Microfluidic setup for quantification of multivalent interactions and massive parallelization of single-molecule force spectroscopy: Unraveling virus-receptor interactions

用于量化多价相互作用和单分子力谱大规模并行化的微流体装置：揭示病毒-受体相互作用

• 批准号: 364654521
• 负责人: Dr. Stephan Block
• 金额: --
• 依托单位: Abteilung Organische Chemie
• 依托单位国家: 德国
• 项目类别: Independent Junior Research Groups
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/364654521?language=en
• 关键词: Microfluidic; setup; quantification; multivalent; interactions

Multivalent interactions are observed in a multitude of biological processes like the attachment of pathogens (e.g., viruses) to cells. A quantitative understanding of such processes requires the quantification of the underlying multivalent interaction on the single-interaction level, which is a yet unresolved issue. To address this challenge, we established in the previous funding period two assays based on microfluidics and total internal reflection fluorescence (TIRF) microscopy, which enabled us to probe weak and multivalent (virus-membrane) interactions with single-molecule resolution and high data throughput. We demonstrated that the mobility of membrane-bound viruses provides a measure for the virus valency (i.e., the number of single interactions engaged within a multivalent virus-membrane interaction), which enabled to determine key binding properties, such as the rate of virus attachment, a qualitative measure for the distribution of the virus valency as well as the valency-dependent rate of virus release from the membrane. Although these investigations yielded new fundamental insights on the properties of virus-membrane interactions, an accurate quantification of virus valency was not yet possible. This limitation is caused by a lack of experimental data on the impact of the valency value on the mobility of particles linked to a supported lipid bilayer (SLB). This aspect of membrane hydrodynamics is theoretically very well established, but was so far not experimentally confirmed. In the final period of this project, we aim to use recently established methods and systems to perform dedicated mobility studies, which will enable to quantitatively determine the valency-mobility relationship of SLB-bound nanoparticles (proteins and viruses). To this end, we will combine microfluidics, TIRF-based single-molecule localization microscopy, and single particle tracking (SPT) to probe the motion of the subunit B of cholera toxin (CTxB), of virus-like particles of the polyomavirus SV40 and of influenza A viruses in absence and presence of an applied shear force. A dual labeling approach, in which receptors as well as nanoparticles are fluorescently labeled, will enable to simultaneously determine the valency (by stepwise bleaching) and mobility (by SPT) of individual SLB-bound nanoparticles. Application of a well-defined hydrodynamic shear force will enable to induce release of individual receptors (engaged in a multivalent interaction) and hence to quantify the off-rate of receptors in dependence of the valency.Taken together, both approaches will provide novel information about hydrodynamics of supported membranes and allow for a quantitative determination of the valency of multivalent interactions. Although applied here mainly to viruses, the presented methodology can, in principle, be extended to any type of multivalent interaction that is formed at interfaces (i.e., to infectious agents in general).

在许多生物过程中观察到多价相互作用，如病原体的附着（例如，病毒）到细胞。定量了解这些过程需要量化的基础上的单相互作用水平的多价相互作用，这是一个尚未解决的问题。为了应对这一挑战，我们在上一个资助期建立了两种基于微流体和全内反射荧光（TIRF）显微镜的检测方法，使我们能够以单分子分辨率和高数据吞吐量探测弱和多价（病毒-膜）相互作用。我们证明了膜结合病毒的移动性提供了病毒效价的量度（即，在多价病毒-膜相互作用中参与的单一相互作用的数量），这使得能够确定关键的结合特性，例如病毒附着速率、病毒价态分布的定性测量以及病毒从膜释放的价态依赖性速率。虽然这些研究对病毒-膜相互作用的性质产生了新的基本见解，但病毒价的准确定量尚不可能。这种限制是由于缺乏实验数据的影响的价值上的粒子连接到支持的脂质双层（SLB）的流动性。膜流体动力学的这一方面在理论上是非常完善的，但到目前为止还没有实验证实。在该项目的最后阶段，我们的目标是使用最近建立的方法和系统进行专门的流动性研究，这将能够定量确定SLB结合纳米颗粒（蛋白质和病毒）的价态-流动性关系。为此，我们将结合联合收割机微流控，TIRF为基础的单分子定位显微镜，和单粒子跟踪（SPT）探测霍乱毒素（CTX B）的亚基B的运动，多瘤病毒SV 40的病毒样颗粒和A型流感病毒在不存在和存在的情况下施加剪切力。双重标记方法，其中受体以及纳米颗粒被荧光标记，将能够同时确定单个SLB结合的纳米颗粒的价态（通过逐步漂白）和迁移率（通过SPT）。应用一个定义明确的流体动力学剪切力将能够诱导释放的个别受体（从事多价相互作用），因此，以定量的解离速率的受体依赖的valency. Together，这两种方法将提供新的信息支持膜的流体动力学，并允许多价相互作用的化合价的定量测定。虽然这里主要应用于病毒，但所提出的方法原则上可以扩展到在界面处形成的任何类型的多价相互作用（即，感染性病原体）。