How Do We Know when Single-Molecule Force Spectroscopy Really Tests Single Bonds?

Abstract

Single-molecule force spectroscopy makes it possible to measure the mechanical strength of single noncovalent receptor-ligand-type bonds. A major challenge in this technique is to ensure that measurements reflect bonds between single biomolecules because the molecules cannot be directly observed. This perspective evaluates different methodologies for identifying and reducing the contribution of multiple molecule interactions to single-molecule measurements to help the reader design experiments or assess publications in the single-molecule force spectroscopy field. We apply our analysis to the large body of literature that purports to measure the strength of single bonds between biotin and streptavidin as a demonstration that measurements are only reproducible when the most reliable methods for ensuring single molecules are used.

TCR–pMHC kinetics under force in a cell-free system show no intrinsic catch bond, but a minimal encounter duration before binding

“…Measurement of the number of bonds observed at different clamp forces relative to the number of contact events is therefore needed to differentiate the 2 cases, as is done with the laminar flow chamber. Second, to demonstrate single bond measurements, BFP and atomic force microscopy rely on ensuring that there is only a low proportion of binding events (classically less than 10%) relative to the total number of cell-surface or surface-surface contacts during the experiment: the proportion of double binding events is then the square of the proportion of single events, i.e., less than 1% (42). While this argument does indicate that the minimal observable binding event predominates under these conditions, it does not prove that this event corresponds to a single molecular bond: the minimal observable binding event could comprise multiple molecular interactions.…”

“…While this argument does indicate that the minimal observable binding event predominates under these conditions, it does not prove that this event corresponds to a single molecular bond: the minimal observable binding event could comprise multiple molecular interactions. The fact that a single TCR-pMHC interaction is measurable by the method is indeed an assumption in studies using low adhesion probabilities to demonstrate single molecular binding (42). Therefore, we believe that the laminar flow chamber uses currently the most stringent criteria to demonstrate measurement of single molecular bonds.…”

Nanobody-antigen catch-bond reveals NK cell mechanosensitivity

“…The Laminar Flow Chamber is a method of choice for rapid measurement of both association and dissociation kinetics of ligand-receptor bonds tethered at surfaces. The criteria of single bond assessment is very stringent, whereas alternative single bond techniques like AFM often rely only on a maximum of 10% of binding events observed (33). Applied flow limits the encounter duration between receptor on the microsphere and ligand on the underlying surface to the millisecond range.…”

Force Spectroscopy and Beyond: Innovations and Opportunities

“…Moreover, specific linkages can be made distinguishable by embedding precalibrated, well-characterized single-molecule behaviors as markers. With such a confirmation signature, one can identify single molecules with high fidelity ( 10 , 11 , 12 , 13 ). There also is a perpetual need for improving anchors.…”