Single Molecule High-throughput Footprinting of Small and Large DNA Ligands

Overview

Journal Nat Commun

Specialty Biology

Date 2017 Aug 22

PMID 28824174

Citations 14

Authors

Maria Manosas

Joan Camunas-Soler

Vincent Croquette

Felix Ritort

Affiliations

Soon will be listed here.

Abstract

Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a challenge, particularly for small drugs where labeling or sequencing methods do not perform well. Here, we present a fast and accurate method based on parallelized single molecule magnetic tweezers to detect the sequence selectivity and characterize the thermodynamics and kinetics of binding in a single assay. Mechanical manipulation of DNA hairpins with an engineered sequence is used to detect ligand binding as blocking events during DNA unzipping, allowing determination of ligand selectivity both for small drugs and large proteins with nearly base-pair resolution in an unbiased fashion. The assay allows investigation of subtle details such as the effect of flanking sequences or binding cooperativity. Unzipping assays on hairpin substrates with an optimized flat free energy landscape containing all binding motifs allows determination of the ligand mechanical footprint, recognition site, and binding orientation.Mapping the sequence specificity of DNA ligands remains a challenge, particularly for small drugs. Here the authors develop a parallelized single molecule magnetic tweezers approach using engineered DNA hairpins that can detect sequence selectivity, thermodynamics and kinetics of binding for small drugs and large proteins.

Citing Articles

Stacking correlation length in single-stranded DNA.

Viader-Godoy X, Manosas M, Ritort F Nucleic Acids Res. 2024; 52(21):13243-13254.

PMID: 39460618 PMC: 11602145. DOI: 10.1093/nar/gkae934.

Exploring TRF2-Dependent DNA Distortion Through Single-DNA Manipulation Studies.

Zhao X, Vogirala V, Liu M, Zhou Y, Rhodes D, Sandin S Commun Biol. 2024; 7(1):148.

PMID: 38310140 PMC: 10838314. DOI: 10.1038/s42003-024-05838-x.

DNA-binding proteins studied by mechanical manipulation and AFM imaging of single DNA molecules.

Zhao X, Liu X, Yan J Biophys Rep. 2023; 8(4):212-224.

PMID: 37288005 PMC: 10185484. DOI: 10.52601/bpr.2022.220015.

A Multiplexable Plasmonic Hairpin-DNA Sensor Based On Target-specific Tether Dynamics.

van Dongen J, Spoelstra L, Berendsen J, Loessberg-Zahl J, Eijkel J, Segerink L ACS Sens. 2021; 6(12):4297-4303.

PMID: 34851614 PMC: 8715532. DOI: 10.1021/acssensors.1c02097.

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay.

Huang W, Yan J Biophys J. 2021; 120(16):3235-3236.

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