Thermodynamics and kinetics of DNA-ligand binding probed by DNA overstretching

描述(申请人提供)：DNA的结构转变和DNA结合配体在DNA上的对接是分子生物学中的基本过程。了解和控制这些过程对于合理设计抗癌和抗艾滋病毒等传染病的新药至关重要。使用光学和磁性镊子以及原子力显微镜的单分子力测量极大地扩展了我们对核酸和蛋白质的了解。具体地说，用光镊仪拉伸单个DNA分子可以诱导DNA双链的两条解开。拉伸引起的DNA双螺旋的结构和热力学变化以一种可控制和可测量的方式改变了与DNA结合配体的相互作用。因此，DNA和DNA-配体相互作用的单分子作用力测量为定量研究与DNA螺旋失稳和DNA-配体结合相关的一系列重要生理现象提供了前所未有的机会。尽管单分子作用力实验取得了进展，但对生物分子对机械应力的结构和热力学响应的了解很少，限制了此类实验对螺旋失稳和DNA与配体结合的洞察。力诱导熔化建模的一个众所周知的困难是这个过程跨越的长度和时间尺度的巨大范围，以及计算伴随的熵变化的困难。为了更好地理解从原子模型到大分子模型的跨越长度和时间尺度所涉及的基本生物物理，我们发展了一个新的DNA拉伸和小配体与拉伸DNA结合的多尺度模型。这个系统在实验上具有很好的特性，并且足够简单，以便于在全原子分子动力学(MD)模拟和粗粒度模型(如波兰-Scheraga模型)方面进行化学精确的生物物理建模。此外，在DNA结合配体存在的情况下拉伸DNA可以在现实的、生物学相关的条件下产生DNA配体结合的信息，这是其他方法无法获得的。在这个项目中，我们通过多管齐下的方法研究放线菌素D(ActD)的结合，ActD是一种针对DNA复制和转录的抗癌抗生素药物。在原子尺度上，化学精确的分子动力学模拟将被用来研究ActD与短DNA低聚物的结合机制和动力学；对于用于拉伸实验的长DNA，我们的多尺度模型将被用来计算平衡力-伸展关系，该关系可以直接用实验数据来验证。该项目将增强我们对基础生物物理学中多尺度现象的了解，并为ActD的抗癌功能提供新的见解。

DESCRIPTION (provided by applicant): Structural transitions of DNA and docking of DNA-binding ligands on DNA are fundamental processes in molecular biology. Understanding and controlling these processes are essential to the rational design of novel drugs against cancer and infectious diseases such as HIV. Single-molecule force measurements using optical and magnetic tweezers and atomic force microscopy have dramatically expanded our knowledge of nucleic acids and proteins. Specifically, stretching single DNA molecules by an optical tweezers instrument can induce the unwinding of the two strands of the DNA duplex. The induced structural and thermodynamic changes in the DNA double helix upon stretching alter interactions with DNA-binding ligands in a controllable and measurable way. Therefore single-molecule force measurements of DNA and DNA-ligand interactions provide an unprecedented opportunity for quantitative study of a wide range of physiologically important phenomena associated with DNA helix-destabilization and DNA-ligand binding. In spite of the progress made in single-molecule force experiments, a poor understanding of the structural and thermodynamic response of biomolecules to mechanical stress has limited the insight that such experiments have provided into helix destabilization and DNA-ligand binding. A notorious difficulty for modeling force-induced melting is the vast range of length and time scales spanned by the process, and by the difficulty in computing the accompanying change in entropy. To better understand the fundamental biophysics involved in crossing length and times scales from the atomistic to the macromolecular model, we develop a novel multiscale model for DNA stretching and small ligand binding to stretched DNA. This system is well characterized experimentally and sufficiently simple to facilitate chemically accurate biophysical modeling in terms of all-atom molecular dynamics (MD) simulations and coarse-grained models such as the Poland-Scheraga model. Moreover, DNA stretching in the presence of DNA binding ligands yields information about DNA ligand binding under realistic, biologically relevant conditions that cannot be obtained by other methods. In this project we study the binding of Actinomycin D (ActD), an anticancer antibiotic drug that targets DNA replication and transcription, by a multipronged approach. On the atomistic scale, chemically accurate MD simulations will be used to study the binding mechanism and dynamics of ActD to short DNA oligomers; for long DNA as used in stretching experiments, our multiscale model will be used to compute equilibrium force-extension relations that can be directly validated with experimental data. The project will both enhance our knowledge of multiscale phenomena in fundamental biophysics and provide new insight into the anticancer function of ActD.