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bioRxiv · 10.1101/2023.12.08.570798

Kinetics and dynamics of single-molecule multivalent interactions revealed by plasmon-enhanced fluorescence

Okholm, K. R.·Nooteboom, S. W.·Lamberti, V.·Dey, S.·Zijlstra, P.·Sutherland, D. S.
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Abstract

Multivalency as an interaction principle is widely utilized in nature. It enables specific and strong binding by multiple weak interactions through enhanced avidity and is a core process in immune recognition and cellular signaling and a current concept in drug design. Rapid binding and unbinding of monovalent constituent interactions during multivalent binding creates dynamics that require a single-molecule approach to be studied. Here, we use the high signals from plasmon enhanced fluorescence of nanoparticles to extract binding kinetics and dynamics of multivalent interactions on the single-molecule level and in real-time. We study mono-, bi-and trivalent binding interactions using a DNA Holliday Junction as a model construct with programmable valency. Furthermore, we introduce a model framework for binding kinetics that involves the binding restriction during multivalent interactions to take into account the structural conformation of multivalent molecules allowing quantitative comparison. We used this approach to explore how length and flexibility of the DNA ligands affect binding restriction and binding strength, where overall binding strength decreased with spacer length. For trivalent systems increasing spacer length was found to activate binding in the trivalent state giving insight into the design of multivalent drug or targeting moieties. Interestingly we could exploit the rapidly decaying near fields of the plasmon that induce a strong dependence of the signal to position of the fluorophore to observe binding dynamics during single multivalent binding events.

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Okholm, K. R., Nooteboom, S. W., Lamberti, V., Dey, S., Zijlstra, P., Sutherland, D. S.. 2023-12-08. Kinetics and dynamics of single-molecule multivalent interactions revealed by plasmon-enhanced fluorescence. https://doi.org/10.1101/2023.12.08.570798

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