Force-modulated FRET for resolving biomolecular motion and bonding

该项目将开发一种新的技术，以比目前的方法更精确地测量生物学中大分子相互作用的强度。这种新工具由使用超声波来破裂相互作用和荧光显微镜来检测破裂的独特组合组成。例如，核糖体合成蛋白质的过程将受到无与伦比的详细监测。核糖体是一种复杂的大分子机器，它“读取”从DNA转录的信息，并相应地制造其产品。然而，人们并不完全了解它是如何沿着“信息”阅读说明的，有时还会滑出框架制造另一种产品。这一过程的动态图像，结合我们现在对机器结构的了解，将极大地增强我们对这台机器如何工作以及故障的理解。进一步的应用，以了解蛋白质如何区分细胞表面，以识别不同的目标是设想。研究活动将为研究生、本科生和高中生创造丰富的培训机会。特别是，将鼓励代表人数不足的群体的学生参加。此外，这项研究将通过开发一门专注于现代物理、化学和生物技术的研究生水平的新课程来加强教育。被称为力调制荧光共振能量转移(FmFRET)显微镜的独特成像技术将为核酸提供亚纳米运动分辨率，为非共价键提供亚皮牛顿力分辨率。FmFRET技术将首次将力光谱的概念与FRET检测相结合：通过施加亚皮牛顿分辨率的声辐射力，大分子相互作用将根据其解离力被精确解析并被检测为荧光信号。由于不同长度的核酸双链体可以通过它们的解离力来分解，这一技术将能够以亚核苷酸分辨率精确地区分核酸在功能大分子复合体中的位置，这是任何其他技术都无法实现的。从力谱的角度来看，荧光检测将比磁检测更快、更灵敏、更容易实现。此外，这项新技术将比单纯依赖软件拟合方法的微粒光学成像更精确和更稳健。这项工作由分子生物学(分子和细胞生物科学，BIO)和化学测量和成像(CHE)计划资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The project will develop a new technique to measure the strength of macromolecular interactions in biology with higher precision than current methods. The new tool consists of a unique combination of using ultrasound to rupture the interactions and fluorescence microscopy for detection of the rupture. For example, one process that will be monitored in unparalleled detail is that of protein synthesis by the ribosome. The ribosome is a complex macromolecular machine that "reads" information transcribed from DNA and manufactures its product accordingly. Yet just how it steps along the "message" to read the instructions, and sometime slips out of frame to manufacture an alternate product, are not fully understood. A dynamic picture of the process, combined with what we now know about the structure of the machine, will greatly enhance our understanding how this machine functions, and misfunctions. Further applications to understand how proteins distinguish among cells surfaces to recognize a distinct target are envisioned. The research activities will generate a wealth of training opportunities for graduate students, undergraduate students, and high school students. In particular, students in underrepresented groups will be encouraged to participate. In addition, the research will enhance education via the development of a new course at the graduate level that focuses on modern physical, chemical, and biological techniques.The unique imaging technique, termed as force-modulated fluorescent resonance energy transfer (fmFRET) microscopy, will provide sub-nanometer motion resolution for nucleic acids and sub-piconewton force resolution for non-covalent bonds. The fmFRET technique will integrate the concept of force spectroscopy with FRET detection for the first time: by applying acoustic radiation force with sub-piconewton resolution, the macromolecular interactions will be precisely resolved based on their dissociation forces and detected as fluorescence signal. Because nucleic acid duplexes of different lengths can be resolved by their dissociation forces, this technique will be able to precisely distinguish the positions of nucleic acids in functioning macromolecular complexes with sub-nucleotide resolution, which cannot be achieved by any other techniques. From the point of view of force spectroscopy, fluorescence detection will be faster, more sensitive, and easier to implement than magnetic detection. Furthermore, the new technique will be more precise and robust than optical imaging of microparticles that relies solely on software fitting methods. This work is funded by Molecular Biophysics (Molecular and Cellular Biosciences, BIO) and Chemical Measurement and Imaging (CHE) programs.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.