Microscale thermophoresis for assessing diverse molecular interactions.

用于评估不同分子相互作用的微尺度热泳。

• 批准号: RTI-2020-00485
• 负责人: Stathopulos, Peter
• 金额: $ 10.93万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=693789
• 关键词: Microscale; thermophoresis; assessing; diverse; molecular

Molecular interactions underlie the life and death signals of all cells. There is a large diversity in the nature of the interacting molecules controlling these processes. For example, binding of single ions can radically alter the conformation and function of small proteins. In contrast, translation machinery is made up of dozens of ribosomal proteins, nucleic acids and ions, forming an enormous 4.3 MDa complex in higher eukaryotes. There is also a wide variability in the strengths of these interactions, which typically range from ~nM to mM affinity in nature. ******Measuring molecular interaction affinities provides insights on the energies, relative probabilities and lifetimes, central to developing new research tools and drugs as well as bioprocess optimization in industry. Numerous approaches and specialized equipment have been engineered to characterize molecular interactions such as isothermal titration calorimetry (ITC), surface plasmon resonance spectroscopy (SPR), analytical ultracentrifugation (AUC) and nuclear magnetic resonance (NMR) spectroscopy. However, these techniques have clear shortcomings when considering the diverse affinities and interaction types in nature. ITC and AUC require pure samples at relatively high concentrations; SPR demands sample immobilization; NMR uses large amounts of sample. Additionally, these approaches are technically demanding, limiting their use by researchers and trainees with varied expertise. Finally, many of these approaches are partial in the strengths of the interactions detected, typically measuring sub-mM affinities. ******This multi-applicant research proposal aims to acquire funds to purchase a microscale thermophoresis (MST) unit, which can overcome these limitations and be used to meet the goals of several NSERC-funded research programs at Western. Remarkably, MST can measure affinities ranging from ~nM mM, assess interactions in complex heterogeneous mixtures and detect interactions ranging from small ions to enormous complexes. Further, the system requires only miniscule volumes (~4 microL) and sample amounts (~pmol), is rapid (~minutes) and is technically straightforward, making it widely accessible to multiple disciplines. Currently, there are no MST units available at Western, highlighting an urgent need to enhance our capabilities in assessing diverse molecular interactions. ******The proposed MST unit will immediately benefit the 6 NSERC-funded research programs of the applicants. Nevertheless, the instrument will also be available for use by individuals in the Faculty of Science, Engineering and Schulich School of Medicine and Dentistry. Ultimately, trainees from diverse fields will be exposed to this modern instrumentation that is simple to use and provides essential data for understanding the respective systems under study. Thus, the acquisition of an MST unit will provide both tangible research and training benefits with applicability in industry, academia and health-related fields.

分子间的相互作用是所有细胞的生与死信号的基础。控制这些过程的相互作用分子的性质存在很大的多样性。例如，单个离子的结合可以从根本上改变小蛋白质的构象和功能。相比之下，翻译机器是由几十个核糖体蛋白质，核酸和离子，形成一个巨大的4.3 MDa复杂的高等真核生物。这些相互作用的强度也有很大的变化，自然界中的亲和力通常在~nM至mM的范围内。** 测量分子相互作用亲和力提供了对能量，相对概率和寿命的见解，这对于开发新的研究工具和药物以及工业中的生物过程优化至关重要。已经设计了许多方法和专用设备来表征分子相互作用，例如等温滴定量热法（ITC）、表面等离子体共振光谱（SPR）、分析超离心（AUC）和核磁共振（NMR）光谱。然而，这些技术有明显的缺点时，考虑到不同的亲和力和相互作用类型的性质。ITC和AUC需要相对高浓度的纯样品; SPR需要样品固定; NMR使用大量样品。此外，这些方法在技术上要求很高，限制了具有各种专门知识的研究人员和受训人员的使用。最后，这些方法中的许多在检测到的相互作用的强度方面是部分的，通常测量亚mM亲和力。** 这个多申请人的研究提案旨在获得资金购买一个微型热泳（MST）单位，它可以克服这些限制，并用于满足几个NSERC资助的研究计划在西方的目标。值得注意的是，MST可以测量范围从~nM mM的亲和力，评估复杂的异质混合物中的相互作用，并检测从小离子到巨大复合物的相互作用。此外，该系统只需要极小的体积（~4微升）和样品量（~pmol），快速（~分钟），技术简单，使其广泛适用于多个学科。目前，在西方没有MST单位，突出了迫切需要提高我们评估不同分子相互作用的能力。** 拟议的MST单位将立即有利于申请人的6个NSERC资助的研究项目。尽管如此，该仪器也将可供科学，工程和医学和牙科Schulich学院的个人使用。最终，来自不同领域的学员将接触到这种现代化的仪器，它使用简单，并为理解所研究的各个系统提供必要的数据。因此，购买MST单位将提供切实的研究和培训效益，适用于工业、学术界和卫生相关领域。