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DNA Melting at Surfaces and Applications to Thermodynamic Analysis of DNA/Ligand Interactions.

机译：表面的DNA熔化及其在DNA /配体相互作用的热力学分析中的应用。

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摘要

Mode of action of many antibiotic, antiviral and anticancer drugs is based on binding of these compounds to DNA leading to interference with DNA replication and transcription. Diverse solution methods exist for analysis of interactions between DNA and small molecule ligands; however, multiplexing such assays remains challenging. Surface approaches present a feasible platform for detecting DNA-ligand interactions at the scales of hundreds to thousands sequences through parallelization in array format resulting in increased efficiency and reduced costs of assays. This thesis describes a surface approach to analysis of DNA ligand binding thermodynamics, which were monitored through electrochemical detection of DNA melting transitions. Surface-bound DNA "probe" molecules were immersed in a sample medium containing complementary "target" DNA bearing an electroactive label. The extent of probe-target hybridization was recorded as a function of temperature with electrochemical methods to generate a DNA melting curve. Equilibrium operation was confirmed through superimposition of DNA hybridization/denaturation transitions triggered by cooling and heating. In the presence of a minor groove binding ligand, the drug netropsin, the DNA melting transition was shifted to higher temperature, indicating duplex stabilization by the ligand. As a result, thermodynamic profile of drug binding to duplex DNA was extracted via analyzing the drug-induced DNA thermal stabilization. Free energies acquired from the surface melting scans were in good agreement with those from solution measurements validating surface approach for drug-DNA binding detection. Multiplexing of surface assays can be accomplished through implementation of multiple DNA sequences on microelectrode arrays manufactured via photolithography. This approach can be also applied to covalently binding drugs, e.g., cisplatin, that typically reduce DNA melting temperature due to distortion of helical structure.
机译：许多抗生素，抗病毒和抗癌药物的作用方式基于这些化合物与DNA的结合，从而导致干扰DNA复制和转录。存在用于分析DNA与小分子配体之间相互作用的多种溶液方法。然而，多重分析仍然具有挑战性。表面方法提供了一种可行的平台，可通过以阵列形式并行化来检测数百至数千个序列的DNA-配体相互作用，从而提高了效率并降低了测定成本。本文介绍了一种表面方法，用于分析DNA配体结合的热力学，通过电化学检测DNA熔解转变进行监测。将表面结合的DNA“探针”分子浸入包含带有电活性标记的互补“靶标” DNA的样品介质中。用电化学方法记录探针-靶标杂交的程度与温度的关系，以产生DNA解链曲线。通过叠加由冷却和加热触发的DNA杂交/变性过渡来确认平衡操作。在小沟结合配体药物netropsin的存在下，DNA熔解转变移至更高的温度，表明该配体具有双链体稳定性。结果，通过分析药物诱导的DNA热稳定性来提取药物与双链体DNA结合的热力学特征。从表面熔解扫描获得的自由能与溶液测量的自由能非常吻合，溶液测量验证了用于药物-DNA结合检测的表面方法。通过在通过光刻技术制造的微电极阵列上实施多个DNA序列，可以完成表面分析的多路复用。该方法也可用于共价结合药物，例如顺铂，其通常由于螺旋结构的扭曲而降低DNA解链温度。

著录项

  • 作者

    Belozerova, Irina.;

  • 作者单位

    Polytechnic Institute of New York University.;

  • 授予单位 Polytechnic Institute of New York University.;
  • 学科 Engineering Biomedical.;Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 181 p.
  • 总页数 181
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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