Investigation of energetics of sharp DNA bending

遗传信息由 DNA 携带，DNA 是一种受物理定律支配的聚合分子。强烈弯曲和 扭曲的 DNA 与许多基因组过程相关，包括包装、转录、修复和编辑， 这表明这些过程受到 DNA 固有变形能力的帮助。因此，变形能力改变 由于损伤或突变而导致的 DNA 缺失会扰乱基因组的调控状态，从而增加 对疾病的易感性。了解 DNA 的变形能力如何随碱基序列变化可以提供 遗传变异和细胞生理学之间缺失的联系。 DNA 是碱基对阶梯的双螺旋梯 与主要和次要凹槽。这种凹槽不对称性赋予 DNA 不对称的可弯曲性和弯曲性 扭转耦合，确实是 DNA 独有的特性，但这些特性尚未被彻底研究 实验手段。单个碱基对步骤的极端弯曲或扭曲可能会导致碱基对的巨大变化 三维DNA构象，但热力学和序列依赖性极端 由于缺乏实验方法，弯曲性和扭曲性仍然很大程度上未知。变形碱基对 步骤可能会影响酶和转录因子与 DNA 相互作用的方式，但测试这个想法需要很好的时间 碱基对阶梯变形的控制。 PI 通过结合使用短 DNA 和 粘性末端和基于表面的单分子测定。在本 R01 的第一个资助期内，循环和 解环率是通过不同长度和碱基序列的 DNA 分子测量的，包括 碱基不匹配。这些研究的结果阐明了环形成的动力学，并帮助我们 设计新方法来量化 DNA 的不对称弯曲性和弯曲扭转耦合。此外， 他们揭示了 DNA 环的几何形状，可以测量个体的弯曲和扭转刚度 碱基对步骤。 基于这些关键结果和第一个 R01 的见解，该提案将测量极端变形能力 DNA 并研究其对 DNA 靶向蛋白动力学的影响。实验方法 是将单分子 FRET 与不同几何形状的小 DNA 环相结合。四个具体目标是 提出：目标1，量化DNA的弯曲不对称性和弯曲扭转耦合；目标 2，量化弯曲 不同弯曲角度下错配碱基对的刚度；目标 3，测量同轴堆叠和拆垛 单个碱基对步骤的速率；目标 4，测量 Cas12（一种 RNA 引导的 DNA）的反应动力学 在弯曲和扭曲的 DNA 基质上靶向 CRISPR 系统的蛋白质。这些研究将揭示 DNA 的一般力学-功能关系。

Genetic information is carried by DNA, a polymeric molecule governed by the laws of physics. Strongly bent and twisted DNA is associated with many genomic processes including packaging, transcription, repair, and editing, which suggests that these processes are aided by the intrinsic deformability of DNA. Hence, altered deformability of DNA due to damage or mutation can perturb the regulatory state of the genome, thus increasing the susceptibility to disease. Understanding how deformability of DNA changes with base sequence can thus provide a missing link between genetic variation and cell physiology. DNA is a double helical ladder of base pair steps with the major and minor grooves. This groove asymmetry confers DNA with asymmetric bendability and bend- twist coupling, properties truly unique to DNA, but these properties have not been thoroughly investigated by experimental means. Extreme bending or twisting of a single base pair step can lead to large changes in the three-dimensional DNA conformation, but the thermodynamics and sequence dependence of extreme bendability and twistability remain largely unknown due to the lack of experimental methods. Deformed base pair steps will likely affect how enzymes and transcription factors interact with DNA, but testing this idea requires fine control of base-pair step deformation. The PI has investigated the thermodynamics of strong DNA bending by the combined use of short DNA with sticky ends and surface-based single-molecule assays. During the first funding period of this R01, looping and unlooping rates were measured from DNA molecules of different lengths and base sequences including mismatched bases. The results from these studies elucidated the kinetics of loop formation and helped us to design new approaches to quantifying asymmetric bendability and bend-twist coupling of DNA. Furthermore, they revealed DNA loop geometries that enable measurement of the bending and twist stiffness of individual base pair steps. Building upon these key results and insights from the first R01, this proposal will measure extreme deformability of DNA and investigate its consequence on the kinetics of a DNA targeting protein. The experimental approach is to combine singe-molecule FRET with small DNA loops of different geometries. Four specific aims are proposed: Aim 1, quantifying the bending asymmetry and bend-twist coupling of DNA; Aim 2, quantifying bending stiffness of mismatched base pairs at different bending angles; Aim 3, measuring coaxial stacking and unstacking rates of individual base pair steps; and Aim 4, measuring the reaction kinetics of Cas12, an RNA-guided DNA targeting protein of the CRISPR system, on curved and twisted DNA substrates. These studies will shed light on the generic mechanics-function relationship of DNA.