Single-molecule microscopy study of nano-systems

In this work, techniques were developed and used to study the properties of molecules on a single-molecule level. Single-molecule techniques have the major advantage, that in contrast to ensemble measurements, they allow a detailed insight on the distribution and dynamics of single molecules without averaging over subpopulations. The use of Total Internal Reflection Fluorescence Microscopy (TIRFM) in combination with single-pair Förster Resonance Energy Transfer (spFRET) and Alternating Laser Excitation (ALEX) allows the identification of molecular-states by making quantitative measurements of distances in the Ångström range. The development of highly sensitive photon detectors and the use of versatile labeling techniques with photostable (synthetic or genetically-encoded) fluorophores, extended the application of TIRF microscopy to in vitro and live-cell experiments. Despite reducing the complexity of biological systems down to the single-molecule level, functions of individual molecules and interactions between them can be very sophisticated and challenging to analyze. Using information theory based methods, e.g. HMM, the dynamics extracted from single-molecule data was used to illuminate protein interactions and functions. The highly regulated process of gene transcription plays a central role in living organisms. The TATA-box Binding Protein (TBP) is a Transcription Factor (TF) that mediates the formation of the Pre-Initiation Complex (PIC). The lifetime of TBP at the promoter site is controlled by the Modulator of transcription 1 (Mot1), an essential TBP-associated ATPase involved in repression and in activation of transcription. Based on ensemble measurements, various models for the mechanism of Mot1 have been proposed. However, little is known about how Mot1 liberates TBP from DNA. Using TIRF microscopy, the conformation and interaction of Mot1 with the TBP/DNA complex were monitored by spFRET. In contrast to the current understanding of how Mot1 works, Mot1 bound to the TBP/DNA complex is not able to directly disrupt the TBP/DNA complexes by ATP hydrolysis. Instead, Mot1’s ATPase activity induces a conformational change in the complex. The nature of this changed, "primed", conformation is the change of the bending dynamics of the DNA. The results presented in this work suggest a model in which this primed conformation is a destabilized TBP/DNA complex. The interaction with an additional Mot1 molecule is required in order to liberate TBP from DNA. The effect of Mot1 on the DNA dynamics is TBP binding orientation specific. Mot1 effects on the DNA bending dynamics are strongest for molecules where TBP is bound in the inverted binding orientation. The specificity of Mot1’s regulation of DNA bending dynamics suggests that Mot1 preferably "primes" TBP bound in the inverted binding orientation. The mechanistic insight into the interaction of Mot1 with the TBP/DNA complex serves as a framework for understanding the role of Mot1 in gene up- and down-regulation. In a second project, the same single-molecule techniques were used to fabricate and evaluate self-assembled optically controllable, nanodevices. Based on the specificity of Watson-Crick base pairing, DNA was used as a scaffold to position different fluorophores with nanometer accuracy. The functionality of these nanodevices was expanded by making them optically addressable by incorporation of the switchable fluorescent protein Dronpa. Two functions have been demonstrated: Signal enhancement using Optical Lock-In Detection (OLID) and pH sensing in a live-cell environment

Toward precision medicine with nanopore technology

Currently, when patients are diagnosed with cancer, they often receive a treatment based on the type and stage of the tumor. However, different patients may respond to the same treatment differently, due to the variation in their genomic alteration profile. Thus, it is essential to understand the effect of genomic alterations on cancer drug efficiency and engineer devices to monitor these changes for therapeutic response prediction. Nanopore-based detection technology features devices containing a nanometer-scale pore embedded in a thin membrane that can be utilized for DNA sequencing, biosensing, and detection of biological or chemical modifications on single molecules. Overall, this project aims to evaluate the capability of the biological nanopore, alpha-hemolysin, as a biosensor for genetic and epigenetic biomarkers of cancer. Specifically, we utilized the nanopore to (1) study the effect of point mutations on C-kit1 G-quadruplex formation and its response to CX-5461 cancer drug; (2) evaluate the nanopore\u27s ability to detect cytosine methylation in label-dependent and label-independent manners; and (3) detect circulating-tumor DNA collected from lung cancer patients\u27 plasma for disease detection and treatment response monitoring. Compared to conventional techniques, nanopore assays offer increased flexibility and much shorter processing time

Nuclear export of single native mRNA molecules observed via light sheet fluorescence microscopy and transcriptional regulation of BR2.1 during heat-shock

