Amplifying Intermolecular Events by Streptavidin-Induced Proximity

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Weak biomolecular interactions are crucial for cellular functions but difficult to study due to their transient nature.
• Existing methods struggle to capture and analyze these fleeting molecular events effectively.

Purpose of the Study:

• To present a generalizable method, streptavidin-induced proximity (SIP), for overcoming the challenges in studying weak biomolecular interactions.
• To demonstrate SIP's capability in enhancing molecular association and enabling quantitative characterization of transient complexes.

Main Methods:

• Anchoring molecular partners to streptavidin hosts to achieve constrained proximity and stoichiometry.
• Utilizing single-molecule experiments to quantitatively characterize DNA hybridization reactions under SIP conditions.
• Applying SIP to study the interaction between Cas9-RNA and noncognate DNA substrates.

Main Results:

• SIP significantly increases the effective molarity of binding partners (∼10–30 μM) compared to solution conditions.
• Enhanced molecular association was observed, reflected in both ensemble and single-molecule measurements (e.g., residence time).
• SIP successfully enabled the observation and characterization of previously challenging unstable complexes, such as Cas9-RNA with noncognate DNA.

Conclusions:

• Streptavidin-induced proximity (SIP) is a conceptually simple and robust approach to enhance the study of weak biomolecular interactions.
• SIP effectively increases the probability and duration of molecular associations, making transient events observable.
• This method provides a powerful tool for quantitative analysis of previously unseeable molecular interactions, advancing biological research.