Hundahl, A. C. | Search bioRxiv

bioRxiv2025

In-solution Characterization of Biomolecular Interaction Kinetics under Native Conditions

Characterizing the kinetics of biomolecular interactions is fundamental for understanding biological mechanisms, devel-oping novel drugs for advancing healthcare and for optimizing processes in protein engineering. Although modern sur-face-based methods have advanced our understanding of protein-protein and protein-ligand kinetics, they rely on immobilized samples, preventing the study of interactions under native conditions and leading to an incomplete understanding. In this work, we propose a paradigm shift by introducing a new method based on flow-induced dispersion analysis to study interaction kinetics while keeping biomolecules in solution, eliminating the need for surface immobilization and thereby preserving molecular mobility and avoiding structural constraints. The method examines reactions outside equilibrium conditions by inducing a rapid concentration change in one of the binding partners (C-Jump) in a controlled microfluidic environment. Notably, it operates without buffer restrictions and requires only minimal sample quantities. We demonstrate C-Jumps capability by accurately determining the association and dissociation rates of both protein-protein and protein-small molecule interactions. Furthermore, we validate its robustness by measuring the rates of a protein-protein interaction in human serum as well as a protein-small molecule interaction in-solution and label free. This under-lines C-Jumps broad applicability for studying biomolecular interactions under native conditions, offering a powerful tool for advancing protein engineering and drug discovery, as well as enabling the characterization of previously inaccessible interactions. TABLE OF CONTENTThis work presents C-Jump, a breakthrough method for the direct measurement of kinetic rates in solution, closely mimicking physiological conditions. Utilizing flow-induced dispersion analysis, C-Jump determines rate constants with high accuracy for protein-protein and protein- small molecule interactions. Free from buffer constraints and requiring only nanograms of protein, it can operate label-free, transforming biomolecular interaction studies across biophysics, chemistry, and medicine. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/645748v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@11385ceorg.highwire.dtl.DTLVardef@b5d88forg.highwire.dtl.DTLVardef@9cc2cforg.highwire.dtl.DTLVardef@1b9d17c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Continuous Titration Based Method For Rapid In-solution Analysis Of Non-covalent Interactions

Development of new drugs typically involves the identification and validation of molecular inhibitors or promotors of endogenous biological processes. The identification of ligands that can bind the target of interest is typically achieved by screening large libraries of small molecules, using analytical methods that only provide yes/no answers. These methods are only qualitative and often associated with unacceptable amounts of false positives and negatives. Quantitative methods are in general more accurate but time intensive. This is mainly due to repeating measurements of a dilution series in order to generate a titration curve and measure the dissociation constant (Kd). In this work, we introduce Continuous Titration Based Spectral Related Intensity Change (cSPRING), that combines Taylor dispersion analysis (TDA) with ratiometric fluorescence detection to measure a Kd in a single experiment. cSPRING is an in-solution method that reduces the sample preparation time 8-fold and requires only nanograms of protein. We show a good agreement of cSPRING with other quantitative methods for three well-known protein-small molecule interactions with binding affinities ranging from the low nanomolar to high micromolar. In addition, we show that cSPRING is able to measure binding affinities in under a minute, highlighting its efficiency and potential for screening applications.

biophysics↗