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Studies of stretching and immobilization of DNA molecules and their interactions with proteins at the single-molecule level.

机译：DNA分子的拉伸和固定化及其与单分子水平蛋白质的相互作用的研究。

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In this dissertation, the degrees of adsorption and stretching of DNA onto surfaces achieved by various stretching methods that use fluid flows are compared and the processes by which proteins find target sequences along these DNA molecules and either carry out cleavage or transcription are directly observed by fluorescence microscopy. DNA molecules either combed or spin-stretched onto hydrophobic surfaces stretch to a greater degree, but fewer are deposited, at pH 8.0 than at lower pH. At pH 8.0, DNA adhesion occurs primarily only at the DNA extremities, and avoids trapped regions of incompletely stretched DNA. A new technique, protein-assisted DNA immobilization (PADI), is developed to immobilize and stretch, but not overstretch, DNA molecules inside a micro/nanochannel at physiological pH. The biological activity of the immobilized DNA molecules is confirmed by digesting the DNA with restriction enzymes in the microchannel. Single molecule transcription, which has stringent requirements on the immobilized DNA with respect to surface interactions and stretched lengths, is also successfully demonstrated on DNA molecules immobilized by PADI.; The one dimensional Brownian motion and transcription elongation of T7 RNA polymerase along aligned DNA molecules bound to substrates by molecular combing are directly visualized. Fluorescent antibodies are used to label T7 RNA polymerase and a shear flow is applied to convect proteins orthogonally to the DNA alignment direction, permitting observation and estimation of the protein diffusivity along the DNA at the single-molecule level. The ID diffusion coefficient varies from molecule to molecule over a large range, suggesting that the individual proteins have distinct diffusivities. From the measured dependence of the rate of transcription on concentration of nucleotide triphosphate, the combed DNA molecules capable of interacting with proteins are inferred to be under an average tension of 25 pN.
机译：在本文中，比较了通过使用流体流动的各种拉伸方法获得的DNA在表面上的吸附和拉伸程度，并且通过荧光直接观察了蛋白质沿着这些DNA分子找到靶序列并进行切割或转录的过程。显微镜检查。梳理或旋转拉伸到疏水表面上的DNA分子在pH 8.0时的拉伸程度更大，但比在较低pH值下沉积的更少。在pH 8.0时，DNA粘附主要仅发生在DNA末端，并避免捕获不完全拉伸的DNA的区域。开发了一种新技术，即蛋白质辅助DNA固定（PADI），可以在生理pH值下固定并拉伸微/纳米通道内的DNA分子，但不能过度拉伸。通过在微通道中用限制酶消化DNA，可以确认固定化DNA分子的生物学活性。在表面相互作用和拉伸长度方面对固定的DNA有严格要求的单分子转录也已在PADI固定的DNA分子上成功证明。 T7 RNA聚合酶沿着通过分子梳理与底物结合的对齐DNA分子的一维布朗运动和转录伸长率直接可见。荧光抗体用于标记T7 RNA聚合酶，并施加剪切流使蛋白质与DNA排列方向垂直对流，从而可以观察和估计蛋白质在单分子水平上沿DNA的扩散性。分子之间的ID扩散系数在很大范围内变化，这表明单个蛋白质具有不同的扩散性。从测得的转录速率对三磷酸核苷酸浓度的依赖性，推断出能够与蛋白质相互作用的精梳DNA分子处于25 pN的平均张力下。

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