In-Solution Characterization of Biomolecular Interaction Kinetics under Native Conditions

Area of Science:

• Biochemistry
• Biophysics
• Chemical Kinetics

Background:

• Accurate characterization of biomolecular interaction kinetics is crucial for drug discovery, protein engineering, and understanding biological mechanisms.
• Current surface-based methods for studying kinetics require sample immobilization, which can alter molecular behavior and limit applicability under native conditions.
• There is a need for methods that can measure interaction kinetics in solution, preserving native molecular states and mobility.

Purpose of the Study:

• To introduce a novel method, flow-induced dispersion analysis (C-Jump), for studying biomolecular interaction kinetics in solution.
• To eliminate the need for surface immobilization, thereby preserving molecular mobility and avoiding structural constraints.
• To demonstrate the method's capability in accurately determining association and dissociation rates for various biomolecular interactions.

Main Methods:

• Utilizing flow-induced dispersion analysis with a rapid concentration change technique (C-Jump) in a microfluidic environment.
• Examining reactions outside equilibrium conditions by inducing a concentration jump in one binding partner.
• Operating without buffer restrictions and requiring minimal sample quantities.

Main Results:

• Accurately determined association and dissociation rates for protein-protein and protein-small molecule interactions.
• Demonstrated robustness by measuring interaction kinetics in human serum and in-solution, label-free.
• Validated C-Jump's broad applicability for studying biomolecular interactions under native conditions.

Conclusions:

• C-Jump offers a paradigm shift in studying biomolecular interaction kinetics by keeping molecules in solution.
• The method provides a powerful tool for advancing protein engineering and drug discovery.
• C-Jump enables the characterization of previously inaccessible biomolecular interactions under native conditions.