Optical single molecule characterisation of natural and synthetic polymers through nanopores.

Nanopore techniques are now widely used to sequence DNA, RNA and even oligopeptide molecules at the base pair level by measuring the ionic current. In order to build a more versatile characterisation system, optical methods for the detection of a single molecule translocating through a nanopore have been developed, achieving very promising results. In this work, we developed a series of tools to interpret the optical signals in terms of the physical behaviour of various types of natural and synthetic polymers, with high throughput. We show that the measurement of the characteristic time of a translocation event gives access to the apparent molecular weight of an object, and allows us to quantify the concentration ratio of two DNA samples of different molecular weights in solution. Using the same tools for smaller synthetic polymers, we were able to obtain information about their molecular weight distribution depending on the synthesis method.

Competition between transcription and loop extrusion modulates promoter and enhancer dynamics.

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the spatiotemporal arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find a close link between chromatin mobility and transcriptional status: active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

Competition between transcription and loop extrusion modulates promoter and enhancer dynamics.

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the 4D arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find that alterations in chromatin mobility, not promoter-enhancer distance, is more informative about transcriptional status. Active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

Experimental study of a nanoscale translocation ratchet.

Despite an extensive theoretical and numerical background, the translocation ratchet mechanism, which is fundamental for the transmembrane transport of biomolecules, has never been experimentally reproduced at the nanoscale. Only the Sec61 and bacterial type IV pilus pores were experimentally shown to exhibit a translocation ratchet mechanism. Here we designed a synthetic translocation ratchet and quantified its efficiency as a nanopump. We measured the translocation frequency of DNA molecules through nanoporous membranes and showed that polycations at the trans side accelerated the translocation in a ratchet-like fashion. We investigated the ratchet efficiency according to geometrical and kinetic parameters and observed the ratchet to be only dependent on the size of the DNA molecule with a power law [Formula: see text]. A threshold length of 3 kbp was observed, below which the ratchet did not operate. We interpreted this threshold in a DNA looping model, which quantitatively explained our results.

Optical Quantification by Nanopores of Viruses, Extracellular Vesicles, and Nanoparticles.

Nanopores combined with optical approaches can be used to detect viral particles. In this work, we demonstrate the ability of hydrodynamical driving and optical sensing to identify and quantify viral particles in a biological sample. We have developed a simple and rapid method which requires only fluorescent labeling of the particles and can therefore be applied to a wide range of virus type. The system operates in real time and at the single particle level while providing a low error on concentration (4%) and a low limit of detection of 105 particles/mL for an acquisition time of 60 s with the ability to increase the acquisition time to achieve a lower limit.

The extruded non-template strand determines the architecture of R-loops.

Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensional conformations characterized by distinct shapes and volumes, which we call R-loop objects. Interestingly, we show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.