Nucleosomes condense DNA into higher order chromatin structures and the position of nucleosomes play an important role in the regulating of transcription. Current methods can only map ensemble...Show moreNucleosomes condense DNA into higher order chromatin structures and the position of nucleosomes play an important role in the regulating of transcription. Current methods can only map ensemble averaged nucleosome profiles. Here we make use of the property that nucleosomes protect the DNA against methylation, induced by methyltransferase enzymes. Nanopore sequencing is subsequently used to detect methylated bases on single DNA molecules. This makes a single molecule approach possible for detecting nucleosomes. We use the Nucleosome-Unmethylated-Score(NUS) to map nucleosomes on single DNA molecules. The NUS was tested by mapping nucleosome occupancy on high nucleosome affinity 601 arrays. The NUS was further used to analyse active and silent genes in Saccharomyces cerevisiae rDNA and to characterise nucleosome remodelling in reconstituted chromatin by whole cell extract (WCE) proteins.The found nucleosome occupancy was comparable with in vivo positions observed from ensemble averaged data. Nucleosome detection on single molecules advances our understanding of chromatin structure and its role in transcription regulation.Show less
The structure of chromatin plays a vital role in the regulation of gene expression. In this thesis, the mechanical properties of chromatin are investigated by measuring force-extension curves with...Show moreThe structure of chromatin plays a vital role in the regulation of gene expression. In this thesis, the mechanical properties of chromatin are investigated by measuring force-extension curves with Magnetic Tweezers. Two models are re-introduced that accurately describe these curves. Upon adding linker histone H1, a protein that can bind to individual nucleosomes, stabilization of nucleosome unstacking up to 10 pN was observed. Varying the Nucleosome Repeat Length (NRL) of the chromatin fibers with linker histones resulted in different degrees of stabilization: 168NRL fibers have a higher energy barrier for unstacking than 197NRL fibers. This could be caused by increased nucleosome stiffness at lower NRLs. This difference could have an important effect on transcription regulation in vivo.Show less
DNA, the carrier of genetic information is compacted into nucleosomes, which regulate access to that DNA. These nucleosomes are themselves folded into a higher order structure called chromatin....Show moreDNA, the carrier of genetic information is compacted into nucleosomes, which regulate access to that DNA. These nucleosomes are themselves folded into a higher order structure called chromatin. Little is known of the effect of this chromatin structure on the conformational dynamics of nucleosomes. Here we introduce a single-pair F¨orster Resonance Energy Transfer (spFRET) method that allows for quantitative measurement of nucleosome structure in folded fibers through both Fluorescence Correlation Spectroscopy (FCS) and burst analysis. Preliminary experiments determined optimal measurement concentrations and methods of excitation. However, measurements on reconstituted chromatin fibers showed poor signal-to-noise. We propose several improvements to enable the study of chromatin dynamics, such as nucleosome breathing. We expect the work outlined in this thesis to contribute to greater understanding of both nucleosome and chromatin structure, and how these regulate the accessibility of DNA to other molecules and proteins.Show less
Force spectroscopy on single molecules is a technique that is used to study the mechanical and structural properties of biomolecules. A relatively new force spectroscopy technique is acoustic force...Show moreForce spectroscopy on single molecules is a technique that is used to study the mechanical and structural properties of biomolecules. A relatively new force spectroscopy technique is acoustic force spectroscopy (AFS), which exploits acoustic waves to generate a wide range of forces with high pulling rates. A piezoelectric transducer is used to generate acoustic waves at specific resonance frequencies. Acoustic standing waves exert forces on micro-particles and pull them to acoustic pressure nodes. However, commercially available AFS flow cells are expensive and users find them nontrivial to operate. Here we present a new AFS flow cell design where we placed the piezoelectric transducer directly on top of the fluid channel of the flow cell to reduce the loss of acoustic pressure between the transducer and fluid. We used 3D finite element method simulations and found that the acoustic pressure profile is more complex than expected due to acoustic variations in the xy-plane. However, we found that the dimensions of the flow cell can be optimised to minimise acoustic variations in the xy-plane, demonstrating that more homogeneous pulling forces can be achieved. We performed AFS measurements on the new flow cell design, but we were not able to demonstrate an acoustic force. We anticipate this study helps to further optimise the AFS flow cell design, resulting in maximised pulling forces with minimal variations in the xy-plane.Show less
We introduce an open source, freely available algorithm for automatic correlation of fluorescence and electron microscopy images in Correlative Light-Electron Microscopy (CLEM). With a minimal...Show moreWe introduce an open source, freely available algorithm for automatic correlation of fluorescence and electron microscopy images in Correlative Light-Electron Microscopy (CLEM). With a minimal amount of manual input, two custom modules perform an automatic identification of the fluorescent nuclei and the watershed nucleus of a TEM image and hence calculate the necessary affine transformation for accurate correlation. The process can be performed on a group of pre-processed images and allows for an efficient solution around the data handling bottleneck encountered in the technique.Show less
