Abstract
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.
Highlights
DNA is the center of action in cells: proteins bind to specific sequences, RNA-polymerases move along and transcribe genes, DNA is modified and wrapped around nucleosomes
Inspired by the work of Noom et al [8], we developed a method that allows label-free high-accuracy detection of proteins bound to DNA by the use of a scanning loop formed by one DNA molecule that is looped around another [9]
Two optical traps were generated by splitting a beam into two orthogonally polarized beams, which could be independently steered in x- and y-direction by acousto-optical deflectors (AODs)
Summary
DNA is the center of action in cells: proteins bind to specific sequences, RNA-polymerases move along and transcribe genes, DNA is modified and wrapped around nucleosomes. An array of single-molecule techniques, which allow for precise control and detection of individual DNA molecules and proteins, have made it possible to determine many of the intrinsic properties of DNA and associated proteins [2,3]. Optical tweezers have been used to monitor the movement of single proteins along DNA in buffer, but rely on labeling of the proteins for optical or mechanical detection [7]. Inspired by the work of Noom et al [8], we developed a method that allows label-free high-accuracy detection of proteins bound to DNA by the use of a scanning loop formed by one DNA molecule that is looped around another [9]. Upon encountering a bound protein, the sliding loop will be halted and the magnetic bead will be displaced, thereby indicating the position of the protein (Fig. 1C)
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