Investigation of energetics of sharp DNA bending

遗传信息是由DNA携带的，DNA是一种受物理定律支配的聚合物分子。强烈弯曲和 扭曲的DNA与许多基因组过程有关，包括包装、转录、修复和编辑， 这表明，这些过程是由DNA固有的变形性帮助的。因此，改变的变形性 DNA损伤或突变会扰乱基因组的调节状态，从而增加 对疾病易感性。因此，了解DNA的变形性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一环。DNA是碱基对台阶的双螺旋阶梯 有主要和次要的凹槽。这种凹槽的不对称性赋予DNA不对称的可弯曲性和弯曲性- 扭曲耦合，DNA真正独有的性质，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会导致大的变化 DNA的三维构象，但对热力学和序列依赖性的极端 由于缺乏实验方法，弯曲和扭转性能在很大程度上仍不为人所知。变形的碱基对 步骤可能会影响酶和转录因子与DNA的相互作用，但测试这一想法需要精细 碱基对阶跃变形的控制。 PI研究了结合使用短DNA和DNA的强弯曲的热力学 粘性末端和基于表面的单分子分析。在本R01的第一个资金期内，循环和 测量了不同长度和碱基序列的DNA分子的解环率，包括 不匹配的碱基。这些研究的结果阐明了环路形成的动力学，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-扭曲耦合。此外， 他们揭示了DNA环的几何结构，使测量个体的弯曲和扭转硬度成为可能 碱基对步骤。 在第一版R01的这些关键结果和见解的基础上，本提案将测量极端变形性 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与不同几何结构的小DNA环结合起来。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 不同弯曲角度下不匹配碱基对的刚性；目标3，测量同轴堆积和去堆积 单个碱基对步长的速率；以及目标4，测量RNA引导的DNA Cas12的反应动力学 CRISPR系统的靶向蛋白，在弯曲和扭曲的DNA底物上。这些研究将有助于 DNA的类属力学-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.