A variety of solution methods exist for analysis of interactions between small molecule ligands and nucleic acids; however, accomplishing this task economically at the scale of hundreds to thousands of sequences remains challenging. Surface assays offer a prospective solution through array-based multiplexing, capable of mapping out the full sequence context of a DNA/ligand interaction in a single experiment. However, relative to solution assays, accurate quantification of DNA/ligand interactions in a surface format must contend with limited understanding of molecular activities and interactions at a solid-liquid interface. We report a surface adaptation of a solution method in which shifts in duplex stability, induced by ligand binding and quantified from melting transitions, are used for thermodynamic analysis of DNA/ligand interactions. The results are benchmarked against solution calorimetric data. Equilibrium operation is confirmed through superposition of denaturation/hybridization transitions triggered by heating and cooling. The antibiotic compound netropsin, which undergoes electrostatic and sequence-specific minor groove interactions with DNA, is used as a prototypical small molecule. DNA/netropsin interactions are investigated as a function of ionic strength and drug concentration through electrochemical tracing of surface melt transitions. Comparison with solution values finds excellent agreement in free energy, though reliable separation into enthalpic and entropic contributions proves more difficult. The results establish key guidelines for analysis of DNA-ligand interactions via reversible melting denaturation at surfaces.
Thermal denaturation, or melting, measurements are a classic technique for analysis of thermodynamics of nucleic base driven associations in solution, as well as of interactions between nucleic acids and small molecule ligands such as drugs or carcinogens. Performed on surface-immobilized DNA films, this well-established technique can help understand how energetics of surface hybridization relate to those in solution, as well as provide high-throughput platforms for screening of small molecule ligands. Here we describe methods for measuring DNA melting transitions at solid/liquid interfaces with focus on the role of immobilization chemistry, including a common "immobilization-through-self-assembly" approach that is effective at moderate temperatures, and a thermo-stable approach based on polymer-supported DNA monolayers that can be used at elevated temperatures. We also discuss conditions necessary for reversible measurements, as signified by superimposition of the association (cooling) and dissociation (heating) transitions of immobilized DNA strands.
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