In eukaryotic cells, nucleosomes regulate DNA processes such as transcription, replication and repair. The dynamic architecture of nucleosomes is the key for understanding gene regulation. Due to...Show moreIn eukaryotic cells, nucleosomes regulate DNA processes such as transcription, replication and repair. The dynamic architecture of nucleosomes is the key for understanding gene regulation. Due to the compacted structure of nucleosome, these dynamics are a challenging to study. Fluorescence Resonance Energy Transfer (FRET) microscopy combined with a nucleosome immobilization strategy, allows to observe conformational dynamics over long time scales. However, it is not trivial to immobilize nucleosomes properly, avoiding impurities or non-targeted molecules that could perturb measuring nucleosome dynamics. Here we present an optimized sample preparation protocol for studying conformational dynamics in nucleosomes. Using an immobilization strategy based on PLL-g-PEG/PEG-biotin and NeutrAvidin, we found a limited immobilization specificity and structural integrity of nucleosomes, possibly due to the influence of the experimental conditions as the measurements settings and the surface preparation. Nevertheless, we were able to identify some nucleosome FRET dynamics using this technique. Furthermore, an innovative method for cleaning coverslips and quantifying the cleaning quality, open a window of possibilities for automated sample preparation protocols in single molecule studies.Show less
The accessibility of DNA is regulated by means of dynamic folding and unfolding of chromatin fibers. Quantification of chromatin unfolding is a key in understanding the higher order structure of...Show moreThe accessibility of DNA is regulated by means of dynamic folding and unfolding of chromatin fibers. Quantification of chromatin unfolding is a key in understanding the higher order structure of chromatin. Single molecule force spectroscopy is an ideal tool to study chromatin dynamics. The extension of reconstituted chromatin fibers under force was measured using Magnetic Tweezers. Here I show a model to fit discrete states in chromatin unfolding, and use this model to quantify the influence of linker histone H1 on chromatin fibers. Stepsize analysis revealed that linker histone H1 induces cooperative unstacking of nucleosomes at forces less than 10 pN. Chromatin fibers in presence of linker histone H1 must be stretched $4.5\pm0.8$ nm for two nucleosomes to unstack. This model can be used to quantify natively folded chromatin fibers.Show less
Next to its well–known helix structure, double stranded DNA can form alternative structures that might have biological importance. For example, in guanine–rich DNA sites of the c–MYC promotor a...Show moreNext to its well–known helix structure, double stranded DNA can form alternative structures that might have biological importance. For example, in guanine–rich DNA sites of the c–MYC promotor a second order structure called a G–Quadruplex has been found. In the G–Quadruplex, one strand of the DNA forms a stack of 4 interacting guanines. In this thesis we study the formation of G–Quadruplexes in double–stranded DNA using a combination of F¨oster Resonance Energy Transfer (FRET) and multiplex Magnetic Tweezers (MT). Moreover, a two–state model was developed which describes the probability to form a G–Quadruplex in double–stranded DNA. Using this model we calculated how the extension and the FRET efficiency depends on force, twist and the sequence of the DNA. Because the synthesis of double–stranded DNA containing a G–Quadruplex site proved challenging, the experimental data could not be compared to the outcomes of the two–state model. Based on simulations we conclude that adding a 3–bp mismatch to the DNA tether next to the G4 site is required for the formation of a G–Quadruplex in dsDNA. Our findings may be relevant for understanding a link with transcription and/or replication.Show less
In this thesis we are going to study the mechanical properties of a chromatin fiber. Chromatin is the second compaction stage of DNA, after the wrapping of DNA around histones proteins to form...Show moreIn this thesis we are going to study the mechanical properties of a chromatin fiber. Chromatin is the second compaction stage of DNA, after the wrapping of DNA around histones proteins to form nucleosomes. Specifically we are going to analyze how its behaviour under external stresses is going to change with the variation of the linker DNA length, the DNA segment that links two adjacent nucleosomes. We will be able to do it at a single-molecule level thanks to the use of magnetic tweezers, an apparatus that can exert forces and torques directly to individual molecules.Show less
The structure of chromatin fibers has been researched in different salt conditions with Magnetic tweezers. The drag force on the tether as a result of flowing in a new buffer into the flow cell...Show moreThe structure of chromatin fibers has been researched in different salt conditions with Magnetic tweezers. The drag force on the tether as a result of flowing in a new buffer into the flow cell resulted in structural changes of the chromatin and disassembly of the histone octamer. In a new flow cell design, the drag force was eliminated by changing the buffer conditions by diffusion through a permeable membrane made from PEG-DA 400. Furthermore, double stranded DNA was unzipped using Magnetic tweezers and the position of the melting force was correlated with the G-C content calculated from the sequence.Show less
