic and thermodynamic parameters for hybridization on surfaces. 11,24,26 In addition to biosensor technology, this information is important for the continued development of DNA surface technologies where DNA is used as a structural material. 28, 29 The label-free nature of SPR measurements makes the gradient methodology described here versatile for characterizing the temperature dependence of a wide range of binding interactions involving DNA, proteins, and drug molecules.…”

Impedance Biosensor Incorporating a Carboxylate-Terminated Bidentate Thiol for Antibody Immobilization

“…7 For such applications, the utility of Au-alkanethiol SAMs has been limited by instability upon exposure to high temperature, 8,9 ultraviolet light, 10,11 and/or harsh chemical reagents. 12,13 A significant increase in stability has been reported for multidentate relative to monodentate thiol adsorbates, [14][15][16] forming amide bonds to amine groups on the protein surface. Non-specific adsorption was reduced by immersing the antibody-coated electrodes in 0.1% BSA for 1 hour.…”

Surface vs. solution hybridization: effects of salt, temperature, and probe type

“…9 This simplest model considers each probe site to be either in an unhybridized or a hybridized state, with the physical nature of these states assumed uniform for all sites as well as constant in time. As such, a two-state treatment does not explicitly model dispersion in hybridization behavior that may arise from heterogeneity in probe coverage, synthetic uncertainties, chemistry of the solid support, or some other source.…”