Dynamics of Double-Stranded DNA in Confined Geometries

受限几何结构中双链 DNA 的动力学

• 批准号: 1106044
• 负责人: Helmut Strey
• 金额: $ 33万
• 依托单位: SUNY at Stony Brook
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1106044&HistoricalAwards=false
• 关键词: Dynamics; Double; Stranded; DNA; Confined

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.

ID: MPS/DMR/BMAT(7623) 1106044 PI: Strey， Helmut ORG: SUNY Stony BrookTitle：受限几何中的双链DNA动力学知识优点：该建议是由PI实验室在二维腔阵列中双链DNA （ds-DNA）分子扩散的先前工作激发的。这项工作通过荧光成像研究线性DNA通过精确控制（和已知）孔结构的介质的扩散。这种结构是一种周期性的二维六边形球形腔阵列，由短圆孔相互连接。通过对多个单分子轨迹的跟踪，发现当DNA的旋转半径接近于腔直径时，扩散主要是由DNA在腔间的偶发跳跃所主导，这是熵势垒输运理论所预测的机制。跳变对应于允许柔性聚合物通过比平均线圈尺寸小的孔收缩的构型波动。放宽的ds-DNA环的扩散最近与相同长度的线性DNA的扩散进行了比较。观察到圆形分子的扩散速度比同等分子量的线性分子慢2.5 ~ 5.6倍，比同等平均维数的线性分子慢3.7 ~ 10.6倍。这些结果定性地揭示了线性分子在通过空腔之间的孔洞转运时可能会形成环路，但这种事件的概率很低。线性分子的主要扩散模式是末端优先。这一建议更详细地论述了这一段落。聚合物线是由其中一个末端形成的还是由其中一个中间部分形成的？还是两个过程都同样容易？这个问题分为几个阶段来解决，这些阶段独立地解决了聚合物动力学中的重要基础问题：(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和光学显微镜研究裂缝状纳米通道中聚合物的内部动力学，加深我们对侧向受限聚合物的理解。(3)测量线性ds-DNA和缺口圆形DNA在空腔和连接通道之间的分割和跳跃频率，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。更广泛的影响：许多大分子操作、纯化和分离技术依赖于分子水平约束的环境来产生选择性的大分子运动。为了深入了解纳米聚合物约束的热力学和动力学，我们提出了制备流体装置和腔阵列，其中大分子可以通过单分子荧光可视化来检测。该项目还将解决一个非常重要的技术领域，即分离不同的聚合物拓扑结构（例如线性与圆形）。主要的教育目标是培养和指导研究生和本科生，使他们能够从事研究和工程方面的职业。这项研究工作将培养出具有严谨的科学背景、独立思考者、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项目以导师和科学评委的身份接触高中生。其中一些学生，在实验室经历之后，在诸如LISF和英特尔竞赛等科学竞赛中取得了非常成功。此外，PI将主持和设计一个网站，允许与研究界分享纳米制造和纳米/微流体的技术和技巧。这样一个网站将使研究人员和学生学习和分享纳米和微流体的复杂性。