Optimizing single-molecule experimental approaches for the study of complex protein assemblies on DNA

Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BN/Nynke Dekker La

Process for creating a double-stranded polyribonucleotide sequence with terminal overhang, as well as a process for creating a double-stranded polynucleotide construct and an application

The invention relates to a process for creating double-stranded RNA (16) having a terminal overhang. In accordance with the invention a DNA amplification is used for this purpose, followed by a transcription of the amplified DNA. When amplifying the DNA, primer pairs are used and care is taken that additional sequences (18, 19) are present that will ultimately provide the terminal overhangs.Applied Science

Method for determining one or more characterizing features of a macromolecule and an apparatus for carrying out said method

The invention concerns a method and apparatus for determining one or more characterizing features of a macromolecule, in particular torque and/or twist of nucleic acids like DNA, using magnetic fields.BN/BionanoscienceApplied Science

Nanobubbles in solid-state nanopores

Applied Science

Recent insights from single-molecule studies into nucleosome structure and dynamics

Eukaryotic DNA is tightly packed into a hierarchically ordered structure called chromatin in order to fit into the micron-scaled nucleus. The basic unit of chromatin is the nucleosome, which consists of a short piece of DNA wrapped around a core of eight histone proteins. In addition to their role in packaging DNA, nucleosomes impact the regulation of essential nuclear processes such as replication, transcription, and repair by controlling the accessibility of DNA. Thus, knowledge of this fundamental DNA–protein complex is crucial for understanding the mechanisms of gene control. While structural and biochemical studies over the past few decades have provided key insights into both the molecular composition and functional aspects of nucleosomes, these approaches necessarily average over large populations and times. In contrast, single-molecule methods are capable of revealing features of subpopulations and dynamic changes in the structure or function of biomolecules, rendering them a powerful complementary tool for probing mechanistic aspects of DNA–protein interactions. In this review, we highlight how these singlemolecule approaches have recently yielded new insights into nucleosomal and subnucleosomal structures and dynamics.BN/Nynke Dekker La

DNA Sequence Is a Major Determinant of Tetrasome Dynamics

Eukaryotic genomes are hierarchically organized into protein-DNA assemblies for compaction into the nucleus. Nucleosomes, with the (H3-H4)2 tetrasome as a likely intermediate, are highly dynamic in nature by way of several different mechanisms. We have recently shown that tetrasomes spontaneously change the direction of their DNA wrapping between left- and right-handed conformations, which may prevent torque buildup in chromatin during active transcription or replication. DNA sequence has been shown to strongly affect nucleosome positioning throughout chromatin. It is not known, however, whether DNA sequence also impacts the dynamic properties of tetrasomes. To address this question, we examined tetrasomes assembled on a high-affinity DNA sequence using freely orbiting magnetic tweezers. In this context, we also studied the effects of mono- and divalent salts on the flipping dynamics. We found that neither DNA sequence nor altered buffer conditions affect overall tetrasome structure. In contrast, tetrasomes bound to high-affinity DNA sequences showed significantly altered flipping kinetics, predominantly via a reduction in the lifetime of the canonical state of left-handed wrapping. Increased mono- and divalent salt concentrations counteracted this behavior. Thus, our study indicates that high-affinity DNA sequences impact not only the positioning of the nucleosome but that they also endow the subnucleosomal tetrasome with enhanced conformational plasticity. This may provide a means to prevent histone loss upon exposure to torsional stress, thereby contributing to the integrity of chromatin at high-affinity sites.Accepted Author ManuscriptBN/Nynke Dekker La

Method for sensing an analyte in a fluid and sensor unit for such method

The invention provides a method for sensing with a sensor system an analyte in an analyte fluid. The sensor system comprises a micron scale birefringent entity, a laser unit configured to generate polarized laser light, a polarization rotation device, wherein the laser unit and polarization rotation device are configured to rotate at a polarization rotation frequency the polarization of the laser light, and a detection unit. The method comprises feeding the analyte fluid along the micron scale birefringent entity while keeping with the laser light and the polarization rotation device the micron scale birefringent entity in an optical torque trap at a polarization rotation frequency; keeping the polarization rotation frequency at a sub-critical polarization rotation frequency; and measuring with the detection unit downstream of the micron scale birefringent entity the polarization of the laser light, and sensing a perturbation on the polarization of the laser light due to the analyte.BN/BionanoscienceApplied Science