G-quadruplexes are non-canonical secondary DNA structures, that are formed by G-rich sequences. Evidence for their existence in vivo has been increasing, and it is suggested that they have an...Show moreG-quadruplexes are non-canonical secondary DNA structures, that are formed by G-rich sequences. Evidence for their existence in vivo has been increasing, and it is suggested that they have an important function in genome biology. The stabilization of G-quadruplexes promises to be a method to selectively kill cancerous cells. Helicases are a group of enzymes that have been associated with the reshaping of the genome. Some helicases have the specific task to unwind G-quadruplexes, or even specific types thereof. It is pivotal to understand the operating mechanism of these helicases, in order to stabilize G-quadruplexes to battle cancer. In this project, the unwinding of G-quadruplexes by Pif1 & RHAU helicases was studied by smFRET. Based on these results, the operating mechanisms for Pif1 & RHAU are reinterpreted. Before our work is extended to the quantitative characterization of their activity, it needs to be determined to what degree surface interactions influence our measurements.Show less
Complex cellular processes can be characterised using single particle tracking techniques (SPT). ‘Labels’ such as metal nanoparticles are introduced into cells and tracked to reveal molecular...Show moreComplex cellular processes can be characterised using single particle tracking techniques (SPT). ‘Labels’ such as metal nanoparticles are introduced into cells and tracked to reveal molecular trajectories. Many current techniques are based on fluorescence microscopy, and have nanometre-resolution. To accurately probe cellular processes, a technique must also have long-term 3D in-vivo observing capability with a minimal toxic effect. Gold nanorods (GNRs) in two-photon microscopy is a promising technique. GNRs are non-toxic, easily functionalisable, and exhibit a bright two-photon fluorescence. However, the theoretical positional accuracy for this technique is not yet known. Furthermore, the detailed trajectory data present statistical challenges. We have addressed these issues here numerically, using simulated image data. We found the accuracy to be between 5.2nm and 8.9nm for stationary GNRS, dependent on the separation between slices. We also found that diffusive movement of a nanorod lowers the accuracy, at worst case to 31.8nm. We have also investigated optimising the extraction of behaviour properties from MSD plots, and have used Welsh’s test to detect transitions. GNRs in two-photon microscopy has been shown to be a very accurate technique, and its trajectory data can yield accurate behavioural information. The fit of the PSF may be improved, but the techniques already compares well against others.Show less
In eukaryotes, three quarters of the DNA is wrapped around histone proteins, forming a string of nucleosomes. This organization condenses the DNA considerably, and at the same time restricts it...Show moreIn eukaryotes, three quarters of the DNA is wrapped around histone proteins, forming a string of nucleosomes. This organization condenses the DNA considerably, and at the same time restricts it accessibility for DNA binding proteins. Conformational dynamics of the nucleosome, like partial release of the DNA, called nucleosome breathing, plays an important part in regulating this accessibility of the genetic information. To study the wrapping and unwrapping in DNA breathing we followed the Förster Resonance Energy Transfer of a pair of fluorophores placed in the nucleosome, in time. We selected a single nucleosome immobilized on glass from an image acquired by a scanning confocal microscopy. Data acquisition and analysis software was developed to record and process time trace of individual nucleosomes with sub ms resolution. Although we can now reach the required temporal resolution to resolve nucleosome breathing, we did not observe it. A large fraction of the nucleosomes did not show FRET after surface immobilization suggesting partial disassembly, which prevented statistical analysis of large numbers of nucleosomes.Show less
Increasing effort into parallel single molecule force experiments has led to the development of acoustic force spectroscopy (AFS). Acoustic standing waves of MHz frequencies are able to exert...Show moreIncreasing effort into parallel single molecule force experiments has led to the development of acoustic force spectroscopy (AFS). Acoustic standing waves of MHz frequencies are able to exert forces on microspheres, which can be used for the manipulation of single bio-molecules. The performance of acoustic tweezers is greatly dependent on setup design and the proper driving frequency. The need for AFS flow cell design modeling software is apparent. In this thesis we present a physical model for calculating the acoustic frequency response of a flow cell. We have found that an oil immersion objective lowers AFS performance drastically as opposed to an air objective and that the optimal driving frequency lies around 10.3 MHz for our prototype flow cell. Limited by the 10 MHz cap of our AC driver and problems with flow cell assembly yields, we were not able to demonstrate an acoustic pulling force. We need to switch to an air objective and, in order to make AFS work in our laboratory, either a new AC generator needs to be implemented or a piezoelectric transducer with a thickness mode within 8-10 MHz needs to be included.Show less
