Investigation of energetics of sharp DNA bending

描述(申请人提供)：DNA是一种动态分子，它经历了弯曲、扭曲和拉伸等构象变化。DNA的弯曲导致最显著的构象变化，与DNA包装、DNA环和细胞内DNA错配修复有关。这些过程中的任何一个过程中的错误都可能导致人类疾病，因此，了解DNA的内在弯曲特性与人类健康有关。为了了解DNA急剧弯曲发生的频率，我们需要知道它们的能量成本。人们通过测量线性DNA通过热波动形成环的可能性来估计这种能量。根据结果，我们都可以同意将一个500个碱基对长的DNA弯曲成一个圆需要多少能量，这被认为是正常弯曲。但实验数据不同意100个碱基对长的DNA，这需要更尖锐的弯曲才能形成一个圆圈。这些相互矛盾的结果引发了关于DNA如何实现急剧弯曲的不同模型。一种模型表明，双链DNA可以在局部融化成未配对的单链，从而获得更大的灵活性。然而，这种“泡沫”的形成缺乏实验证据。在这个方案中，我们调查了储存的能量 在小到50个碱基对的DNA环中，测试在急剧弯曲区域中DNA弯曲的各种模型。关键的想法是形成一个形状像泪滴的小DNA环，并测量它作为一个环停留多长时间。使用一种名为荧光共振能量转移(FRET)的技术，将在显微镜上观察到环状结构的形成或断裂。循环的寿命将取决于其能量；能量较高的循环将不太稳定。因此，我们可以测试一种经典的DNA聚合物模型，即蠕虫链模型，该模型在急剧弯曲区域内具有准确性。由于泪滴也有不同于圆形的尖端，我们可以在尖端附近放置不同的DNA含量，以比较它们在不同弯曲制度下的弯曲刚性。我们将测量作为温度函数的各种顺序的弯曲刚性以及在正常弯曲和急剧弯曲区域中的失配。我们还将把我们的结果与DNA熔化理论进行比较，以阐明DNA熔化在DNA弯曲中的作用。这项提案将解决DNA生物物理学中一些最具争议性的话题。

DESCRIPTION (provided by applicant): DNA is a dynamic molecule that goes through conformational changes such as bending, twisting, and stretching. Bending of DNA, which leads to most noticeable conformational changes, is associated with DNA packaging, DNA looping and DNA mismatch repair inside a cell. Errors in any of these processes can cause human diseases, and therefore, understanding intrinsic bending properties of DNA is related to human health. To understand how frequently sharp DNA bends can arise, we need to know their energy cost. People have estimated this energy by measuring how likely it is for a linear DNA to form a loop by thermal fluctuation. Based on the results, we can all agree on how much energy is needed to bend a 500 base-pair long DNA into a circle, which is considered as normal bending. But experimental data disagree regarding a 100 base-pair long DNA, which requires sharper bending to form a circle. These contradictory results prompted different models about how DNA achieves sharp bending. One model suggests that double-stranded DNA can locally melt into unpaired single strands and acquire increased flexibility. However, experimental evidence for this "bubble" formation is lacking. In this proposal, we investigate the energy stored in a DNA loop as small as 50 base pairs and test various models of DNA bending in the sharp bending regime. The key idea is to form a small DNA loop shaped like a teardrop, and measure how long it stays as a loop. Loop formation or breakage will be observed on a microscope using a technique called Fluorescence Resonance Energy Transfer (FRET). The lifetime of the loop will depend on its energy; a loop with a higher energy will be less stable. Therefore, we can test a classical polymer model of DNA known as the worm-like chain model with accuracy in the sharp bending regime. Because a teardrop also has a pointy tip unlike a circle, we can place various DNA content near the tip to compare their bending rigidity in different bending regimes. We will measure bending rigidity of various sequences and mismatches in normal and sharp bending regimes as a function of temperature. We will also compare our results against DNA-melting theories to elucidate the role of DNA melting in DNA bending. This proposal will address some of the most controversial topics on DNA biophysics.