Imaging unlabeled proteins on DNA with super-resolution.

Fluorescence microscopy is invaluable to a range of biomolecular analysis approaches. The required labeling of proteins of interest, however, can be challenging and potentially perturb biomolecular functionality as well as cause imaging artefacts and photo bleaching issues. Here, we introduce inverse (super-resolution) imaging of unlabeled proteins bound to DNA.

Sliding sleeves of XRCC4-XLF bridge DNA and connect fragments of broken DNA.

Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. Such breaks are formed, for example, during gene-segment rearrangements in the adaptive immune system or by cancer therapeutic agents. Although the core components of the NHEJ machinery are known, it has remained difficult to assess the specific roles of these components and the dynamics of bringing and holding the fragments of broken DNA together.

Tuning the Music: Acoustic Force Spectroscopy (AFS) 2.0.

AFS is a recently introduced high-throughput single-molecule technique that allows studying structural and mechanochemical properties of many biomolecules in parallel. To further improve the method, we developed a modelling tool to optimize the layer thicknesses, and a calibration method to experimentally validate the modelled force profiles. After optimization, we are able to apply 350pN on 4.

Optical Pushing: A Tool for Parallelized Biomolecule Manipulation.

The ability to measure and manipulate single molecules has greatly advanced the field of biophysics. Yet, the addition of more single-molecule tools that enable one to measure in a parallel fashion is important to diversify the questions that can be addressed. Here we present optical pushing (OP), a single-molecule technique that is used to exert forces on many individual biomolecules tethered to microspheres using a single collimated laser beam.

Acoustic force spectroscopy.

Force spectroscopy has become an indispensable tool to unravel the structural and mechanochemical properties of biomolecules. Here we extend the force spectroscopy toolbox with an acoustic manipulation device that can exert forces from subpiconewtons to hundreds of piconewtons on thousands of biomolecules in parallel, with submillisecond response time and inherent stability. This method can be readily integrated in lab-on-a-chip devices, allowing for cost-effective and massively parallel applications.

Effect of temperature on the intrinsic flexibility of DNA and its interaction with architectural proteins.

The helical structure of double-stranded DNA is destabilized by increasing temperature. Above a critical temperature (the melting temperature), the two strands in duplex DNA become fully separated. Below this temperature, the structural effects are localized.

Mobility analysis of super-resolved proteins on optically stretched DNA: comparing imaging techniques and parameters.

Fluorescence microscopy in conjunction with optical tweezers is well suited to the study of protein mobility on DNA. Here, we evaluate the benefits and drawbacks of super-resolution and conventional imaging techniques for the analysis of one-dimensional (1D) protein diffusion as commonly observed for DNA-binding proteins. In particular, we demonstrate the visualization of DNA-bound proteins using wide-field, confocal, and stimulated emission depletion (STED) microscopy.

STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA.

Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication and repair. We present a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in the presence of high protein concentrations. Our approach combines optical tweezers with multicolor confocal and stimulated emission depletion (STED) fluorescence microscopy.