BRIGE: Fluorescence Based Single Molecule Force Spectroscopy with DNA Nanotechnology

这项扩大参与工程研究启动基金（bridge）为基于荧光的高通量单分子力光谱的纳米级设备的开发提供了资金。该装置将使用最近开发的纳米技术DNA折纸进行组装。DNA折纸技术可以通过程序化的分子自组装，构建出具有空前几何复杂性的纳米级物体。该装置将包括双链DNA束的刚性框架，两个生物分子的附着点，柔性聚合物力探针和荧光分子，作为两个感兴趣的生物分子之间相互作用的读数。力探针将促进生物分子之间的结合，并随后施加已知的力来破坏相互作用。键寿命将监测使用荧光读出，这是适用于高通量数据收集。相互作用寿命将作为力的函数来测量，以确定控制分子相互作用的动力学参数。初步的原理验证实验将探测DNA和RNA碱基配对的相互作用。最终，该设备将被用于研究蛋白质- dna、蛋白质- rna和蛋白质-蛋白质系统。单分子力谱研究为稳定生物分子机制和调节细胞功能的相互作用提供了重要的见解。然而，力谱的广泛应用一直受到笨重昂贵的设备和低通量数据采集的阻碍。如果成功，这项研究将使生物分子机械的经济和高通量力谱研究成为可能。最终目标是开发一种可以包装和分发的设备，广泛应用于基础实验室荧光显微镜。该奖项还将支持建立生物分子机械短期课程，为暑期工程夏令营的高中学生提供，并开发研究研讨会课程，旨在招募代表性不足的工程学生进行研究生水平的研究。

This Broadening Participation Research Initiation Grant in Engineering (BRIGE) provides funding for the development of a nanoscale device for fluorescence-based high throughput single molecule force spectroscopy. The device will be assembled using the recently developed nanotechnology DNA origami. DNA origami enables the construction of nanoscale objects with unprecedented geometric complexity via programmed molecular self-assembly. The device will comprise a stiff framework of bundles of double-stranded DNA, attachment points for two biomolecules, a flexible polymer force probe, and fluorescent molecules to act as a readout of the interaction between the two biomolecules of interest. The force probe will facilitate binding between the biomolecules, and subsequently apply a known force acting to rupture the interaction. The bond lifetime will be monitored using a fluorescence readout, which is amenable to high throughput data collection. Interaction lifetimes will be measured as a function of force to determine kinetic parameters that govern the molecular interaction. Initial proof-of-principle experiments will probe DNA and RNA base-pairing interactions. Ultimately, the device will be implemented to study protein-DNA, protein-RNA, and protein-protein systems.Single molecule force spectroscopy studies have provided critical insight into the interactions that stabilize biomolecular machinery and regulate the function of cells. However, the widespread application of force spectroscopy has been hindered by the need for cumbersome and expensive equipment and low throughput data acquisition. If successful, this research will enable economical and high-throughput force spectroscopy studies of biomolecular machinery. The ultimate goal is to develop a device that can be packaged and distributed for widespread application on basic laboratory fluorescence microscopes. This award will also support the establishment of a biomolecular machinery short course to be offered to high school age students at summer engineering camps and the development of a research seminar course aimed to recruit underrepresented engineering students to pursue graduate level research.