Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers

The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensiti...

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Main Authors:	van Loenhout, Marijn T. J., De Vlaminck, Iwijn, Flebus, Benedetta, den Blanken, Johan F., Zweifel, Ludovit P., Hooning, Koen M., Kerssemakers, Jacob W. J., Dekker, Cees
Format:	Online
Language:	English
Published:	Public Library of Science 2013
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670887/

id	pubmed-3670887
recordtype	oai_dc
spelling	pubmed-36708872013-06-10 Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers van Loenhout, Marijn T. J. De Vlaminck, Iwijn Flebus, Benedetta den Blanken, Johan F. Zweifel, Ludovit P. Hooning, Koen M. Kerssemakers, Jacob W. J. Dekker, Cees Research Article The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ∼17 nm spatial resolution. An offset of 33±5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions. Public Library of Science 2013-06-03 /pmc/articles/PMC3670887/ /pubmed/23755219 http://dx.doi.org/10.1371/journal.pone.0065329 Text en © 2013 van Loenhout et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	van Loenhout, Marijn T. J. De Vlaminck, Iwijn Flebus, Benedetta den Blanken, Johan F. Zweifel, Ludovit P. Hooning, Koen M. Kerssemakers, Jacob W. J. Dekker, Cees
spellingShingle	van Loenhout, Marijn T. J. De Vlaminck, Iwijn Flebus, Benedetta den Blanken, Johan F. Zweifel, Ludovit P. Hooning, Koen M. Kerssemakers, Jacob W. J. Dekker, Cees Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers
author_facet	van Loenhout, Marijn T. J. De Vlaminck, Iwijn Flebus, Benedetta den Blanken, Johan F. Zweifel, Ludovit P. Hooning, Koen M. Kerssemakers, Jacob W. J. Dekker, Cees
author_sort	van Loenhout, Marijn T. J.
title	Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers
title_short	Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers
title_full	Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers
title_fullStr	Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers
title_full_unstemmed	Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers
title_sort	scanning a dna molecule for bound proteins using hybrid magnetic and optical tweezers
description	The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ∼17 nm spatial resolution. An offset of 33±5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.
publisher	Public Library of Science
publishDate	2013
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670887/
_version_	1611983116656705536

Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers

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