Eucaryotes store most of their genetic information in the nucleus. Parts of this information encode the amino acid sequence of proteins. To synthesize a protein according to the nucleotide sequence, first the corresponding DNA-sequence is transcribed by RNA-Polymerase II to mRNA. Subsequently ribosomes translate the mRNA into the correct amino acid sequence. In eucaryotes the ribosomes are localized in the cytoplasm and are separated from the nucleus by the nuclear envelope. On the one hand separation of transcription and translation enables eucaryotes to process the transcript post-transcriptionally, on the other it requires a transport of the mRNA from the nucleoplasm into the cytoplasm. The nucleoplasm is interconnected with the cytoplasm by nuclear pore complexes. Most of the nucleo-cytoplasmic trafficking is facilitated through the nuclear pore complexes. Messenger RNA is exported into the cytoplasm through the nuclear pore complexes, too. During transcription the nascent mRNA is bound by several proteins which are essential e.g. for mRNA processing and export. The complex of the mRNA and its associated proteins is called an mRNP-particle. Fully processed mRNP-particles are able to cross the permeability barrier of nuclear pore complexes. In this thesis the kinetics of the mRNA-export were measured in salivary gland cells of C. tentans at the single molecule level. Therefore, mRNA was labeled by Hrp36, which was bacterially expressed and subsequently covalently linked to a fluorescent dye. Hrp36 associates cotranscriptionally with the nascent mRNA and is part of the mRNP-particle. After microinjection, labeled Hrp36 is transported into the nucleus, via its endogenous M9-shuttle domain. As all mRNP-particles, also the labeled ones, diffuse through the nucleus after transcription is finished and can be imaged by advanced fluorescence microscopy. In this thesis it is shown that the kinetics of the mRNA-export across the nuclear prore complexes follow a broad distribution in the range of 20ms to seconds. Furthermore, only 30% of all mRNP-particles are exported after they engaged an NPC. Fitting the mRNA-export kinetics with a bimodal gamma distribution revealed average export times of t1exp = 76ms, which is governed by multiple rate limiting steps and t2exp = 158ms, which is governed by just a single rate limiting step. Therefore, the translocation of the mRNA across the nuclear pore complex is not rate limiting for protein-biosynthesis which takes on average several minutes. Trajectory analysis of export events =300ms, showed that the mRNA were localized mainly in the nuclear basket during the export process. Here proteins are localized which are crucial for the mRNP-particle quality control. These proteins bind mRNP-particles, which are only partially processed, and thereby inhibit their translocation through the nuclear pore complex until their processing is completed. Assuming that the general reaction scheme is the same for all mRNP-particles and considering the fact that these slow export events show only a single rate limiting reactions step, this export events presumably correspond to mRNP-particles, whose processing were not finished. In addition to the mRNP-particle export kinetics, the Dbp5 interaction kinetics with the nuclear pore complexes were measured. Dbp5isaRNA-helicase, which is essential form RNP- particle export. It is assumed that Dbp5 removes the transport receptors from the mRNA via its helicase activity and thereby inhibit the translocation of mRNA back into the nucleus. The interaction kinetics of Dbp5 showed two interaction times (t1Dbp5 200Hz = 26ms & t2Dbp5 200Hz = 240ms). Due to the low number of observations, the interaction times gained by fitting the data with a bimodal gamma distribution showed a high uncertainty This makes a comparison of this results with the observed mRNA-export kinetics not advisable. In the second part of the thesis a so far unknown regulation mechanism of transcription was studied. First hints to this mechanism were observed by a control experiment during the examination of the mRNA-export kinetics. Transcription can be subdivided into the four stages of initiation, early elongation, stable elongation and termination. It was previously believed that after transition into stable elongation the transcription process is either completed or terminated prematurely. The results of this thesis give evidence that the transcription process in salivary gland cells of C. tentans can be halted temporally at the stage of stable elongation by applying a heat-shock to the larvae. The halted transcription processes can be resumed after heat-shock is released. Since RNA-polymerase II is highly conserved throughout eucaryotes, it seems very likely that this regulatory mechanism is not limited to C. tentans . The transcription halt during stable elongation described here, shows that eucaryotes have a more direct and far-ranging access to transcription as believed. This direct control of transcription significantly increases the temporal dynamic of transcriptional regulation

The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts

In response to intracellular signals in Gram-negative bacteria, translational riboswitches—commonly embedded in messenger RNAs (mRNAs)—regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ_1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by ‘bursts’ of probe binding associated with increased SD sequence accessibility. Addition of preQ_1 decreases the lifetime of the SD’s high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation

