Enhanced Site-Specific Anchoring of Biomolecules for Single-Molecule Force Spectroscopy

Abstract

Single-molecule force spectroscopy (SMFS) provides unique insights into the mechanical properties and dynamics of biomolecules. In atomic force microscopy (AFM)-based SMFS, non-specific interactions (i.e., passive adsorption) is often used to couple biomolecules to surfaces and AFM tips. However, non-specific interactions lead to artifacts in SMFS data and decrease throughput. Building on prior efforts by other groups, we developed an efficient, hetero-bifunctional coupling of biomolecules via copper-free click chemistry to a PEG-coated substrate and by a strong, but reversible streptavidin-biotin linkage to AFM tips. PEG coating reduces non-specific interactions between the biomolecule and the surfaces (both sample surfaces and AFM tips). Importantly, our protocol substantially reduces the labor involved in functionalizing AFM tips and sample surfaces. Functionalized coverslips can be coated with protein or DNA even weeks after surface modification. Moreover, protein-coated tips and surfaces were reused over multiple days and DNA-coated surfaces were reused over 2 weeks. The use of a streptavidin-biotin bond to attach the biomolecule to the tip facilitates rapid tip-molecule coupling and provides a strong (but reversible) connection between the biotinylated biomolecule and the AFM tip. The reversible bond leaves the biomolecule on the surface and the tip can re-engage the same molecule for repeated unfolding. The overall combination of PEG-coated surfaces, site-specific coupling, and streptavidin-coated tips yielded a dramatic improvement the acquisition rate of high-quality AFM-based SMFS data. In particular, we measured the full dynamic force spectrum of a single, individual polyprotein (NuG2) in just 2 hours and the resulting biophysical parameters agreed with prior literature. We demonstrated the site-specific coupling for stretching individual DNA molecules with both optical traps and AFM. Hence, while this site-specific coupling protocol was developed primarily for AFM-based SMFS, this technique is also applicable to a wide range of surface-coupled, single-molecule assays.