Single molecule approaches of nucleic acids conformational changes One of the primordial questions today in biology is: how does DNA regulate its own metabolism? In other words, what are the mechanisms that enables specific genes to be transcribed while others are silenced? Indeed, apart from storing genetic information, nucleic acids provide regulatory structures to which proteins bind/unbind, creating a versatile regulatory framework. The characterization of this regulation at the molecular level requires a combination of various experimental and theoretical studies. For the last 20 years, this field has greatly benefited from single-molecule approaches that complement conventional biochemical and biophysical techniques. In these approaches, naked nucleic acids or nucleoprotein complexes are investigated one-at-a-time, either through observation with high resolution imaging techniques such as electron and atomic force microscopies or single molecule FRET, indirect monitoring of the conformation with Tethered Particle Motion, or through manipulation with magnetic or optical tweezers. Thanks to these techniques, we have now access to dynamics events that tend to be blurred in traditional biochemical bulk experiments [1]. Also, we have the ability to characterize inhomogenous specimens whereas biochemical techniques generally measure the average behavior of the members of a population. With their growing popularity, more and more labs are now equipped with such apparatus that enable to investigate at the same time structure, dynamics, forces and motions and goes more deeply into the physics of gene expression [2,3]. What structures are formed in nucleic acids during the cell life and what are their kinetics? What sort of deformation the DNA molecule is undergoing upon transcription, replication and recombination? Do the forces exerted on the nucleic acids during these processes range within pN (thermal fluctuation, entropic forces), tens of pN (produced by some powerful molecular motors), hundreds of pN (non-covalent interactions such as van der Waals, hydrogen and ionic bonds) or thousands of pN (covalent bonds)? To address these issues (among others), new single molecule developments are achieved. Thus, fast and high throughput acquisitions are now possible in vitro. The combination of several single molecule techniques gives access to correlative characterization of the nucleoprotein complexes such as structural and mechanical characteristics (rigidity, bending, torsion, stretching) of the nucleic acid. In vivo, new labelling techniques and super resolution imaging methods have made possible to measure the dynamic organization of the DNA within the nucleus. Finally, new modelling of DNA in vitro and in vivo has led to improve the quality of the characteristics extracted from single molecule data and offers new predictive tools for single molecule exploration of DNA and chromosome. This issue provides some example of these new developments. Two main devices have been traditionally used in biology to look at samples at nanometer-scale resolution: electron microscope and atomic force microscope (AFM), but only AFM enables direct imaging of biomo-lecules in their physiological environment. In his contribution [4], Endo describes how the DNA origami technology allows the precise placement of target molecules and the subsequent visualization of their dynamic movements in a subsecond resolution using high-speed AFM (HS-AFM). With such an approach, DNA conformational changes including G-quad-ruplex formation and disruption and B-Z transition are visualized, along with some enzymatic reactions such as DNA recombination. Beside direct visualization tools, single-molecule Forster resonance energy transfer (smFRET) appears also as a powerful technique for nanometer-scale studies of single molecules under biologically relevant conditions. Schärfen and Schlierf explain how to implement this technique and provide examples of its applications such as conformational changes studies of DNA secondary structures induced by the single-stranded DNA binding protein SSB and its competition with DnaA [5]. Further demonstrating about the potential of smFRET, Michalet and colleagues detail a high-throughput smFRET approach that can be used to increase the temporal resolution from minutes to seconds and show how, combined with microfluidics, this approach can enable real-time kinetic studies of binding/unbinding events and conformational changes [6]. Allemand and colleagues demonstrate the use of tethered particle motion (TPM) and magnetic tweezers to analyse the behavior of individual DNA molecules in the absence or under the application of a force and/or a torque [7]. The fabrication of dedicated nanoarrays by soft nanolithography has recently enabled the massive parallelization of these techniques, increasing their potential for various single molecule investigations. These techniques however provide only raw data such as the tracked particle amplitude of movement, from which relevant information about DNA conformations or states must be recovered. For this, Manghi and colleagues have developed theoretical tools reviewed in their paper [8]. Endly, Ivanov and Bryant describe some advanced single-molecule methods developed to track multiple degrees of freedom in nucleic acids and nucleoprotein complexes at high resolution and to enable better manipulation of the system under investigation [9]. Combining magnetic or optical tweezers with FRET have indeed demonstrated results unattainable by either technique alone. With all these contributions, this special issue provide an updated snapshot of past and ongoing developments single molecule measurements and manipulations to further decipher regulatory mechanisms at task in the nucleus.