Examining Biomolecular Interactions using Microscale Thermophoresis

使用微尺度热泳检查生物分子相互作用

• 批准号: 458470-2014
• 负责人: Moraes, Trevor
• 金额: $ 10.93万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2013
• 资助国家: 加拿大
• 起止时间: 2013-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=541935
• 关键词: Examining; Biomolecular; Interactions; using; Microscale

As part of a team of 10 structural biologists at the University of Toronto (spanning several departments and faculties), we use biophysical tools such as Atomic Force Microscopy, NMR spectroscopy, X- ray crystallography and electron cryomicroscopy to analyze the biomolecular interactions between small molecules (including ions, toxins, ligands, etc), proteins, and nucleic acids. Herein we request NSERC RTI funds for the purchase of an innovative instrument, Monolith NT, that uses microscale thermophoresis to measure the affinities between a fluorescent (or intrinsic Trp Fluorescent) ligand and an analyte. Nanotemper technologies created the Monolith NT detector to monitor the change in migration rate of a fluorescent probe upon interaction with an additional molecule to inform on the affinity between two molecules. This method detects changes in the hydration shell, charge or size of molecules and thus allows measurement of a wide range of biomolecular interactions under close-to-native conditions: Label-Free and immobilization-Free, and in any buffer or complex bioliquid. The method also requires very low concentrations of protein (nM) and low sample consumption of less than 4 µl per sample and can measure a high dynamic range of dissociation constants between sub-nM to mM. Currently this instrument would be the first of its kind in Canada and one of only a handful in North America (as of Oct 15, 2013). Each of the 10 researchers has an NSERC Discovery Grant that primary deals with the interactions between proteins, small molecules, lipids, nucleic acids or ions. This instrument will not only benefit the training their ~74 HQP but will also increase their productivity. Furthermore the instrument fits well with this teams' goals towards an NSERC-CREATE grant on biomolecular interactions that is being developed for the upcoming competition.

作为多伦多大学 10 名结构生物学家（跨多个院系）团队的一员，我们使用原子力显微镜、核磁共振波谱、X 射线晶体学和电子冷冻显微镜等生物物理工具来分析小分子（包括离子、毒素、配体等）、蛋白质和核酸之间的生物分子相互作用。在此，我们请求 NSERC RTI 提供资金购买创新仪器 Monolith NT，该仪器使用微尺度热泳来测量荧光（或内在色氨酸荧光）配体与分析物之间的亲和力。 Nanotemper 技术创建了 Monolith NT 检测器，用于监测荧光探针与其他分子相互作用时迁移速率的变化，从而了解两个分子之间的亲和力。该方法可检测分子的水合壳、电荷或大小的变化，从而可以在接近天然条件下测量各种生物分子相互作用：无标记和无固定化，以及在任何缓冲液或复杂生物液体中。该方法还需要非常低的蛋白质浓度 (nM) 和每个样品少于 4 µl 的低样品消耗，并且可以测量亚 nM 到 mM 之间的高动态解离常数范围。 目前，该仪器将是加拿大首个此类仪器，也是北美仅有的少数仪器之一（截至 2013 年 10 月 15 日）。这 10 名研究人员中的每一位都获得了 NSERC 发现基金，主要研究蛋白质、小分子、脂质、核酸或离子之间的相互作用。该仪器不仅有利于培训他们的 ~74 HQP，而且还可以提高他们的生产力。此外，该仪器非常符合该团队的目标，即获得 NSERC-CREATE 生物分子相互作用资助，该资助正在为即将到来的比赛而开发。