Single molecule high-throughput footprinting of small and large DNA ligands

Maria Manosas,Vincent Croquette,Joan Camunas-Soler,Felix Ritort

doi:10.1038/s41467-017-00379-w

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.

Highlights

To cite this version: Maria Manosas, Joan Camunas-Soler, Vincent Croquette, Felix Ritort
Since ligands that bind to DNA locally stabilize the double helix structure, their binding along the DNA hairpin can be detected as kinetic roadblocks observed during the unzipping reaction
We present a method to determine the selectivity of small and large ligands binding to nucleic acids with near one bp resolution, based on the mechanical unzipping of DNA hairpins using a parallelized magnetic tweezers set-up

Summary

Introduction

To cite this version: Maria Manosas, Joan Camunas-Soler, Vincent Croquette, Felix Ritort. HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. 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. Many natural and synthetic drugs used in chemotherapies bind DNA, changing its structure and interfering with such processes These drugs are mostly small ligands that target short DNA sequences (typically 2–5 bp) via minor or major groove binding, intercalation and/or alkylating activity[1,2,3]. Determining the selectivity and affinity of ligands (both small drugs and large proteins) to different DNA sequences is key to understand fundamental biological processes and for its potential applications in drug development[4, 5]

Methods

Results

Conclusion