Dynamics of Double-Stranded DNA in Confined Geometries

ID：MPS/dmr/bmat(7623)1106044 PI：Strey，Helmut ORG：纽约州立大学石溪分校标题：受限几何中双链DNA的动力学内部优点：这项建议是由PI实验室以前关于双链DNA分子在二维空腔阵列中扩散的工作推动的。这项工作通过荧光成像研究了线性DNA在具有精确控制(和已知)孔结构的介质中的扩散。这种结构是由短圆孔相互连接的球腔的周期性、二维六角形阵列。跟踪许多单分子轨迹发现，当DNA的回转半径接近空腔直径时，扩散主要由DNA在空腔之间的零星跳跃所主导，这是由熵势垒输运理论预测的一种机制。跳跃对应于构型波动，它允许柔性聚合物通过比平均线圈尺寸小的孔收缩。最近，人们将松弛的双链DNA环的扩散与相同长度的线性DNA的扩散进行了比较。观察到，环状分子的扩散速度比相同分子量的线形分子慢2.5~5.6倍，比相同平均尺寸的线形分子慢3.7~10.6倍。这样的结果定性地揭示了线性分子在通过空穴之间的孔的转移过程中可能会形成环，但发生这种事件的概率很低。对于线性分子，主要的扩散方式是末端优先。这项提案更详细地阐述了这一段落。聚合物线是用一端穿的，还是用中间段的一段绕线，还是两种工艺都同样方便？这个问题分几个阶段来解决，这些阶段独立地解决了聚合物动力学中的重要基本问题：(1)创建不同分子量的双色末端标记分子，这将使使用荧光相关光谱研究内部和溶液聚合物动力学成为可能。(2)利用FCS和光学显微镜研究缝隙状纳米通道中聚合物的内部动力学，加深对横向受限聚合物的理解。(3)测量线性双链DNA和缺口环状DNA在空腔和连接通道之间的分配和跳跃频率，作为DNA长度和阵列尺寸(高度、空腔直径、收缩宽度和长度)的函数。(4)表征双标记DNA在腔阵列中的穿线动力学。BROADER的影响：许多大分子操作、纯化和分离技术依赖于分子水平限制的环境来产生选择性的大分子运动。为了加深对纳米尺度聚合物约束的热力学和动力学的理解，人们准备了可以通过单分子荧光显示来检测大分子的流体器件和空腔阵列。该项目还将解决分离不同聚合物拓扑(例如线形和圆形)的一个非常重要的技术领域。主要的教育目标是培养和指导研究生和本科生，使他们能够在研究和工程方面追求自己的职业生涯。这项研究工作将培养出具有严谨的科学背景、独立思考并了解知识产权和现实世界应用的学生。此外，PI将继续通过石溪西蒙斯计划接触高中生，担任导师和科学评委。其中一些学生在实验室经历后，在LISF和英特尔竞赛等科学竞赛中取得了非常成功的成绩。此外，PI将主持和设计一个网站，允许与研究社区分享纳米制造和纳米/微流体方面的技术和诀窍。这样一个网站将使研究人员和学生能够学习和分享纳米和微流体的复杂之处。

ID: MPS/DMR/BMAT(7623) 1106044 PI: Strey, Helmut ORG: SUNY Stony BrookTitle: Dynamics of double-stranded DNA in confined geometriesINTELLECTUAL MERIT: This proposal is motivated by previous work from the PI's lab on the diffusion of double-stranded DNA (ds-DNA) molecules in 2-dimensional cavity arrays. This work investigated by fluorescence imaging the diffusion of linear DNA through a medium of precisely controlled (and known) pore structure. This structure was a periodic, two-dimensional hexagonal array of spherical cavities interconnected by short circular holes. Tracking many single molecule trajectories, it was found that, for DNA radius of gyration approaching the cavity diameter, diffusion is dominated by the sporadic hopping of DNA between cavities, a mechanism predicted by the entropic barriers transport theory. Hopping corresponds to configurational fluctuations that allow passage of a flexible polymer through a pore constriction smaller than the average coil size. The diffusion of relaxed ds-DNA circles has recently been compared with that of linear DNA of the same length. It is observed that circular molecules diffuse from 2.5 to 5.6 times slower than corresponding linear molecules of the same molecular weight, and 3.7 to 10.6 times slower than corresponding linear molecules of the same average dimension. Such results qualitatively reveal that linear molecules may form loops during translocation through holes between cavities, but the probability of such events is low. The predominant mode of diffusion for linear molecules is end first. This proposal addresses this passage in more detail. Does a polymer thread by one of its ends or loop by one of its mid-segments or do both processes occur with equal facility? This question is addressed in several stages that independently address important fundamental questions in polymer dynamics: (1) Create 2-color end-labeled molecules of varying molecular weights that will enable the study of internal and solution polymer dynamics using fluorescence correlation spectroscopy. (2) Study internal polymer dynamics in slit-like nanochannels to deepen our understanding of laterally confined polymers using FCS and optical microscopy. (3) Measure partitioning and hopping frequencies of linear ds-DNA and nicked circular DNA between cavities and connecting channels as a function DNA length and the array dimensions (height, cavity diameter, constriction width, and length). (4) Characterize the threading dynamics of double-labeled DNA in cavity arrays.BROADER IMPACTS: Many technologies for macromolecular manipulation, purification, and separation rely on an environment of molecular level constraints to create selective macromolecular motion. It is proposed to develop a deeper understanding of the thermodynamics and dynamics of nanoscale polymer confinement by preparing fluidic devices and cavity arrays in which macromolecules can be examined by single molecule fluorescence visualization. The project will also address a very important technological area of separating different polymer topologies (e.g. linear vs. circular). The main educational goal is to train and mentor graduate and undergraduate students to enable them to pursue their career in research and engineering. This research effort will produce students that have a rigorous science background, are independent thinkers, and have an understanding of intellectual property and real world applications. In addition, the PI will continue to reach out to high-school students through the Stony Brook Simons program as mentor and science judge. Some of those students, after their lab experience, have been very successful in science competitions such as LISF and the Intel competition. In addition, the PI will host and design a website that allows sharing of techniques and tricks in nanofabrication and nano/microfluidics with the research community. Such a website will enable researchers and students to learn and share the intricacies of nano- and microfluidics.