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

为了研究复杂的生物系统，生物化学家通常采取一种简单化的实验方法：生物分子被单独提纯并在试管中重组，并测量它们的相互作用。尽管这些集合实验是生化研究的基石，可以揭示许多关于生物分子功能的信息，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性。然而，由于溶液包含数十亿个分子，被测量的过程可能会变得不同步。一个蛋白质群体可以有不同的活性(静态无序)，或者单个蛋白质可以随时间改变其活性(动态无序)。此外，一些物理性质很难操纵，例如作用在生物分子上的力。为了克服这些限制，科学家们可以转向另一套方法，单分子生物物理学。在这些方法中，集合反应被简化为较少数量的相互作用伙伴(例如，单个DNA与一个或多个蛋白质相互作用)和用于操纵和/或观察过程的探针。此次Alert Equipment竞标的目标是将这种方法应用于基本的生物学问题，将尖端的单分子显微镜带到布里斯托尔大学的沃尔夫森生物成像设施(WBF)。我们想要资助的仪器被称为C-TRAP-一种结合了光学镊子和共聚焦荧光显微镜的仪器。光学镊子使用聚焦的激光束来捕获一个小颗粒，通常是1微米的乳胶珠。来自激光的光子具有动量，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，珠子上产生相等和相反的力，从而将粒子困住。因此，移动激光焦点将导致珠子在3D中相应的运动。C-陷阱可以同时产生多达四个这样的陷阱。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，还可以从被困珠子的位移测量对它们的作用力。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。为了观察单个蛋白质与DNA的相互作用，C-TRAP集成了共焦扫描激光，可以激发多达3种颜色的荧光分子。然后，我们可以制作三种不同的蛋白质在一个DNA分子上移动和相互作用的电影--我们把这称为“视觉生物化学”。同步和无序不再是限制，因为个体的反应可以进行比较。C-Trap是一种非常复杂的仪器，可以将测力与准确的荧光定位相关联，只需最少的用户干预。它的相对易用性使其成为像WBF这样的多用户设备的理想工具。为了在WBF中建立这项技术，C-TRAP将首先被一组研究蛋白质与DNA和RNA相互作用的实验室使用。他们将首次能够直接观察：基因是如何表达的(转录)；DNA损伤是如何处理的(DNA修复)；染色体在细胞分裂期间是如何包装的，这种包装如何影响其他蛋白质的获取；RNA产生蛋白质(翻译)；以及像CRISPR-Cas这样的酶在基因编辑过程中如何识别特定的DNA序列。该团队将由研究技术专业人员斯蒂芬·克罗斯协助显微镜操作和数据分析，他是接受过生物物理学培训的WBF的关键成员。他将确保我们从这个工具中获得最大的好处。一旦制定了使用协议，他还将向更广泛的用户群体推广该文书的使用。一个特别重要的目标是通过在跨学科科学方面对我们团队的年轻成员进行培训来增强他们的就业能力。

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.