Department of Chemical & Biomolecular Engineering, Polytechnic Institute of New York University, Brooklyn, 11201, United States.
J Am Chem Soc. 2012 Nov 14;134(45):18667-76. doi: 10.1021/ja3066368. Epub 2012 Oct 30.
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.
存在多种用于分析小分子配体和核酸之间相互作用的解决方案方法；然而，在数百到数千个序列的规模上经济地完成这项任务仍然具有挑战性。表面分析通过基于阵列的多重检测提供了一个有前景的解决方案，能够在单个实验中绘制出 DNA/配体相互作用的完整序列背景。然而，与溶液分析相比，在表面格式中准确量化 DNA/配体相互作用必须应对对固液界面处分子活动和相互作用的有限理解。我们报告了一种溶液方法的表面适应，其中通过配体结合诱导的双链体稳定性变化，并从熔解转变中定量，用于 DNA/配体相互作用的热力学分析。结果与溶液量热数据进行了基准测试。通过加热和冷却触发的变性/杂交转变的叠加来确认平衡操作。作为典型小分子的抗生素化合物 netropsin 与 DNA 发生静电和序列特异性小沟相互作用。通过电化学跟踪表面熔解转变来研究 DNA/netropsin 相互作用作为离子强度和药物浓度的函数。与溶液值的比较发现自由能非常吻合，尽管可靠地分离成焓和熵贡献更具挑战性。这些结果为通过表面可逆熔化变性分析 DNA-配体相互作用建立了关键指南。
