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Probing a label-free local bend in DNA by single-molecule Tethered Particle Motion

Abstract : Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of 1 to 7 6A-tracts, we find that the sequence CA6CGG induces a bend angle of 19 [4] {\deg}. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well.
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Submitted on : Monday, March 22, 2021 - 4:18:27 PM
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Annaël Brunet, Sébastien Chevalier, Nicolas Destainville, Manoel Manghi, Philippe Rousseau, et al.. Probing a label-free local bend in DNA by single-molecule Tethered Particle Motion. Nucleic Acids Research, Oxford University Press, 2015, 43 (11), pp.e72. ⟨10.1093/nar/gkv201⟩. ⟨hal-01174058⟩



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