The physicist's guide to one of biotechnology's hottest new topics: CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) constitute a multi-functional, constantly evolving immune system in bacteria and archaea cells. A heritable, molecular memory is generated of phage, plasmids, or other mobile genetic elements that attempt to attack the cell. This memory is used to recognize and interfere with subsequent invasions from the same genetic elements. This versatile prokaryotic tool has also been used to advance applications in biotechnology. Here we review a large body of CRISPR-Cas research to explore themes of evolution and selection, population dynamics, horizontal gene transfer, specific and cross-reactive interactions, cost and regulation, non-immunological CRISPR functions that boost host cell robustness, as well as applicable mechanisms for efficient and specific genetic engineering. We offer future directions that can be addressed by the physics community. Physical understanding of the CRISPR-Cas system will advance uses in biotechnology, such as developing cell lines and animal models, cell labeling and information storage, combatting antibiotic resistance, and human therapeutics.Comment: 75 pages, 15 figures, Physical Biology (2018

Molecular Mechanisms of Transcription through Single-Molecule Experiments

Transcription represents the first step in gene expression. It is therefore not surprising that transcription is a highly regulated process and its control is essential to understand the flow and processing of information required by the cell to maintain its homeostasis. During transcription, a DNA molecule is copied into RNA molecules that are then used to translate the genetic information into proteins; this logical pattern has been conserved throughout all three kingdoms of life, from Archaea to Eukarya, making it an essential and fundamental cellular process. Even though some viruses that encode their genome in an RNA molecule use it as a template to make mRNA, others synthesize an intermediate DNA molecule from the RNA, a process known as reverse transcription, from which regular transcription of viral genes can then proceed in the host cells

Functional characterization of CRISPR-Cas interactions with DNA

The recent Nobel-prize-winning CRISPR technology has revolutionised the gene-editing field with its ability to precisely modify any target DNA sequence and its relevance in a wide range of applications, from functional characterisation of gene variants in biological sciences to mutation of plant genomes in agriculture. New prospectives for CRISPR-therapeutic applications are also rapidly advancing, particularly for the modification of disease-causing genes and ex-vivo editing of immune cells for cancer treatment. However, off-target editing exists as collateral damage and it represents a significant hurdle to realise CRISPR’s full potential. To date, the scientific community lacks extensive knowledge regarding the impact of the eukaryotic cellular context on Cas nucleases activity, and how this affects Cas efficiency and specificity on human DNA. In this thesis, I use a combination of cellular and single-molecule assays to investigate this specific topic. First, I developed a strategy to demonstrate that Cas9 specificity is diminished by a local distortion of the DNA 3D structure in human cells. Next, by using the CRISPR system as a tool for gene expression regulation, I investigated the correlation between target transcription in cells and Cas9 editing efficiency. Together this first part of my work suggests that in vivo processes, which occur in eukaryotic cells and destabilise the DNA structure, have the potential to induce off-targets. However, this effect is highly dependent on the target itself and its genomic context. Finally, I extended my study to two other CRISPR-Cas systems: Cas12a and a new engineered Cas (AZ-Cas9). By applying single-molecule technologies, I contributed to the characterization of these nucleases, and obtained new information about the mechanisms underlining the DNA target search, the binding and cleavage kinetics and the off-target discrimination. These findings fill important knowledge gaps for future applications of these variants, and will be useful for the rational design of new high-fidelity nucleases

Reconciling kinetic and thermodynamic models of bacterial transcription

The study of transcription remains one of the centerpieces of modern biology with implications in settings from development to metabolism to evolution to disease. Precision measurements using a host of different techniques including fluorescence and sequencing readouts have raised the bar for what it means to quantitatively understand transcriptional regulation. In particular our understanding of the simplest genetic circuit is sufficiently refined both experimentally and theoretically that it has become possible to carefully discriminate between different conceptual pictures of how this regulatory system works. This regulatory motif, originally posited by Jacob and Monod in the 1960s, consists of a single transcriptional repressor binding to a promoter site and inhibiting transcription. In this paper, we show how seven distinct models of this so-called simple-repression motif, based both on thermodynamic and kinetic thinking, can be used to derive the predicted levels of gene expression and shed light on the often surprising past success of the thermodynamic models. These different models are then invoked to confront a variety of different data on mean, variance and full gene expression distributions, illustrating the extent to which such models can and cannot be distinguished, and suggesting a two-state model with a distribution of burst sizes as the most potent of the seven for describing the simple-repression motif