Visual biochemistry of protein-nucleic acid interactions using a multi-user single-molecule optical trapping fluorescence microscope.

使用多用户单分子光学捕获荧光显微镜观察蛋白质-核酸相互作用的视觉生物化学。

基本信息

批准号：
BB/W019337/1
负责人：
Mark Dominik Szczelkun
金额：
$ 82.59万
依托单位：
University of Bristol
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2022
资助国家：
英国
起止时间：
2022 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FW019337%2F1
关键词：
Visual biochemistry protein nucleic acid

项目摘要

To study complex biological systems, biochemists often take a reductionist experimental approach: biomolecules are individually purified and recombined "in a test tube" and their interactions measured. Although these ensemble experiments are the cornerstone of biochemical study and can reveal much about biomolecular function, they are often hard to interpret. The mathematical rules used to quantify the interactions rely on synchronisation between molecules assumed to have identical properties. However, because the solutions contain billions of molecules, the processes being measured can become desynchronised. A protein population can have differences in activity (static disorder) or individual proteins may alter their activity with time (dynamic disorder). Additionally, some physical properties are hard to manipulate, such as forces acting on a biomolecule. To overcome these limitations, scientists can turn to another set of approaches, single-molecule biophysics. In these methods, the ensemble reaction is reduced to a smaller number of interacting partners (e.g., a single DNA interacting with one or multiple proteins) and a probe used to manipulate and/or observe the process. The goal of this Alert equipment bid is to apply such approaches to fundamental biological problems by bringing a cutting-edge single-molecule microscope to the Wolfson Bioimaging Facility (WBF) at the University of Bristol.The instrument we want to fund is called a C-trap - a combined optical tweezers and confocal fluorescence microscope. An optical tweezers uses a focussed laser beam to trap a small particle, typically an ~1 micron latex bead. Photons from the laser have momentum, and refraction or reflection caused by the bead changes their path hence changing their momentum. By conservation of energy, equal and opposite forces are produced on the bead, trapping the particle. Accordingly, moving the laser focus will result in corresponding motion of the bead in 3D. The C-trap can produce up to four of these traps simultaneously. By attaching biomolecules to the beads, we can not only move them at will, we can also measure forces acting on them from the displacement of the trapped bead. For example, a molecule of DNA can be tethered between two beads and stretched into different conformations. To observe single proteins interacting with the DNA, the C-trap integrates confocal scanning lasers that can excite fluorescent molecules using up to 3 colours. We can then create movies of 3 different proteins moving and interacting on a single DNA molecule - we term this "visual biochemistry". Synchronisation and disorder are no longer limitations as the individual reactions can be compared. The C-trap is a very sophisticated instrument for correlating force measurements with accurate fluorescent positioning, with minimal user-intervention. Its relative ease-of use makes it an ideal instrument for a multi-user facility such as the WBF.To establish the technique in the WBF, the C-trap will be first used by a group of labs who study protein interactions with DNA and RNA. For the first time they will be able to directly observe: how genes are expressed (transcription); how DNA damage is dealt with (DNA repair); how chromosomes are packaged during cell division and how that packaging influences access by other proteins; the production of proteins from RNA (translation); and how enzymes like CRISPR-Cas recognise specific DNA sequences during gene editing. The team will be assisted in microscope operation and in the analysis of data by a research technical professional, Stephen Cross, a key member of the WBF with biophysics training. He will ensure that we get the most benefit from the instrument. He will also promote the use of the instrument to a wider group of users once protocols for use are established. A particularly important goal is to bolster the employability of the younger members of our teams by training them in interdisciplinary science.

为了研究复杂的生物系统，生物化学家经常采用还原论实验方法：将生物分子单独纯化并“在试管中”重组，并测量它们的相互作用。尽管这些整体实验是生化研究的基石，并且可以揭示许多有关生物分子功能的信息，但它们通常难以解释。用于量化相互作用的数学规则依赖于假设具有相同性质的分子之间的同步。然而，由于溶液含有数十亿个分子，被测量的过程可能会变得不同步。蛋白质群体的活性可能存在差异（静态紊乱），或者单个蛋白质可能会随时间改变其活性（动态紊乱）。此外，一些物理特性难以操纵，例如作用在生物分子上的力。为了克服这些限制，科学家们可以转向另一组方法，即单分子生物物理学。在这些方法中，整体反应被简化为较少数量的相互作用伙伴（例如，单个DNA与一种或多种蛋白质相互作用）和用于操纵和/或观察该过程的探针。此次警报设备招标的目标是通过为布里斯托大学的沃尔夫森生物成像设施 (WBF) 带来尖端的单分子显微镜，将此类方法应用于基本的生物学问题。我们想要资助的仪器称为 C -trap - 组合光学镊子和共焦荧光显微镜。光镊使用聚焦激光束捕获小颗粒，通常是~1微米的乳胶珠。来自激光的光子具有动量，珠子引起的折射或反射会改变它们的路径，从而改变它们的动量。通过能量守恒，珠子上会产生相等且相反的力，从而捕获颗粒。因此，移动激光焦点将导致珠子在 3D 中进行相应的运动。 C 型陷阱最多可以同时产生四个这样的陷阱。通过将生物分子附着在珠子上，我们不仅可以随意移动它们，还可以通过捕获珠子的位移来测量作用在它们上的力。例如，DNA 分子可以被束缚在两个珠子之间并拉伸成不同的构象。为了观察单个蛋白质与 DNA 的相互作用，C-trap 集成了共焦扫描激光器，可以使用多达 3 种颜色激发荧光分子。然后，我们可以制作 3 种不同蛋白质在单个 DNA 分子上移动和相互作用的电影 - 我们称之为“视觉生物化学”。同步和无序不再是限制，因为可以比较各个反应。 C-trap 是一种非常复杂的仪器，可将力测量与精确的荧光定位相关联，并且只需极少的用户干预。它的相对易用性使其成为 WBF 等多用户设施的理想仪器。为了在 WBF 中建立该技术，C-trap 将首先由一组研究蛋白质与 DNA 相互作用的实验室使用，核糖核酸。他们将第一次能够直接观察：基因如何表达（转录）；如何处理 DNA 损伤（DNA 修复）；染色体在细胞分裂过程中如何包装以及该包装如何影响其他蛋白质的访问；从 RNA 生产蛋白质（翻译）；以及 CRISPR-Cas 等酶如何在基因编辑过程中识别特定的 DNA 序列。研究技术专家 Stephen Cross 将协助该团队进行显微镜操作和数据分析，Stephen Cross 是 WBF 的关键成员，接受过生物物理学培训。他将确保我们从该工具中获得最大利益。一旦建立使用协议，他还将向更广泛的用户群体推广该仪器的使用。一个特别重要的目标是通过跨学科科学培训来提高我们团队中年轻成员的就业能力。