DNA Surface Hybridization: Comparison of Theory and Experiment

Abstract

The design and interpretation of surface hybridization assays is complicated by poorly understood aspects of the interfacial environment that cause both kinetic and thermodynamic behaviors to deviate from those in solution. The origins of these differences lie in the additional interactions experienced by hybridizing strands at the surface. In this report, an analysis of surface hybridization equilibria is provided for end-tethered, single-stranded oligonucleotide "probes" hybridizing with similarly sized, single-stranded solution "target" molecules. Theoretical models by Vainrub and Pettitt (Phys. Rev. E 2002, 66, 041905) and by Halperin, Buhot, and Zhulina (Biophys. J. 2004, 86, 718), and an "extended" model that in addition includes a solution-like salt dependence of probe-target dimerization, are compared to experiments as a function of salt concentration and probe coverage. Good agreement with experiment is observed when the DNA volume fraction at the surface remains below approximately 0.25. None of the models, however, can account for strong suppression of hybridization when the volume fraction of DNA approaches 0.3, realizable in the limit of high buffer strength and densely tethered films. Under these conditions, hybridization yields become insensitive to increases in analyte concentration even though many probes remain available to bind targets. These observations are attributed to the onset of packing constraints which, interestingly, become limiting significantly below maximum DNA coverages estimated from ideally efficient hexagonal packing. By delineating conditions under which specific hybridization behaviors are observed, the results advance fundamental knowledge in support of DNA microarray and biosensor applications.

What Controls the “Off/On Switch” in the Toehold-Mediated Strand Displacement Reaction on DNA Conjugated Gold Nanoparticles?

“…We suspect that the attenuation of the DNA hybridization for the toehold domain is related to the large electrostatic repulsion between the negatively charge probes and the highly stretched brush-like conformation for the tethered oligonucleotide strands at high surface probe density on AuNP-1. 30,31,45 These properties induce a high enthalpic impediment for the linkerprotector complex to move close to and insert into the oliogonucleotide layer of DNA-AuNP-1.…”

Temperature Gradient Approach for Rapidly Assessing Sensor Binding Kinetics and Thermodynamics

“…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.…”

Targeting Helicase-Dependent Amplification Products with an Electrochemical Genosensor for Reliable and Sensitive Screening of Genetically Modified Organisms

“…33 This problem would be circumvented with a RP flanking the specific target sequence. On the other hand, the forward primer (FP) not consumed during amplification would compete with target for signaling probe (SP); however working with a SP longer than FP and in a large excess over FP concentration, as usual, the equilibrium could be strongly displaced toward the SP-T duplex formation.…”