Probing a label-free local bend in DNA by single molecule tethered particle motion

Nucleic Acids Research
Annaël BrunetCatherine Tardin

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 one to seven 6A-tracts, we find that the sequence CA6CGG induces a bend angle of 19° ± 4°. 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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Citations

Sep 10, 2015·Langmuir : the ACS Journal of Surfaces and Colloids·Andrew WittmeierJohn G Gibbs
Jun 19, 2016·Proceedings of the National Academy of Sciences of the United States of America·Florian FournesPhilippe Rousseau
Dec 29, 2016·Journal of Chemical Theory and Computation·Jonathan S MitchellJohn H Maddocks
Jan 3, 2018·Nucleic Acids Research·Annaël BrunetCatherine Tardin
Apr 14, 2020·Nucleic Acids Research·Alberto Marin-GonzalezFernando Moreno-Herrero
Feb 21, 2018·Nanoscale·J Madariaga-MarcosF Moreno-Herrero
Feb 6, 2019·Physical Review Letters·Sébastien GuilbaudCatherine Tardin
Jul 16, 2019·Methods : a Companion to Methods in Enzymology·Manoel ManghiAnnaël Brunet
Jul 29, 2019·Methods : a Companion to Methods in Enzymology·Jean-François AllemandLaurence Salomé
Aug 31, 2021·Analytical Chemistry·Yi WangHui Wang

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Methods Mentioned

BETA
electrophoresis
Nuclear
X-ray
Fluorescence
Atomic Force Microscopy
AFM
PCR
chip
NMR
FRET

Software Mentioned

HT
Nanomultiplex

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