De novo fabrication of custom-sequence plasmids for the synthesis of long DNA constructs with extrahelical features

DNA constructs for single-molecule experiments often require specific sequences and/or extrahelical/noncanonical structures to study DNA-processing mechanisms. The precise introduction of such structures requires extensive control of the sequence of the initial DNA substrate. A commonly used substrate in the synthesis of DNA constructs is plasmid DNA. Nevertheless, the controlled introduction of specific sequences and extrahelical/noncanonical structures into plasmids often requires several rounds of cloning on pre-existing plasmids whose sequence one cannot fully control. Here, we describe a simple and efficient way to synthesize 10.1-kb plasmids de novo using synthetic gBlocks that provides full control of the sequence. Using these plasmids, we developed a 1.5-day protocol to assemble 10.1-kb linear DNA constructs with end and internal modifications. As a proof of principle, we synthesize two different DNA constructs with biotinylated ends and one or two internal 3′ single-stranded DNA flaps, characterize them using single-molecule force and fluorescence spectroscopy, and functionally validate them by showing that the eukaryotic replicative helicase Cdc45/Mcm2-7/GINS (CMG) binds the 3′ single-stranded DNA flap and translocates in the expected direction. We anticipate that our approach can be used to synthesize custom-sequence DNA constructs for a variety of force and fluorescence single-molecule spectroscopy experiments to interrogate DNA replication, DNA repair, and transcription.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BN/Nynke Dekker La

Nucleosome Assembly Dynamics Involve Spontaneous Fluctuations in the Handedness of Tetrasomes

DNA wrapping around histone octamers generates nucleosomes, the basic compaction unit of eukaryotic chromatin. Nucleosome stability is carefully tuned to maintain DNA accessibility in transcription, replication, and repair. Using freely orbiting magnetic tweezers, which measure the twist and length of single DNA molecules, we monitor the real-time loading of tetramers or complete histone octamers onto DNA by Nucleosome Assembly Protein-1 (NAP1). Remarkably, we find that tetrasomes exhibit spontaneous flipping between a preferentially occupied left-handed state (?Lk = ?0.73) and a right-handed state (?Lk = +1.0), separated by a free energy difference of 2.3 kBT (1.5 kcal/mol). This flipping occurs without concomitant changes in DNA end-to-end length. The application of weak positive torque converts left-handed tetrasomes into right-handed tetrasomes, whereas nucleosomes display more gradual conformational changes. Our findings reveal unexpected dynamical rearrangements of the nucleosomal structure, suggesting that chromatin can serve as a “twist reservoir,” offering a mechanistic explanation for the regulation of DNA supercoiling in chromatin.BN/BionanoscienceApplied Science

Modification of the histone tetramer at the H3-H3 interface impacts tetrasome conformations and dynamics

Nucleosomes consisting of a short piece of deoxyribonucleic acid (DNA) wrapped around an octamer of histone proteins form the fundamental unit of chromatin in eukaryotes. Their role in DNA compaction comes with regulatory functions that impact essential genomic processes such as replication, transcription, and repair. The assembly of nucleosomes obeys a precise pathway in which tetramers of histones H3 and H4 bind to the DNA first to form tetrasomes, and two dimers of histones H2A and H2B are subsequently incorporated to complete the complex. As viable intermediates, we previously showed that tetrasomes can spontaneously flip between a left-handed and right-handed conformation of DNA-wrapping. To pinpoint the underlying mechanism, here we investigated the role of the H3-H3 interface for tetramer flexibility in the flipping process at the single-molecule level. Using freely orbiting magnetic tweezers, we studied the assembly and structural dynamics of individual tetrasomes modified at the cysteines close to this interaction interface by iodoacetamide (IA) in real time. While such modification did not affect the structural properties of the tetrasomes, it caused a 3-fold change in their flipping kinetics. The results indicate that the IA-modification enhances the conformational plasticity of tetrasomes. Our findings suggest that subnucleosomal dynamics may be employed by chromatin as an intrinsic and adjustable mechanism to regulate DNA supercoiling.Accepted Author ManuscriptBN/Nynke Dekker La