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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，生物化学家经常采取还原论的实验方法：生物分子被单独纯化并在“试管”中重组，并测量它们的相互作用。虽然这些集合实验是生物化学研究的基石，可以揭示很多生物分子的功能，但它们往往很难解释。用于量化相互作用的数学规则依赖于假定具有相同性质的分子之间的同步。然而，由于溶液中含有数十亿个分子，被测量的过程可能会变得不稳定。蛋白质群体可以具有活性差异（静态紊乱），或者单个蛋白质可以随时间改变其活性（动态紊乱）。此外，一些物理特性很难操纵，例如作用在生物分子上的力。为了克服这些限制，科学家可以转向另一组方法，即单分子生物物理学。在这些方法中，系综反应被减少到更少数量的相互作用伴侣（例如，与一种或多种蛋白质相互作用的单个DNA）和用于操纵和/或观察该过程的探针。本次Alert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）提供尖端的单分子显微镜，将这种方法应用于基本的生物学问题。我们希望资助的仪器称为C-陷阱--一种结合了光学镊子和共聚焦荧光显微镜的仪器。光镊使用聚焦激光束捕获小颗粒，通常是约1微米的乳胶珠。来自激光的光子具有动量，并且由珠子引起的折射或反射改变了它们的路径，从而改变了它们的动量。根据能量守恒定律，在珠子上产生大小相等、方向相反的力，从而捕获粒子。因此，移动激光焦点将导致珠在3D中的相应运动。C阱可以同时产生多达四个这样的阱。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，还可以测量作用在它们上的力，这些力来自于被困珠子的位移。例如，一个DNA分子可以被拴在两个珠子之间，并被拉伸成不同的构象。为了观察与DNA相互作用的单个蛋白质，C-trap集成了共聚焦扫描激光器，可以使用多达3种颜色激发荧光分子。然后，我们可以创建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，因为个体反应可以进行比较。C-trap是一种非常复杂的仪器，用于将力测量与准确的荧光定位相关联，最大限度地减少用户干预。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-陷阱将首先由一组研究蛋白质与DNA和RNA相互作用的实验室使用。他们将首次能够直接观察：基因如何表达（转录）; DNA损伤如何处理（DNA修复）;染色体在细胞分裂期间如何包装以及包装如何影响其他蛋白质的访问;从RNA产生蛋白质（翻译）;以及CRISPR-Cas等酶如何在基因编辑期间识别特定的DNA序列。该小组将在显微镜操作和数据分析方面得到研究技术专业人员Stephen Cross的协助，Stephen Cross是WBF的重要成员，受过生物物理学培训。他将确保我们从该文书中获得最大利益。一旦确定使用规程，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对我们团队的年轻成员进行跨学科科学培训来提高他们的就业能力。