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Pré-Publication, Document De Travail Année : 2022

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds

Yong Jian Wang
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Claire Valotteau
Adrien Aimard
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Lorenzo Villanueva
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Dorota Kostrz
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Maryne Follenfant
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Terence Strick
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Patrick Chames
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Felix Rico
Charlie Gosse
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Laurent Limozin

Résumé

Single-molecule data are of great significance in biology, chemistry, and medicine. However, experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are needed. Acoustic force spectroscopy (AFS) is an emerging manipulation technique which generates acoustic waves to apply force in parallel on a large population of microbeads tethered to a surface. We have exploited this configuration on a recently developed modular Junctured-DNA (J-DNA) scaffold designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measured its unbinding kinetics under force at the single-bond level. Special effort was made in analyzing the data in order to identify potential pitfalls. We established a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy for measuring the force dependent rupture of a single domain antibody with its antigen. We get a good agreement with standard measurement at zero force. Our technique offers single molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.
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Dates et versions

hal-03861138 , version 1 (19-11-2022)

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Yong Jian Wang, Claire Valotteau, Adrien Aimard, Lorenzo Villanueva, Dorota Kostrz, et al.. Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds. 2022. ⟨hal-03861138⟩
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