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

Subject Area Biophysics
Term from 2017 to 2024
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 364654521
 
Final Report Year 2024

Final Report Abstract

Multivalent interactions, i.e., the simultaneous engagement of many weak interactions in parallel to yield a strong overall interaction, are observed in a multitude of biological processes like the attachment of infectious agents to cells or the modulation of cell signaling cascades. Accurate quantification of such interactions is complicated by the fact that the individual interaction is typically weak (affinity in the µM to mM range) and therefore maintained only transiently within the multivalent complex, causing the number of engaged interactions (i.e., the valency) to fluctuate considerably over time and within the ensemble. Hence, an accurate quantification of such interactions calls for methods that enable to probe the valency on the level of single interaction complexes as well as for many interaction complexes in parallel (allowing for a statistically relevant characterization of the ensemble). The overarching goal of this project is to establish two assays based on total internal reflection fluorescence (TIRF) microscopy as tools to accurately quantify interactions that consist of multiple weak interactions. In the first assay, the transient interaction of fluorescently labeled viruses with a receptor-containing supported lipid bilayer (SLB) is recorded under equilibrium conditions using TIRF microscopy, which allows to probe the multivalent interaction of hundreds of viruses in parallel while keeping the single-virus resolution. Using this assay, we demonstrated that probing the motion of single, SLB-linked viruses provides a measure for the virus valency and allows to extract the 3 key parameters of this interaction (the virus attachment rate, the virus valency distribution as well as the valency-dependent off-rate distribution). Furthermore, addition of virus inhibitors induced, as expected, changes in the multivalent virusmembrane interaction, which can be quantified accurately by the TIRF-based binding assay, thereby providing a new means to quantify the efficiency of virus inhibitors based on a direct readout for virus binding. In a second assay, TIRF microscopy was combined with microfluidics, thereby enabling to load weak interactions with hydrodynamic shear forces (hydrodynamic force spectroscopy). Owing to the widefield imaging capabilities of TIRF microscopy, this new modality enabled to accurately quantify thousands of macromolecules/interaction complexes of interest in parallel and achieved outstanding accuracies (combining sub-pN force and subnm spatial resolution). To conclude, two new means to quantify interactions were successfully established in this project, both of which enable to accurately extract key information that were not extractable by complementary approaches so far. The applicability and performance of these assays was demonstrated by investigating the properties of multivalent virus-receptor interaction (and their modification upon application of virus inhibitors), the sequential rupture of multivalent interaction as well as sequential unfolding of proteins upon force application.

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