Single-molecule interaction microscopy reveals antibody binding kinetics

Abstract

Single-molecule imaging has provided new insights on weak transient biomolecular interactions with micromolar to millimolar affinity. However, the limited duration of observation has hindered the study of strong and reversible interactions with sub-nanomolar affinity. We report single-molecule interaction microscopy (SMIM), which combines point accumulation for imaging in nanoscale topography (PAINT) with extended imaging durations that enables the study of antibody binding kinetics in the cellular environment. SMIM revealed heterogeneous binding kinetics and the effect of concentration and antibody valency on the association and dissociation rates on antibody-antigen interactions in their cellular environments. We thereby demonstrate SMIM as a versatile single-molecule technique for studying strong, transient biomolecular interactions.

Illuminating amyloid fibrils: Fluorescence-based single-molecule approaches

“…Single-molecule fluorescence methods can be used to detect protein–protein interactions involved in some of the most complex molecular processes that underpin life and disease states. This includes membrane-protein interactions involved in cell-signalling processes [187] , [188] , and DNA-replication machinery [189] . A key example of a cellular process that uniquely benefits from such approaches is the interplay between protein aggregates and molecular chaperones.…”

Technologies for the identification and validation of protein-protein interactions

“…The covalent linker can obstruct the characterization (Chen et al 1994;Fancy 2000) popular methods in the in vivo group are protein-fragment complementation assays (Morell et al 2009), bimolecular fluorescence complementation (Hu et al 2002), Yeast (Fields and Song 1989), Bacterial (Joung et al 2000) and Mammalian (Luo et al 1997) two-hybrid systems, Förster resonance energy transfer (Helms 2008), fluorescence correlation spectroscopy (Elson and Magde 1974), proximity ligation assay (Soderberg et al 2006), rotating cell-based ligand binding assay (Bjorke and Andersson 2006) and single-molecule interaction microscopy (Perera et al 2020), and several methods developed for in vivo PPIs analysis can be used also to study PPIs in vitro.…”

“…SMIM is a method used for the quantitative study of PPIs inside the cell, focusing mostly on antibodies. It is based on the merge of point accumulation for imaging in the nanoscale topography (PAINT) method with extended imaging duration, enabled by inserting non-illuminating intervals between image frames (Perera et al 2020). PAINT is a process that uses fast and transient dyes to capture several fluorescence points at once.…”