Optically Resonant Nanotweezers

光学共振纳米镊子

基本信息

批准号：
8069193
负责人：
David Carl Erickson
金额：
$ 17.82万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2013-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): In this work, we propose a joint project between the Erickson and Chen labs at Cornell University to demonstrate an entirely new approach to the study of weak protein-protein interactions through the development of a single molecule nanophotonic optical trapping and florescence resonant energy transfer (FRET) technique. The molecular system we apply the technique to here is the human copper transport pathway from the intracellular copper chaperone Hah1 to the copper transporting ATPase Wilson disease protein (WDP). Abnormal function of this transport pathway can lead to diseases such as Wilson disease and familial amyotrophic lateral sclerosis. Despite its importance, very limited quantitative information is available on how Hah1 and WDP interact. A major difficulty in obtaining this information is the lack of a single molecule analysis tool which can simultaneously: (1) capture and suspend small molecules in free solution for an indefinite period time (2) effectively "concentrate" the set of molecules of interest to a point where weak protein-protein interactions can be studied and (3) allow rapid modulation of the external environmental conditions (e.g. background ion concentration). The core technological advancement we propose to exploit here in order to meet these requirements is our recently demonstrated optically resonant nanotweezers. The advantage of optical confinement techniques, like optical tweezers, in single molecule analysis is that they can suspend and concentrate targets in dynamically changing background solutions. Fundamentally however, existing optical confinement techniques are limited by diffraction which places a lower bound on the size of dielectric target which can be trapped to about 100nm. We demonstrate here that our planar optically resonant nanotweezers allow us to concentrate the optical energy in such a way that this force can be enhanced so as to trap molecules as small as a few nanometers, bringing us down into the range to make single protein measurements possible. In this work we propose to initially develop the system by trapping a series of larger test proteins (6-8nm) building on our previous work in trapping nucleic acids. After initial development we will conduct a series of single molecule trapping-FRET studies on the Hah1-WDP complex examining how binding interactions respond to changes in Cu1+ ion background concentration. PUBLIC HEALTH RELEVANCE: Metal ions, for example iron and copper, are essential nutrients that can also be toxic if their concentration exceeds the physiological limit. Abnormal function of metal transport molecules can lead to diseases such as Wilson disease, Menkes disease and familial amyotrophic lateral sclerosis. In this work we propose to develop a fundamentally new approach to optically based single molecule analysis and apply it to understanding the function of a series of proteins which control intracellular copper transport.

描述（由申请人提供）：在这项工作中，我们提出了康奈尔大学埃里克森和陈实验室之间的一个联合项目，以通过开发单个分子纳米光学光学诱捕和氟氟此类谐振能量转移（FRET）技术来证明一种全新的研究方法，用于研究弱蛋白质蛋白质相互作用的研究。我们将该技术应用于这里的分子系统是从细胞内铜伴侣HAH1到运输ATPase Wilson病蛋白（WDP）的铜运输途径。该运输途径的异常功能会导致诸如威尔逊病和家族性肌萎缩性侧面硬化症等疾病。尽管它很重要，但对于HAH1和WDP相互作用的方式仍非常有限的定量信息。获得此信息的主要困难是缺乏单个分子分析工具，该工具可以同时可以同时进行：（1）在不确定的时间（2）中捕获和悬浮在自由溶液中的小分子（2）有效地“浓缩”感兴趣的分子集合，以从弱蛋白相互作用进行弱的蛋白质相互作用，并且可以进行外部环境环境的调节（3）。我们建议在此处利用以满足这些要求的核心技术进步是我们最近证明的光学共振纳米wee剂。单分子分析中，光学限制技术（如光晶）的优势在于，它们可以在动态变化的背景解决方案中暂停并集中目标。但是，从根本上讲，现有的光学限制技术受到衍射的限制，该衍射将介电靶标的大小放在可将其捕获到约100nm的介电靶标上。我们在这里证明，我们的平面光共振纳米weee剂使我们能够以一种可以增强该力的方式集中光能能量，从而使我们的小分子捕获小至几纳米的分子，从而使我们进入范围，以使单个蛋白质测量可能。在这项工作中，我们建议最初通过捕获一系列较大的测试蛋白（6-8nm）来开发系统，以诱捕核酸捕获核酸。最初开发后，我们将对HAH1-WDP复合物进行一系列单分子诱捕-FRET研究，以研究结合相互作用如何响应Cu1+离子背景浓度的变化。公共卫生相关性：金属离子，例如铁和铜是必不可少的营养素，如果其浓度超过生理极限，也可能是有毒的。金属转运分子的异常功能会导致疾病，例如威尔逊疾病，Menkes疾病和家族性肌萎缩性侧面硬化症。在这项工作中，我们建议开发一种基本的基于光学的单分子分析的新方法，并将其应用于了解控制细胞内铜转运的一系列蛋白质的功能。