Single-Molecule Force Spectroscopy: Probing Biomolecular Interactions at the Nanoscale

Single-molecule force spectroscopy (SMFS) is a powerful technique that enables the study of biomolecular interactions and mechanical properties at the single-molecule level. By applying and measuring forces on individual molecules, SMFS provides unique insights into the strength, kinetics, and conformational changes of biomolecular systems, such as proteins, nucleic acids, and their complexes.

Several techniques have been developed to perform SMFS experiments, each with its own advantages and limitations:

Atomic Force Microscopy-based SMFS is widely used due to its high force sensitivity and spatial resolution. In this technique, a biomolecule is attached between a surface and an AFM cantilever. As the cantilever is retracted, the force acting on the molecule is measured by monitoring the deflection of the cantilever. AFM-based SMFS has been extensively used to study protein unfolding, receptor-ligand interactions, and the mechanical properties of nucleic acids.

Optical tweezers use focused laser beams to trap and manipulate microscopic objects, such as beads attached to biomolecules. By moving the trapped bead relative to a fixed surface, forces can be applied to the tethered molecule. Optical tweezers offer high force and spatial resolution, making them suitable for studying the mechanical properties of nucleic acids, motor proteins, and protein-protein interactions.

Magnetic tweezers employ magnetic fields to apply forces on paramagnetic beads attached to biomolecules. The force is controlled by varying the distance between the bead and a permanent magnet or electromagnet. Magnetic tweezers are particularly useful for studying DNA-protein interactions, DNA supercoiling, and the torsional properties of biomolecules.

SMFS has found numerous applications in the study of biomolecular systems:

SMFS has been instrumental in elucidating the folding and unfolding pathways of proteins. By applying forces to individual protein molecules, researchers can study the stability, kinetics, and intermediates involved in the folding process. SMFS has revealed the existence of multiple unfolding pathways, the role of mechanical stability in protein function, and the effects of mutations on protein folding.

SMFS allows the direct measurement of the strength and kinetics of receptor-ligand interactions at the single-molecule level. By attaching a receptor to a surface and a ligand to a force probe, the unbinding force and dissociation rate of the complex can be determined. This information is crucial for understanding the specificity and affinity of biomolecular recognition processes, such as antibody-antigen interactions and cell adhesion.

SMFS has been extensively used to study the mechanical properties of DNA and RNA. By stretching individual nucleic acid molecules, researchers can investigate their elasticity, persistence length, and the formation of secondary structures, such as hairpins and G-quadruplexes. SMFS has also shed light on the interactions between nucleic acids and proteins, including DNA-binding proteins and enzymes involved in DNA replication and repair.

SMFS has emerged as a powerful tool in the field of mechanobiology, which studies the role of mechanical forces in biological processes. By applying forces to cells and their components, researchers can investigate how cells sense and respond to mechanical stimuli. SMFS has been used to study the mechanics of cell adhesion molecules, the unfolding of mechanosensitive proteins, and the force-induced activation of signaling pathways.

Despite the remarkable advances in SMFS, several challenges remain to be addressed. One of the main challenges is the complex and heterogeneous nature of biological samples, which can lead to variability in the obtained force measurements. Improving sample preparation techniques and developing standardized protocols are crucial for enhancing the reproducibility and comparability of SMFS experiments.

Future directions in SMFS will focus on integrating SMFS with other techniques, such as fluorescence microscopy and mass spectrometry, to obtain complementary information on the structure and dynamics of biomolecules. The development of high-throughput SMFS methods will enable the screening of large numbers of molecules and the identification of rare events. Additionally, the application of SMFS to study the mechanics of more complex biological systems, such as membrane proteins and cellular organelles, will provide new insights into the role of mechanical forces in cellular processes.

ACS Omega, Force Spectroscopy on Single Molecules of Life

Nature Reviews Physics, Single-Molecule Force Spectroscopy: A Tool for Basic and Applied Science

Methods In Molecular Biology, Single-Molecule Force Spectroscopy: Experiments, Analysis, and Simulations