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Transient incomplete separation of species with close diffusivity to study the stability of affinity complexes

Séminaire Chimie IBMM/ED459

Dr. Sergey N. Krylov (Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Canada)

Le Jeudi 06 Octobre 2022 à 16h

CNRS Pôle Chimie Balard Recherche, salle de réunion N3J15, 3e étage (1919 route de Mende)

Date de début : 2022-10-06 16:00:00

Date de fin : 2022-10-06 17:30:00

Lieu : CNRS bâtiment Balard salle N3J15 (3e étage | accès contrôlé)

Intervenant : Dr. Sergey N. Krylov

Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Canada

Large molecules can be generically separated from small ones, though partially and temporarily, in a pressure-driven flow inside a capillary. This transient incomplete separation has been only applied to species with diffusion coefficients different by at least an order of magnitude.[1–5] Here, we demonstrate, for the first time, analytical utility of transient incomplete separation for species with close diffusion coefficients. First, we prove in silico that even a small difference in diffusivity can lead to detectable transient incomplete separation of species. Second, we use computer simulation to prove that such separation can be used for reliable determination of equilibrium dissociation constant (Kd) of complexes composed of similar-size molecules. Finally, we demonstrate experimentally the use of this separation for accurate determination of Kd value for a protein-aptamer complex. We conclude that “accurate constant via transient incomplete separation” (ACTIS) can serve as a reference method for affinity characterization of protein–aptamer binding in solution.


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2. R. Umehara, M. Harada, T. Okada. J. Sep. Sci. 2009, 32, 472–478.

3. N. Sisavath, J.-L. Rukundo. J. C. Y. Le Blanc, V. A. Galievsky, J. Bao, S. Kochmann, A. S. Stasheuski, S. N. Krylov. Angew. Chem. Int. Ed. 2019, 58, 6635−6639.

4. J.-L. Rukundo, J. C. Y. Le Blanc, S. Kochmann, S. N. Krylov. Anal. Chem. 2020, 92, 11973−11980.

5. J.-L. Rukundo, S. Kochmann, T. Y. Wang, N. A. Ivanov, J. C. Y. Le Blanc, B. I. Gorin, S. N. Krylov. Anal. Chem. 2021, 93, 11654–11659.

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