Examining Biomolecular Interactions using Microscale Thermophoresis

作为多伦多大学10名结构生物学家团队的一员（跨越多个系和学院），我们使用原子力显微镜、核磁共振光谱、X射线晶体学和电子低温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，我们要求NSERC RTI资金用于购买一种创新仪器，Monolith NT，该仪器使用微尺度热泳来测量荧光（或内在Trp荧光）配体和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后迁移率的变化，以了解两个分子之间的亲和力。该方法检测分子的水化壳、电荷或大小的变化，从而允许在接近天然的条件下测量广泛的生物分子相互作用：无标记和无固定化，以及在任何缓冲液或复杂的生物液体中。该方法还需要非常低浓度的蛋白质（nM）和每个样品小于4 μl的低样品消耗，并且可以测量亚nM至mM之间的高动态范围的解离常数。目前，该仪器将是加拿大的第一台此类仪器，也是北美为数不多的仪器之一（截至2013年10月15日）。这10名研究人员中的每一位都有一个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，而且还将提高他们的生产力。此外，该仪器非常符合该团队的目标，该目标是为即将到来的比赛开发的生物分子相互作用的NSERC-CREATE资助。

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.