In Situ Kinetics of Solution-Phase Biomolecular Reactions and Interactions through Single-Molecule Displacement...

Area of Science:

• Biochemistry
• Chemical Kinetics
• Single-Molecule Biophysics

Background:

• Quantifying reaction kinetics of biomolecules in solution is crucial for understanding biological processes.
• Traditional methods often require large sample volumes or lack temporal resolution.
• Developing high-throughput, sensitive techniques is essential for advancing biochemical research.

Purpose of the Study:

• To present a novel framework for quantifying in situ reaction kinetics of bio-(macro)-molecules in solution.
• To enable kinetic analysis at high temporal resolution using single-molecule displacement statistics.
• To demonstrate the applicability of the method to diverse reaction types, including bimolecular reactions and antibody binding.

Main Methods:

• Utilized single-molecule displacement/diffusivity mapping (SMdM) to measure transient displacements of freely diffusing molecules.
• Employed a two-component diffusion model to fit time-dependent displacement distributions and quantify reactant/product fractions.
• Applied the method to fluorescently tagged reactants and unlabeled reactants across various concentrations.

Main Results:

• Successfully quantified second-order reaction rate constants for strain-promoted azide-alkyne cycloaddition and NHS ester aminolysis.
• Determined first-order hydrolysis side-reaction rate constants for NHS ester aminolysis.
• Quantified association and dissociation rate constants for monoclonal antibodies and observed complex formation for polyclonal antibodies.

Conclusions:

• The developed SMdM framework provides an effective means to quantify in situ reaction kinetics of biomolecules in solution.
• The approach offers intuitive, molecular-size-linked readouts and is adaptable to diverse reaction types and conditions.
• This single-molecule displacement-based method advances the study of reaction dynamics in microliter-scale samples.