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， 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，这些过程是由DNA的内在变形能力的帮助。因此，变形能力的改变 由于损伤或突变而导致的DNA的减少可以扰乱基因组的调节状态，从而增加 对疾病的易感性。因此，了解DNA的变形性如何随碱基序列而变化可以提供 遗传变异和细胞生理学之间缺失的一环DNA是一种双螺旋梯状结构， 有主凹槽和次凹槽。这种沟的不对称性赋予DNA不对称的可弯曲性和弯曲。 扭曲耦合，真正独特的性质，DNA，但这些性质还没有得到彻底的研究， 实验手段。单个碱基对步骤的极端弯曲或扭曲可以导致在细胞中的大的变化。 三维DNA构象，但极端的热力学和序列依赖性 由于缺乏实验方法，可弯曲性和可扭转性在很大程度上仍然是未知的。变形碱基对 步骤可能会影响酶和转录因子如何与DNA相互作用，但测试这一想法需要精细的 控制碱基对阶跃变形。 PI已经研究了通过结合使用短DNA与 粘性末端和基于表面的单分子测定。在本R 01的第一个供资期内，循环和 从不同长度和碱基序列的DNA分子中测量解环速率，包括 不匹配的碱基这些研究的结果阐明了环形成的动力学，并帮助我们 设计新的方法来量化DNA的不对称弯曲性和弯曲-扭转耦合。此外，委员会认为， 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。 基于第一个R 01的这些关键结果和见解，该提案将测量极端变形能力 的DNA和研究其后果的动力学的DNA靶向蛋白。实验方法 是将联合收割机单分子FRET与不同几何形状小DNA环结合。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，测量同轴堆积和非堆积 目标4，测量Cas 12的反应动力学，Cas 12是一种RNA引导的DNA CRISPR系统的靶向蛋白，在弯曲和扭曲的DNA底物上。这些研究将揭示 DNA的一般机械-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.