Weak interactions between biomolecules play important roles in many cellular functions. Structural and kinetic analyses of these interactions, however, have been hindered by the transient nature of such events. Here, we pointed out a general approach to overcome this obstacle─anchoring the molecular partners to streptavidin hosts─and achieved constrained proximity and stoichiometry for the sought-after molecular coupling. We elaborated this idea through a series of DNA hybridization reactions and quantitatively characterized them using single-molecule experiments. Compared to a nominally 1 μM solution, for example, the streptavidin-induced proximity (SIP) amounted to an effective molarity of ∼10-30 μM for the binding partners. There was also a significantly increased proportion of molecular association, manifested in both ensemble population and single-molecule residence time. As an application example, we showed how SIP enabled the observation and quantitative characterization of an unstable complex between Cas9-RNA and noncognate DNA substrates, interactions that had been challenging to characterize previously. Conceptually simple and implementationally robust, SIP was shown to considerably enhance the efficacy in capturing weak interactions and, as demonstrated here, could empower scientists to see the otherwise unseeable.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry