Tesis sobre el tema "Single-molecule biophysic"

DNA bending plays a vital role in numerous cellular activities such as transcription, viral packaging, and nucleosome formation. Therefore, understanding the physics of DNA bending at the length scales relevant to these processes is one of the main keys to the quantitative description of life. However, previous studies provide a divided picture on how DNA should be modeled in strong bending condition relevant to biology. My thesis is devoted to answering how far an elastic rod model can be applied to DNA. We consider several subtle features that could potentially lead to the break-down of the worm-like chain model, such as local bendedness of the sequence and large bending angles. We used single-molecule Fluorescence Resonance Energy Transfer to track looping and unlooping of single DNA molecules in real time. We compared the measured looping and unlooping rates with theoretical predictions of the worm-like chain model. We found that the intrinsic curvature of the sequence affects the looping propensity of short DNA and an extended worm-like chain model including the helical parameters of individual base pairs could adequately explain our measurements. For DNA with random sequence and negligible curvature, we discovered that the worm-like chain model could explain the stability of small DNA loops only down to a critical loop size. Below the critical loop size, the bending stress stored in the DNA loop became less sensitive to loop size, indicative of softened dsDNA. The critical loop size is sensitive to salt condition, especially to magnesium at mM concentrations. This finding enabled us to explain several contrasting results in the past and shed new light on the energetics of DNA bending.

Inner-ear mechanotransduction relies on tip links, fine protein filaments made of cadherin-23 and protocadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link cadherins are thought to form a heterotetrameric complex, with two cadherin-23 molecules forming the upper part of the filament and two protocadherin-15 molecules forming the lower end. The interaction between cadherin-23 and protocadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. We have developed molecular tools to perform single-molecule force spectroscopy on the tip-link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of cadherin-23 and protocadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip-link-functionalized nanoswitches are designed to provide a signature force-extension profile, which allows us to identify single-molecule rupture events that result from applying force. Using this system, we have been able to measure the cadherin-23-protocadherin-15 single-molecule force-dependent off rate, as well as the concentration-dependent on rate for a single pair of these proteins. The rates suggest that a single bond is inadequate to withstand physiological forces for physiological times, but we construct a new model for tip-link dynamics which greatly alters our understanding of tip-link function and explains the necessity for a two-filament tip link.
Medical Sciences

Super-resolution microscopy is a relatively new and rapidly growing field. Development has been largely technology-driven, with high power lasers, higher resolution CCD cameras, and increasing computing power all enabling new biological questions to be explored. Single-molecule imaging is the tool of choice for studying systems where heterogeneity is present; ensemble methods can average away the interesting behaviour and lead to false conclusions. This thesis develops and optimises bespoke fluorescence microscopy for application to three biological questions, each pushing a limit of super-resolution imaging. Super-resolution imaging of lambda DNA labelled with the intercalating dye YOYO-1 and the minor groove binder SYTO-13 at localisation precisions of 40nm and 62nm respectively has been achieved in preparation for combined fluorescence imaging and magneto-optical tweezers experiments. The combination of these two methods is challenging as both operate with low tolerances.\ Single-molecule tracking was used to measure the diffusion coefficients of the chemokines CXCL13 and CCL19 at extremely high temporal resolution. Single-molecule imaging was found to have advantages over the ensemble techniques of FRAP and FCS for measuring the diffusion coefficient of the test molecule; Alexa Fluor 647 labelled bovine serum albumin. The diffusion coefficients of the two chemokines, CXCL13 and CCL19 were found by single particle tracking at sub-millisecond timescales in a collagen matrix to be 6.2±0.3µm2s-1 and 8.4±0.2µm2s-1. Further, CXCL13 was tracked in B cell follicle regions of ex vivo lymph node tissue sections at ~2 millisecond timescales, giving a diffusion coefficient of 6.6±0.4µm2s-1.\ Fluorescence microscopy was used to elucidate the stoichiometry of YOYO-1 on DNA origami tiles after treatment with low temperature plasma. Undamaged tiles were found to have a mean stoichiometry of 67.4±25.2 YOYO-1 molecules and a model of LTP damage to DNA origami tiles was proposed.

In this thesis, single-molecule experiments using LOT are employed to extract accurate information about the thermodynamics and kinetics of various molecular systems, with special emphasis on the elastic properties of single-stranded DNA (ssDNA). The thesis is divided in three parts. Part I provides a general description of the research field as well as the main theoretical framework for the basic concepts that will be developed in parts II and III. In Chapter 2 the miniTweezers and the experimental setup used throughout the thesis is described, as well as the physical basis of its working mechanisms, introducing the phenomenon of optical trapping. Chapter 3 contains a brief introduction of the biomolecules of study in this thesis, with an explanation of their historical discoveries, as well as their structure and function. The main focus of this chapter is on ssDNA, which is the main object of study of the thesis. Chapter 4 introduces the polymer models that are widely used in describing the elasticity of nucleic acids and proteins. Specifically, the Freely-Jointed Chain and Worm-Like Chain models are presented. Part II deals with the elasticity of single-stranded DNA. This is the main part of the thesis, and it includes chapters 5-7. Chapter 5 is about the study of the elasticity of ideal ssDNA chain, i.e. the one that can be modelled as ideal polymers (presented in Chapter 4). The study of the elasticity of different DNA sequences is presented. The blocking-splint oligo technique is described, a experimental technique developed for studying the elasticity of short (tens of bases) DNA molecules. This study shows the need of using extensible models to succesfully describe ssDNA elasticity over a large range of forces, which explains the previous discrepancies on the elastic parameters obtained in different studies. We also provide an explanation for the required extensibility of the model: a transition experienced at the nucleotide level: a change in DNA sugar pucker conformation. A simple two-states model is introduced and preeliminary results regarding its energetics are presented. The characterization of the ssDNA elasticity is central for the works developed in the following chapters. Chapter 6 studies the stacking-unstacking transition for ssDNA, previously observed for certain sequences (mainly purine-rich ones). Several molecules, with different degrees of stacking, are studied by obtaining their force extension curves (FECs). A cooperative helix-coil model including heterogeneity is developed and used to fit the obtained FECs, allowing to obtain elastic parameters to describe the stacked chain. The salt dependence of the unstacking transition is also measured by studying two of the sequences by varying the salt concentration over two decades. The free energy of formation of dsDNA duplexes depends on the salt concentration. The obtained salt dependence on the stacking free-energy of ssDNA provides a possible explanation for the salt dependence of duplex formation. Chapter 7 deals with the non-specific structures that arise at low forces and high salt concentration when pulling ssDNA molecules longer than $\sim 100$ bases. A helix-coil model with cooperativity is proposed and used to extract some mean-field characteristics of these structures. 8 different sequences are studied, characterizing their elasticity and deviation from the ideal elastic behaviour. The results for a $14$kb molecule for 3 decades of varying \ce{NaCl} and \ce{MgCl2} are also shown. All experimental FECs are fitted to the helix-coil model. The model can be used to predict the formation of secondary structures at zero force. A comparison between the predicted structures from the model and those obtained from Mfold is also investigated. Part III contains two studies which also need of the correct determination of ssDNA elasticity. In Chapter 8, we study the interaction between the RecQ helicase from E. coli and DNA, i.e. how the RecQ unwinds double-stranded DNA molecules, releasing single-stranded DNA. We obtain some of its kinematic properties as well as study the entropy production of the system using the Fluctuation Theorem. In Chapter 9, the effect of DNA mismatches, i.e. non complementary base pairing, on the stability of DNA is studied. To do so, two types of experiments on several DNA sequences are performed: stretching and releasing the molecule by moving the optical trap (pulling experiments) and monitoring the folding/unfolding of the molecule passively (hopping experiments).
En aquesta tesi hem realitzat experiments fent servir pinces òptiques per tal d’extreure informació precisa sobre les propietats termodinàmiques i cinètiques de diferents sistemes moleculars, posant especial èmfasi en les propietats elàstiques de la cadena simple d’ADN (ssDNA, pel seu acrònim en anglès). La tesi es troba dividida en tres parts. A la primera part s’introdueix de forma general el camp de recerca dels experiments de molècula única, així com s’expliquen els conceptes més bàsics que es desenvoluparan en les parts II i III. La configuració experimental emprada al llarg de tota la tesi, les pinces òptiques, s’introdueix al capítol 2. Per a fer-ho, s’expliquen els principis físics de funcionament de les pinces, que es basen en l’atrapament òptic. Breument, la focalització d’un feix de llum d’alta intensitat permet atrapar i exercir forces en micropartícules dielèctriques (pilotes fetes de plàstic de la mida d’un bacteri), que són recobertes químicament de manera que la molècula d’estudi pot estirar-se, de forma individual, repetides vegades. El capítol 3 conté una breu introducció a les biomolècules que apareixen en aquesta tesi, amb una breu explicació de la seva descoberta, així com la seva estructura i funció (íntimament relacionades). Ens centrem en la descripció de la ssDNA que és el principal objecte d’estudi de la tesi. Al capítol 4 s’introdueixen els models de polímers que s’empren habitualment per a descriure l’elasticitat d’àcids nucleics i proteïnes. En concret, es descriuen els models de la Freely-Jointed Chain i la Worm-Like Chain. La Part II tracta de l’elasticitat de la ssDNA, i inclou els capítols 5, 6 i 7. El capítol 5 es basa en la caracterització de l’elasticitat de la cadena ideal de ssDNA, és a dir, aquella que pot ser modelitzada pels polímers ideals introduïts en el capítol 4. S’estudia l’elasticitat de diferents seqüències de ssDNA, introduint un nou mètode experimental, blocking-splint oligo, per tal d’ampliar el rang de forces estudiat habitualment en molècules curtes (d’una longitud de desenes de bases) de ssDNA. L’estudi mostra la necessitat d’emprar models elàstics extensibles per a la correcte caracterització de l’elasticitat de ssDNA, que explica les discrepàncies existents entre els paràmetres elàstics trobats a la literatura. També hipotetitzem que l’extensibilitat del model pot ser explicada gràcies a la transició experimentada a nivell de nucleòtids: el canvi que experimenta la distància interfosfat de l’ADN es veu modificada segons quina sigui la configuració de l’anell de desoxiribosa. Tot i que és un fenomen molt més conegut en la cadena doble d’ADN, l’apilament-desapilament de bases també s’ha observat en certes seqüències de ssDNA (especialment les que són riques en contingut de purines). Al capítol 6 s’estudien quatre molècules amb un grau d’apilament diferent a partir de les seves corbes força-extensió (FECs). Es desenvolupa un model helix-coil (hèlix-cabdell) per tal d’ajustar les FECs, fet que permet d’obtenir, indirectament, les propietats elàstiques de la cadena apilada. També s’estudia la dependència d’aquesta transició variant la concentració de sal dels experiments en més de dos ordres de magnitud. A través d’aquests experiments, trobem una dependència amb la concentració de sal de l’energia lliure de formació de l’apilament de la ssDNA, fet que ens permet explicar, parcialment, la dependència que es troba en la literatura per la hibridació de la cadena doble d’ADN. El capítol 7 tracta de la formació d’estructures no específiques que apareixen a forces baixes i a concentració de sal alta per a molècules de ssDNA de més de ~100bases. Es proposa un model helix-coil amb cooperativitat per tal de caracteritzar propietats de camp mitjà de les estructures estudiades. S’estudien vuit seqüències diferents, entre 120 i ~14000 bases, i es caracteritza el seu desviament respecte de la corba elàstica ideal amb el model. També s’estudia la dependència de l’estructura secundària de la ssDNA en funció de la concentració de la sal. Analitzant experiments variant la concentració de MgCl2 i NaCl, aconseguim reproduir les FECs a partir de fer dependre els paràmetres del model amb la sal. Finalment, el model desenvolupat ens permet predir la formació d’estructura secundària a força zero (fet que no podem detectar directament a partir d’experiments d’espectroscopia de forces). Es comparen les previsions del model amb les trobades per Mfold, trobant una compatibilitat per als resultats per a molècules de de menys de 1000 bases. La darrera part se centra en col·laboracions que he fet durant a tesi i que necessiten una determinació precisa de les propietats elàstiques de la ssDNA. Al capítol 8 s’estudia la interacció entre l’helicasa del bacteri E. coli i l’ADN, que s’encarrega d’obrir la cadena doble d’ADN, alliberant ssDNA. S’extreuen les seves propietats cinètiques, com la velocitat de translocació – obtenim, independentment de la força aplicada, d’uns 50bp/s, d’acord amb la literatura –. També n’estudiem les seves propietats termodinàmiques, a partir del Teorema de Fluctuació. Finalment, al capítol 9 s’estudien els efectes de certs defectes en molècules d’ADN. A partir d’experiments fora de l’equilibri s’extrau la penalització que suposa per a la hibridització d’ADN la presència d’aquestes bases no complementàries (és a dir, que no són enllaços de A-T o G-C).

This thesis describes signal analysis methods for single-molecule fluorescence data. The primary factor motivating method development is the need to distinguish single-molecule FRET fluctuations due to conformational dynamics from fluctuations due to distance-independent FRET changes. Single-molecule Förster resonance energy transfer (FRET) promises a distinct advantage compared to alternative biochemical methods in its potential to relate biomolecular structure to function. Standard measurements assume that the mean transfer efficiency between two fluorescent probes, a donor and an acceptor, corresponds to the mean donor-acceptor distance, thus providing structural information. Accordingly, measurement analysis assumes that mean transfer efficiency fluctuations entail mean donor-acceptor distance fluctuations. Detecting such fluctuations is important in resolving molecular dynamics, as molecular function often necessitates structural changes. A problem arises, however, in that factors other than donor-acceptor distance changes may induce mean transfer efficiency fluctuations. We refer to these factors as distance-independent FRET changes. We present analysis methods to detect distance-independent photophysical dynamics and to determine their correlation with distance-dependent FRET dynamics. First, we review a theory of photon statistics and show how we can use the theory to detect FRET fluctuations. Second, we extend the theory to alternating laser excitation (ALEx) measurements and demonstrate how fluorophore stoichiometry, a measure of fluorophore brightness, reports on distance-independent photophysical dynamics. Next, we provide a measure to determine the extent to which stoichiometry fluctuations account for FRET dynamics. Finally, we use a framework similar to the preceding along with recent advances in the theory of total internal reflection fluorescence (TIRF) microscopy FRET measurements to detect TIRF FRET fluctuations which occur on a timescale faster than the measurement temporal resolution. We validate our methods with simulations and demonstrate their utility in delineating RNA polymerase open complex conformational dynamics.

Rotaviruses are the single most common cause of fatal and severe childhood diarrhoeal illness worldwide (>125 million cases annually). Rotavirus shares structural and functional features with many viruses, such as the presence of segmented double-stranded RNA genomes selectively and tightly packed with a conserved number of transcription complexes in icosahedral capsids. Nascent transcripts exit the capsid through 12 channels, but it is unknown whether these channels specialise in specific transcripts or simply act as general exit conduits; a detailed description of this process is needed for understanding viral replication and genomic organisation. To test these opposing models, a novel single-molecule assay was developed for the capture and identification (CID) of newly synthesised specific RNA transcripts. CID combines the hybridisation of transcripts with biotinylated and FRET compatible labelled ssDNAs with the implementation of recent developments in single molecule fluorescence such as alternating laser excitation (ALEX) and total internal reflection fluorescence (TIRF) microscopy. CID identifies and quantifies specific transcripts of rotavirus based on a FRET/Stoichiometry (E*/S) value of the hybridised labelled probes. I used CID to pull down the capsid on the surface slide and identify partially extruded transcripts of three different segments 2, 6 and 11. The findings presented in this thesis support a model in which each channel specialises in extruding transcripts of a specific segment, that in turn is linked to a single transcription complex. The method can be extended to study other transcription systems including E.coli, and can be further developed as a potential diagnostic tool.

Single-molecule Förster resonance energy transfer (smFRET) has emerged as an important tool for studying biological reactions. This thesis describes smFRET investigations into the mechanism of bacterial transcription initiation. We developed protocols to immobilize RNAP-DNA initiation complexes using vesicles and antibodies. We used these techniques to show that the transcription bubble conformation in immobilized complexes exhibits inter-molecular heterogeneity. We observed large FRET changes that we attribute to transcription bubble opening and closing dynamics. We found that σ⁷⁰ region 3.2 (σR3.2) influences the kinetics of the bubble dynamics, which supports proposals that σR3.2 interacts with the transcription bubble template strand. We extended our investigations to RNA synthesis and were able to observe abortive initiation cycles directly. We observed RNAP pausing and backtracking for the first time in transcription initiation. We obtained data suggesting that σR3.2 stabilises short RNAs at the active centre and forms a barrier to the extension of RNAs longer than 5-nt in length. We extended our abortive initiation assay to observe signal changes that we attribute to promoter escape. Our data revealed the number of abortive cycles that occur prior to escape, the kinetics of promoter escape, and pausing events that may have some regulatory function. We investigated the conformational dynamics of the RNAP β clamp and observed dynamic conformational changes between clamp-open and clamp-closed states. Our work confirms proposals that the clamp remains stably closed once the open complex (RPO) is formed. We investigated what affect the antibiotics Myxopyronin and Lipiarmycin have on the clamp conformation. Our results revealed that Myxopyronin traps the clamp in a closed conformation, while Lipiarmycin traps it in an open conformation. Overall, we made a number of novel observations that we believe advance our understanding of the mechanism of transcription. We hope that the discoveries reported here will direct future research efforts into RNAP function.

Förster resonance energy transfer (FRET) has become an important tool for studying biochemical reactions at the single-molecule level, despite its increasing maturity there is an on-going effort to improve and expand the technique. This thesis presents methods for extending conventional two-colour single-molecule FRET measurements; by expanding the range and applicability of single-molecule fluorescence methods a greater variety of biological reactions can be studied, in greater detail than previously possible. To circumvent the complexities of multi-colour FRET measurements and extend the range of observable distances I developed and characterised a new single-molecule fluorescence method termed tethered fluorophore motion (TFM). TFM is based on the existing technique of tethered particle motion (TPM) which relies on Brownian motion of a particle, attached to a surface by DNA, to probe the effective length of the DNA tether. TFM takes this concept and applies it at the single-fluorophore level, allowing simultaneous measurement of other fluorescence observables such as FRET and protein induced fluorescence enhancement (PIFE). Having developed TFM I combined it with FRET to study site-specific recombinase proteins at the single-molecule level, in greater detail than possible by either technique alone. Studying the model tyrosine recombinase Cre, I extend and clarify previous ensemble observations regarding the order of DNA strand exchange, as well as uncovering a previously unobserved complex conformation and molecular heterogeneity. Finally, I used TFM-FRET to study the more complex XerCD recombination system and its interaction with the DNA translocase FtsK. I made observations, for the first time, of synaptic complex formation and of recombination at the single-molecule level, and these suggest intriguing and unexpected intermediates in the recombination reaction. I also combine TFM with PIFE to investigate the mechanism of DNA looping by FtsK. The introduction of TFM, and its combination with other fluorescence techniques, allows observation of complex protein-DNA interactions from a variety of perspectives and will help expand the repertoire and applicability of single-molecule biophysical experiments.

DNA replication is a fundamental cellular process. However, the structure and dynamics of the eukaryotic DNA replication machinery remain poorly understood. A soluble extract system prepared from Xenopus eggs recapitulates eukaryotic DNA replication outside of a cell on a variety of DNA templates. This system has been used to reveal many aspects of DNA replication using a variety of ensemble biochemical techniques. Single-molecule fluorescence imaging is a powerful tool to dissect biochemical mechanisms. By immobilizing or confining a substrate, its interaction with individual, soluble, fluorescently-labeled reactants can be imaged over time and without the need for synchrony. These molecular movies reveal binding parameters of the reactant and any population heterogeneity. Moreover, if the experiments are imaged in wide-field format, the location or motion of the labeled species along the substrate can be followed with nanometer accuracy. This dissertation describes the use and development of novel single-molecule fluorescence imaging techniques to study eukaryotic DNA replication. A biophysical characterization of a replication fork protein, PCNA, revealed both helical and non-helical sliding modes along DNA. Previous experiments demonstrate that the egg extracts efficiently replicate surface-immobilized linear DNA. This finding suggested replication of DNA could be followed as motion of the replication fork along the extended DNA. However, individual proteins bound at the replication fork could not be visualized in the wide-field due to the background from the high concentration of the fluorescent protein needed to compete with the extract’s endogenous protein. To overcome this concentration barrier, I have developed a wide-field technique that enables sensitive detection of single molecules at micromolar concentrations of the labeled protein of interest. The acronym for this method, PhADE, denotes three essential steps: (1) Localized PhotoActivation of fluorescence at the immobilized substrate, (2) Diffusion of unbound fluorescent molecules to reduce the background and (3) Excitation and imaging of the substrate-bound molecules. PhADE imaging of flap endonuclease I (Fen1) during replication revealed the time-evolved pattern of replication initiation, elongation and termination and the kinetics of Fen1 exchange during Okazaki fragment maturation. In the future, PhADE will enable the elucidation of the dynamic events at the eukaryotic DNA replication fork. PhADE will also be broadly applicable to the investigation of other complex biochemical process and low affinity interactions. It will be especially useful to those researchers wishing to correlate motion with binding events.

I present two applications of fluorescence spectroscopy in biophysics. The first is an instrument, the anti-Brownian electrokinetic (ABEL) trap, which is capable of trapping individual small molecules in aqueous solution at room temperature. The second is an investigation of the bending mechanics of double-stranded DNA using a novel DNA structure called a "molecular vise". Both projects take advantage of the sensitivity and specificity of fluorescence spectroscopy, and both benefit from the interplay of experimental work with theoretical and computational modeling. The ABEL trap uses fluorescence microscopy to track a freely diffusing particle, and applies real-time electrokinetic feedback forces to oppose observed motion. Small molecules are difficult to trap because they diffuse quickly and because their fluorescence emission is typically weak. I describe the experimental and algorithmic approaches that enabled small-molecule fluorophores to be trapped at room temperature. I additionally derive and discuss the theory of the molecules' behavior in the trap; this mathematical work informed the design of the trapping algorithm and additionally enabled trapped molecules to be distinguished on the basis of their diffusion coefficient and electrokinetic mobility. Molecular vises are DNA hairpins that use the free energy of hybridization to exert a compressive force on a sub-persistence length segment of double-stranded DNA. In response to the applied force, this "target strand" may either remain straight or bend, depending on its flexibility and length. Experimentally, the conformation can be monitored via Förster resonance energy transfer (FRET) between appended fluorophores. The experimental results quantitatively matched the predictions of the classic wormlike chain (WLC) model of DNA elasticity at low-to-moderate salt concentrations. Higher ionic strength induced an apparent softening of the DNA which was best accounted for by a high-curvature "kinked" state. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA and provides a platform for studying the effects of chemical modifications and small-molecule or protein binding on these properties.

The measurement of Förster resonance energy transfer (FRET) at the single-molecule level provides a powerful method for monitoring the structural dynamics of biomolecular systems in real time. These single-molecule FRET (smFRET) assays enable the characterization of the transient intermediates that form during enzymatic processes, providing information about the mechanism and regulation of the enzymes involved. In this dissertation, I develop and use smFRET assays to study two processes driven by motor proteins—reverse transcription and chromatin remodeling—and reveal novel features of their mechanism and regulation. Reverse transcription of the human immunodeficiency virus genome initiates from a cellular tRNA primer that is bound to a specific sequence on the viral RNA (vRNA). During initiation, reverse transcriptase (RT) exhibits a slow mode of synthesis characterized by pauses at specific locations, and RT transitions to a faster mode of synthesis after the extension of the tRNA primer by six nucleotides. By using smFRET to examine how RT interacts with the tRNA-vRNA substrate, we found that RT binds to its substrate in either an active or inactive orientation and samples the two orientations during a single binding event. The equilibrium between these two orientations is a major factor influencing the activity and pausing of the enzyme, and a specific RNA secondary structure in the vRNA substrate modulates the binding mode of RT, determining the locations of the pauses and the transition to the faster mode of synthesis. These results provide a mechanistic explanation for the changes in RT activity observed during initiation and show how the dynamics of a ribonucleoprotein complex can regulate enzymatic activity. ISWI family chromatin remodelers are another family of motor enzymes regulated by nucleic acid structures. These enzymes are involved in creating evenly spaced nucleosome arrays, and this nucleosome spacing activity arises from the regulation of the enzymes’ catalytic activity by the amount of linker DNA present on the nucleosome. We use smFRET and other biochemical assays to monitor intermediates of the remodeling reaction and examine various remodeler mutants in order to elucidate the mechanism of this regulation. These experiments led to the discovery of an allosteric mechanism by which one subunit of the ISWI remodeling complex communicates the presence of linker DNA to the the catalytic subunit by modulating the availability of the histone H4 tail. These results provide a mechanistic explanation for the nucleosome spacing activity of the ISWI chromatin remodelers. Like the ISWI chromatin remodelers, the SWI/SNF family chromatin remodelers can also reposition nucleosomes, but they may do so by a different mechanism. To investigate the mechanism by which these remodelers move DNA around the nucleosome, we used smFRET to monitor the structural dynamics of nucleosomes during remodeling by the SWI/SNF enzymes. Our results are consistent with movement of the DNA along its canonical path without substantial lifting of DNA off the edges of the nucleosome or displacement of the H2A-H2B dimer. We observe DNA translocation in 1-2 bp increments at both edges of the nucleosome, which suggests that the motion of DNA at the edges of the nucleosome is driven directly by the action of the ATPase near the dyad of the nucleosome.
Biophysics

DNA replication is a fundamental process that is primarily regulated at the initiation step. In higher eukaryotes, the location and properties of replication origins are not well understood. Existing genome-wide approaches to map origins—such as nascent strand abundance mapping, Okazaki fragment mapping, or chromatin immunoprecipitation-based assays—average the behavior of a population of cells. However, due to cell-to-cell variability in origin usage, single molecule techniques are necessary to investigate the actual behavior of a cell. Here, I investigate the feasibility of using three single molecule, genome-wide technologies to map origins of replication. The Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing technology, the BioNano Genomics Irys optical mapping technology, and the Oxford Nanopore Technologies MinION nanopore sequencing technology are promising approaches that can advance our understanding of DNA replication in higher eukaryotes.

Observations of the position of a microscopic bead attached to a single kinesin protein moving along a microtubule contains detailed information about the position of the kinesin as a function of time, although this information remains obscured because of the fluctuations of the bead. The theory of hidden Markov models suggests a possible theoretical framework to analyze these data with an explicit stochastic model describing the kinesin cycle and the attached bead. We model the mechanical cycle of kinesin using a discrete time Markov chain on a periodic lattice, representing the microtubule, and model the position of the bead using an Ornstein-Uhlenbeck autoregressive process. We adapt the standard machinery of hidden Markov models to derive the likelihood of this model using a reference measure, and use the Expectation-Maximization (EM) algorithm to estimate model parameters. Simulated data sets indicate that the method does have potential to better analyze kinesin-bead experiments. However, analysis of the experimental data of Visscher et al. (1999) indicates that current data sets still lack the time resolution to extract significant information about intermediate states. Considerations for future experimental designs are suggested to allow better hidden Markov model analysis.

Inwardly rectifying potassium (Kir) channels are essential for controlling the excitability of eukaryotic cells, forming a key part of the inter-cellular signalling system in multi-cellular organisms. However, as prokaryotic (KirBac) channels are less technically challenging to study in vitro and have been shown to be directly homologous to eukaryotic channels, they are often studied in lieu of their mammalian counterparts. A vital feature of Kir and KirBac channels is their mechanism for opening and closing, or their gating: this study predominantly features observations of open and/or closed channel populations. A well-characterised member of the KirBac family, KirBac1.1, has been successfully expressed, purified into detergent micelles, and doubly labelled with fluorescent maleimide dyes in order to enable observation of confocal-in-solution Förster Resonance Energy Transfer (FRET) at the single molecule level. Results demonstrate single-molecule FRET signals from KirBac1.1 and therefore represent the first single-molecule FRET observations from a KirBac channel. Perturbation of the open-closed dynamic equilibrium was performed via activatory point mutations, changes in pH, and ligand binding. A protocol for reconstitution into nanodiscs was optimised in order to more closely approximate native conditions, and the single-molecule FRET observations repeated. This thesis presents a comparison between measurements made using the detergent solubilisation system and those made using nanodiscs.

Biology
Ph.D.
The nucleus of eukaryotic cells is a vitally important organelle that sequesters the genetic information of the cell, and protects it with the help of two highly evolved structures, the nuclear envelope (NE) and nuclear pore complexes (NPCs). Together, these two structures mediate the bidirectional trafficking of molecules between the nucleus and cytoplasm by forming a barrier. NE transmembrane proteins (NETs) embedded in either the outer nuclear membrane (ONM) or the inner nuclear membrane (INM) play crucial roles in both nuclear structure and functions, including: genome architecture, epigenetics, transcription, splicing, DNA replication, nuclear structure, organization and positioning. Furthermore, numerous human diseases are associated with mutations and mislocalization of NETs on the NE. There are still many fundamental questions that are unresolved with NETs, but we focused on two major questions: First, the localization and transport rate of NETs, and second, the transport route taken by NETs to reach the INM. Since NETs are involved with many of the mechanisms used to maintain cellular homeostasis, it is important to quantitatively determine the spatial locations of NETs along the NE to fully understand their role in these vital processes. However, there are limited available approaches for this task, and moreover, these methods provide no information about the translocation rates of NETs between the two membranes. Furthermore, while the trafficking of soluble proteins between the cytoplasm and the nucleus has been well studied over the years, the path taken by NETs into the nucleus remains in dispute. At least four distinct models have been proposed to suggest how transmembrane proteins destined for the INM cross the NE through NPC-dependent or NPC-independent mechanisms, based on specific features found on the soluble domains of INM proteins. In order to resolve these two major questions, it is necessary to employ techniques with the capabilities to observe these dynamics at the nanoscale. Current experimental techniques are unable to break the temporal and spatial resolution barriers required to study these phenomena. Therefore, we developed and modified single-molecule techniques to answer these questions. First, to study the distribution of NETs on the NE, we developed a new single-molecule microscopy method called single-point single-molecule fluorescence recovery after photobleaching (smFRAP), which is able to provide spatial resolution <10 nm and, furthermore, provide previously unattainable information about NET translocation rates from the ONM to INM. Secondly, to examine the transport route used by NETs destined for the INM, we used a single-molecule microscopy technique previously developed in our lab called single-point edge-excitation sub-diffraction (SPEED) microscopy, which provides spatio-temporal resolution of <10 nm precision and 0.4 ms detection time. The major findings from my doctoral research work can be classified into two categories: (i) Technical developments to study NETs in vivo, and (ii) biological findings from employing these microscopy techniques. In regards to technical contributions, we created and validated of a new single-molecule microscopy method, smFRAP, to accurately determine the localization and distribution ratios of NETs on both the ONM and INM in live cells. Second, we adapted SPEED microscopy to study transmembrane protein translocation in vivo. My work has also contributed four main biological findings to the field: first, we determined the in vivo translocation rates for lamin-B receptor (LBR), a major INM protein found in the nucleus of cells. Second, we verified the existence of peripheral channels in the scaffolding of NPCs and, for the first time, directly observed the transit of INM proteins through these channels in live cells. Third, our research has elucidated the roles that both the nuclear localization signal (NLS) and intrinsically disordered (ID) domains play in INM protein transport. Finally, my work has elucidated which transport routes are used by NETs destined to localize in the INM.
Temple University--Theses

Each organism has to maintain the integrity of its genetic code, which is stored in its DNA. This is achieved by strongly controlled and regulated cellular processes such as DNA replication, -repair and -recombination. An essential element of these processes is the unwinding of the duplex strands of the DNA helix. This biochemical reaction is catalyzed by helicases that use the energy of nucleoside triphophate (NTP) hydrolysis. Although all helicases comprise highly conserved domains in their amino acid sequence, they exhibit large variations regarding for example their structure, their function and their target nucleic acid structures. The main objective of this thesis is to obtain insight into the DNA unwinding mechanisms of three helicases from two different organisms. These helicase vary in their structures and are involved in different pathways of DNA metabolism. In particular the replicative, hexameric helicase Large Tumor-Antigen (T-Antigen) from Simian virus 40 and the DNA repair helicases RecQ2 and RecQ3 from Arabidopsis thaliana are studied. To observe DNA unwinding by these helicases in real-time on the single molecule level, a biophysical technique, called magnetic tweezers, was applied. This technique allows to stretch single DNA molecules attached to magnetic particles. Simultaneously one can measure the DNA end-to-end distance. Special DNA hairpin templates allowed to characterize different parameters of the DNA unwinding reaction such as the unwinding velocity, the length of unwound DNA (processivity) or the influence of forces. From this mechanistic models about the functions of the helicases could be obtained. T-Antigen is found to be one of the slowest and most processive helicases known so far. In contrast to prokaryotic helicases, the unwinding velocity of T-Antigen shows a weak dependence on the applied force. Since current physical models for the unwinding velocity fail to describe the data an alternative model is developed. The investigated RecQ helicases are found to unwind and close short stretches of DNA in a repetitive fashion. This activity is shown for the first time under external forces. The experiments revealed that the repetitive DNA unwinding is based on the ability of both enzymes to switch from one single DNA strand to the other. Although RecQ2 and RecQ3 perform repetitive DNA unwinding, both enzymes differ largely in the measured DNA unwinding properties. Most importantly, while RecQ2 is a classical helicase that unwinds DNA, RecQ3 mostly rewinds DNA duplexes. These different properties may reflect different specific tasks of the helicases during DNA repair processes. To obtain high spatial resolution in DNA unwinding experiments, the experimental methods were optimized. An improved and more stable magnetic tweezers setup with sub-nanometer resolution was built. Additionally, different methods to prepare various DNA templates for helicase experiments were developed. Furthermore, the torsional stability of magnetic particles within an external field was investigated. The results led to selection rules for DNA-microsphere constructs that allow high resolution measurements
Jeder Organismus ist bestrebt, die genetischen Informationen intakt zu halten, die in seiner DNA gespeichert sind. Dies wird durch präzise gesteuerte zelluläre Prozesse wie DNA-Replikation, -Reparatur und -Rekombination verwirklicht. Ein wesentlicher Schritt ist dabei das Entwinden von DNA-Doppelsträngen zu Einzelsträngen. Diese chemische Reaktion wird von Helikasen durch die Hydrolyse von Nukleosidtriphosphaten katalysiert. Obwohl bei allen Helikasen bestimmte Aminosäuresequenzen hoch konserviert sind, können sie sich in Eigenschaften wie Struktur, Funktion oder DNA Substratspezifität stark unterscheiden. Gegenstand der vorliegenden Arbeit ist es, die Entwindungsmechanismen von drei verschieden Helikasen aus zwei unterschiedlichen Organismen zu untersuchen, die sich in ihrer Struktur sowie ihrer Funktion unterscheiden. Es handelt sich dabei um die replikative, hexamerische Helikase Large Tumor-Antigen (T-Antigen) vom Simian-Virus 40 und die DNA-Reparatur-Helikasen RecQ2 und RecQ3 der Pflanze Arabidopsis thaliana. Um DNA-Entwindung in Echtzeit zu untersuchen, wird eine biophysikalische Einzelmolekültechnik, die \"Magnetische Pinzette\", verwendet. Mit dieser Technik kann man ein DNA-Molekül, das an ein magnetisches Partikel gebunden ist, strecken und gleichzeitig dessen Gesamtlänge messen. Mit speziellen DNA-Konstrukten kann man so bestimmte Eigenschaften der Helikasen bei der DNA-Entwindung, wie z.B. Geschwindigkeit, Länge der entwundenen DNA (Prozessivität) oder den Einfluß von Kraft, ermitteln. Es wird gezeigt, dass T-Antigen eine der langsamsten und prozessivsten Helikasen ist. Im Gegensatz zu prokaryotischen Helikasen ist die Entwindungsgeschwindigkeit von T-Antigen kaum kraftabhängig. Aktuelle Modelle sagen dieses Verhalten nicht vorraus, weshalb ein alternatives Modell entwickelt wird. Die untersuchten RecQ-Helikasen zeigen ein Entwindungsverhalten bei dem permanent kurze Abschnitte von DNA entwunden und wieder zusammengeführt werden. Dieses Verhalten wird hier zum ersten Mal unter dem Einfluß externer Kräfte gemessen. Es wird gezeigt, dass die permanente Entwindung auf die Fähigkeit beider Helikasen, von einem einzelen DNA-Strang auf den anderen zu wechseln, zurückzuführen ist. Obwohl RecQ2 und RecQ3 beide das Verhalten des permanenten Entwindens aufzeigen, unterscheiden sie sich stark in anderen Eigenschaften. Der gravierendste Unterschied ist, dass RecQ2 wie eine klassische Helikase die DNA entwindet, während RecQ3 eher bestrebt ist, die DNA-Einzelstränge wieder zusammenzuführen. Die unterschiedlichen Eigenschaften könnten die verschieden Aufgaben beider Helikasen während DNA-Reparaturprozessen widerspiegeln. Weiterhin werden die experimentellen Methoden optimiert, um möglichst hohe Auflösungen der Daten zu erreichen. Dazu zählen der Aufbau einer verbesserten und stabileren \"Magnetischen Pinzette\" mit sub-nanometer Auflösung und die Entwicklung neuer Methoden, um DNA Konstrukte herzustellen. Außerdem wird die Torsions\\-steifigkeit von magnetischen Partikeln in externen magnetischen Feldern untersucht. Dabei finden sich Auswahlkriterien für DNA-gebundene magnetische Partikel, durch die eine hohe Auflösung erreicht wird

Molecular interactions between cellular components such as proteins and nucleic acids govern the fundamental processes of living systems. Technological advancements in the past decade have allowed the characterization of these molecular interactions at the single-molecule level with high temporal and spatial resolution. Simultaneously, progress in computer simulation has enabled theoretical research at the atomistic level, assisting in the interpretation of experimental results. This thesis combines single-molecule force spectroscopy and simulation to explore inter- and intra-molecular interactions. Specifically, we investigate the interaction between RecA and DNA to elucidate the underlying molecular mechanism of the DNA homologous recombination process. We also evaluate the stability of the von Willebrand Factor (vWF) A2 domain to determine the molecular origins of von Willebrand Diseases (vWD). This thesis also describes the development and application of a new single-molecule technique that combines the centrifuge force microscope (CFM) with DNA self-assembled mechanical switches to enable massively parallel repeating force measurements of molecular interactions.
Engineering and Applied Sciences - Applied Physics

Since the discovery of deoxyribonucleic acid (DNA) over 140 years ago, this biomolecule still remains one the most studied macromolecules in nature. The modifications and interactions associated with this duplex biopolymer have been shown to play fundamental roles in cellular machinery. This research project exploited sets of single-molecule detection techniques in parallel with conventional molecular biology methodologies to study i) chemical modification (methylation) of the DNA and its functional role and ii) electrostatic interaction between two homologous DNA duplexes. In this project solid-state nanopores were utilised as a novel approach to probe structural and conformational changes of linear and circular DNA. To begin with, the effect of DNA methylation level in breast cancer cell-lines was investigated. Using solid-state nanopore sensors and a methyl specific antibody (5'-mc), the methylated and unmethylated regions of FOXA1 (a gene associated with breast cancer development) promoter were differentiated. Simultaneously, the methylation level of this gene was evaluated in various breast cancer cell-lines and confirmed the impact of DNA methylation in gene silencing. In addition, using atomic force microscopy analysis, the binding affinity of the antibody to the methylated DNA was determined Furthermore, by employing the same methodologies, the presence of an electrostatic recognition step in homologous segments of a bacteria plasmid within the framework of Kornyshev-Leikin theory was investigated. However to this end, the verification of this model was inconclusive. Nevertheless it was serendipitously found that the plasmid with homologous regions was dimerised and then formed a single loop. This finding would be the motivation behind further experiments to gain a better understanding of the possible sequence dependence of the DNA topology and configuration during cloning and amplification procedures. Furthermore, using a combination of various techniques, the biophysical properties of the monomeric and dimeric plasmids were characterised. Overall, the combined findings of the mentioned projects provided remarkable insights on the molecular biophysics of DNA-DNA and DNA-protein interactions within the framework of the central dogma of molecular biology.

Single molecules and single cells are the fundamental building blocks in biology. Facilitated by the advancement of technology, quantitative single-molecule and single-cell measurements provide a unique perspective toward many biological systems by revealing individual stochasticity and population heterogeneity. Taking advantage of these approaches, we studied chromosome organization and gene expression in bacteria and discovered new biophysical mechanisms: chromosome organization by a nucleoid-associated protein in live bacteria, and transcriptional bursting by the regulation of DNA supercoiling in bacteria.

In this work a system-level approach was taken to the single-molecule fluorescence microscopy of living cells. This primarily involved the unification of relevant information within appropriately structured artefacts that were used to inform and enhance experimentation. Initially the diversity of emerging single-molecule techniques was reviewed and presented with a novel article structure to suit the purpose of designing an experiment (Harriman and Leake 2011). Techniques were grouped by the type of information they could access, rather than the standard organisation centred on the techniques themselves. A bespoke microscope was conceived and built with reference to knowledge and tools from the fields of Architecture and Systems-Engineering. The microscope layout would enable multiple experiment types through independent control of multiple illumination beams. A technique was developed enabling the prescription of evanescent field penetration depth for each incident beam. The various empirical and theoretical results that are used to understand and modify a microscopy experiment were integrated into an internally consistent simulation model (Harriman and Leake. 2013). This was used to inform the selection of experimental components and parameters and ultimately acquire higher data quality as measured by functions such as signal-to-noise ratio (SNR). The combined experimental system of microscope and simulation model was applied in two live-cell investigations. In Escherichia coli, the spatial distribution of membrane bound proteins was investigated and a novel technique was applied to the analysis of colocalisation. Results indicate that NADH dehydrogenase and ATP synthase follow uncorrelated trajectories. This supports the hypothesis of spatial decoupling of molecules that energise the membrane and molecules that use membrane energy. In human carcinoma cells, the mechanism of ligand-receptor binding was investigated. Data was collected prior to and periodically after the addition of ligands, and fluorescence images were acquired of both ligands and receptors. Analyses based on single particle tracking are currently being carried out by a collaborator to extract information on stoichiometry and dynamics at the single-molecule level.

Genetic information is encoded in the long sequence of bases which form DNA, which is replicated during cell division by enzymes known as DNA Polymerases. Polymerases replicate DNA extremely accurately to avoid errors which can cause cell death and diseases such as cancer, although the mechanisms behind these extraordinary fidelities are not well understood. A large conformational change in the protein, in which the “fingers" subdomain closes around an incoming nucleotide, is thought to be implicated in these fidelity mechanisms. Here we present an assay to monitor this conformational change in single polymerase molecules, in real-time. We achieve this using total-internal-reflection-fluorescence (TIRF) microscopy to monitor the fluorescence resonance energy transfer (FRET) of an intra-protein dye labelled DNA Polymerase I (KF) as it binds to surface-immobilised DNA. Initially, we investigated the polymerase fingers-conformations during the pre-chemistry polymerisation reaction, resolving forward and backward rates which would be challenging to observe using ensemble techniques. These observations confirmed that KF closes rapidly around complementary nucleotide, but we discovered that the reverse step, fingers-opening, is particularly slow relative to chemistry. These finger kinetics act to remove the influence of the reaction rate-limiting step on fidelity, surprising given decades of investigations have focused on the rate-limiting step as the key determinant of fidelity. We also use our kinetic measurements to quantify contributions of different reaction steps to the macroscopic error rate of the polymerase. Subsequently, we developed our assay to investigate the fingers-conformations across the entire DNA polymerisation reaction. We observed single-nucleotide incorporations, and processive DNA polymerisation at high and low nucleotide concentrations, which suggested heterogeneous nucleotide incorporation rates. The observations demonstrated that the post-chemistry slow step that limits processive polymerisation occurs before post-chemistry fingers-opening, or is accounted for by post-chemistry fingers-opening. We observe a correlation in turn-over kinetics and binary complex kinetics, suggesting that turn-over rates could be limited by the intrinsic dynamics of the binary complex, as seen in other protein systems, although more work is needed on this.

Biology
Ph.D.
Nuclear pore complexes (NPCs) are large macromolecular gateways embedded in the nuclear envelope of Eukaryotic cells that serve to regulate bi-directional trafficking of particles to and from the nucleus. NPCs have been described as creating a selectively permeable barrier mediating the nuclear export of key endogenous cargoes such as mRNA, and pre-ribosomal subunits as well as allow for the nuclear import of nuclear proteins and some viral particles. Remarkably, other particles that are not qualified for nucleocytoplasmic transport are repelled from the NPC, unable to translocate. The NPC is made up of over 30 unique proteins, each present in multiples of eight copies. The two primary protein components of the NPC can be simplified as scaffold nucleoporins which form the main structure of the NPC and the phenylalanine-glycine (FG) motif containing nucleoporins (FG-Nups) which anchor to the scaffold and together create the permeability barrier within the pore. Advances in fluorescence microscopy techniques including single-molecule and super-resolution microscopy have made it possible to label and visualize the dynamic components of the NPC as well as track the rapid nucleocytoplasmic transport process of importing and exporting cargoes. The focus of this dissertation will be on live cell fluorescence microscopy application in probing the dynamic components of the NPC as well as tracking the processes of nucleocytoplasmic transport.
Temple University--Theses

The p53 protein has been widely studied since its discovery as a tumour suppressor protein in 1989 with applications in cancer diagnostics and anti-cancer therapy. p53 has been found to be mutated in 50% of human cancers. Therefore, understanding the properties, mechanisms and mutations of the protein is undoubtedly of great importance in the pursuit of understanding and treating cancer. In this thesis, single molecule methods such as atomic force microscopy (AFM) and solid-state nanopores were applied to investigate the binding interaction between p53 protein and DNA. The focus of this research was to examine p53 binding to different DNA molecules with or without specific binding sites and to distinguish between bare DNA and different p53-DNA complexes in a label-free manner. Firstly, AFM was utilised to examine DNA and p53 individually to determine the size and characteristics of the analytes separately. Thereafter, successful binding of p53 to DNA was confirmed and statistics were obtained with respect to the size and position of p53 protein along the DNA samples. Next, solid-state nanopores were employed to attempt ultrafast single molecule sensing without the need for labels or immobilisation of the DNA and DNA-p53 complexes. Successful single molecule detection was achieved with nanopipette sensors. Further experiments using low-noise planar nanopore devices resulted in successful discrimination between bare DNA and DNA-p53 complexes in a label-free manner. This new development in the p53 field provides a novel method of detecting the tumour suppressor protein p53 binding to DNA. However, the nanopore characteristics for DNA-p53 complexes with different DNA sequences were within experimental error of each other. This study provides a framework for future development towards the detection and distinction of different p53 interactions with DNA.

The covalent chemistry of individual reactants bound within a protein nanopore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. An efficient method to incorporate unnatural amino acid is semisynthesis, which allows site-specific modification with a chemically-defined functional group. However, relatively little work has been done on engineered membrane proteins. This deficiency stems from attributes inherent to proteins that interact with lipid bilayer, notably the poor solubility in aqueous buffer. In the present work, four different derivatives α-hemolysin (αHL) monomer were obtained either by two- or three-way native chemical ligation. The semisynthetic αHL monomers were successfully refolded to heptameric pores and used as nanoreactors to study single-molecule chemistry. The semisynthetic pores show similar biophysical properties to native αHL pores obtained from an in vitro transcription and translation technique. Interestingly, when αHL pores with one semisynthetic subunit containing a terminal alkyne group were used to study Cu(I)-catalyzed azide-alkyne cycloaddition, a long-lived intermediate in the reaction was directly observed.

Engineered protein nanopores can be used to investigate a wide range of dynamic processes in real time and at the single-molecule level, for example covalent bond making and breaking or the interaction of ligands with their cognate binding sites. The detection of such processes is accomplished by monitoring the current carried by ions through the pore in an applied potential, which is modulated as molecules of interest interact with engineered binding sites within the pore. In contrast to ensemble measurements, where the behaviour of individual molecules is obscured by averaging, single-channel recordings can identify short-lived intermediates and rare reaction pathways, thereby adding to our understanding of fundamental processes in chemistry and biology. The goal of my thesis work was to engineer alpha-hemolysin (αHL) pores to gain insight into such processes. Chapter 1 provides an overview of common techniques used to study single- molecule processes, in particular single channel recordings. General techniques to engineer ion channels and pores are presented, followed by examples of how the alpha-HL pore has been engineered to monitor dynamic processes at the single- molecule level. Chapter 2 describes how alpha-HL pores can be chemically modifeed with a tridentate "half-chelator" ligand. Single channel recordings show that this modifeed pore can be used to determine rates of chelation and the stability of divalent metal ion complexes. The modifeed pore can also be used as a stochastic sensor for the detection of different divalent metal ions in solution. Chapter 3 investigates the chelate-cooperativity between two half-chelator ligands installed in close proximity in the alpha-HL pore, as they form a full complex with a single Zn²⁺ ion. The single channel recordings reveal a two step process, in which the Zn²⁺ ion must fiferst bind to one of the two half-chelators, before the second one completes the complex. The rate constants for all the major steps of the process are determined and the extent of cooperativity between the half-chelators is quantifeed. Chapter 4 demonstrates that genetically encoded subunit dimers of alpha-HL can be used to control the subunit arrangement in the heptameric pore. Although techniques exist to prepare heteroheptameric pores, pores containing more than one type of modifeed subunit are not commonly used because it is impossible to distinguish between the permutations of the pore. By using subunit dimers, heptamers in which two defefined subunits are adjacent to each other can be formed, which increases the range of structures that can be obtained from engineered protein nanopores. Chapter 5 explores the possibility of following the nuclease activity of a metal complex in the alpha-HL pore at the single-molecule level. The Rh(III) complex [Rh(bpy)2phzi]²⁺ binds strongly to CC mismatches in dsDNA, and on activation with UV light promotes the cleavage of one of the two strands. To follow this reaction by single channel recording, a piece of dsDNA with the bound Rh-complex was immobilised in the HL pore and the single current changes under UV irradiation were monitored. The preliminary data indicate that the rate of the photocleavage reaction can be measured.

Gene regulation is vital to the success of all living organisms. Understanding this complex process is crucial to our knowledge of how cells function and how in some cases they can lead to debilitating or even fatal disease. In this thesis I focus on a set of DNA-binding proteins known as transcription factors (TFs), proteins fundamental to the process of gene regulation at the level of transcription. I develop assays and techniques for the detection and quantitation of TFs in vitro and in vivo as well as a method for TF encapsulation and release. The advantages of the TF detection assays in this thesis are made possible through the use of single-molecule (sm) fluorescence. This methodology enables detection of individually labeled molecules allowing discrimination of sample heterogeneities inaccessible with ensemble techniques. Here I present two different TF assays based on two sm observables: relative probe stoichiometry and Förster resonance energy transfer (FRET). The first assay design, based on stoichiometry, detects TFs using TF-dependent coincidence of two distinctly labelled DNA ‘half-sites’. I demonstrate sensitive detection (~ pM) in solution and on surfaces, multiplexed detection of multiple TFs, and detection in cell lysates. A kinetic model of the system is also developed, verified experimentally and used to quantify TF concentrations without the need for a calibration curve. The second assay design, based on FRET, is a novel approach to TF detection using TFmediated DNA bending. TFs are detected by bending the sensor and monitored with FRET at the single-molecule or ensemble level. I demonstrate TF detection in purifed form and expressed in cell lysates. As this sensor was designed for use in vivo, methods to hinder nuclease degradation are explored. For TF detection in vivo, I describe a successful strategy to internalise fluorescently labeled molecules into live E.coli. Viability and internalisation efficiency are characterised and ensemble measurements with FRET standards are demonstrated. Importantly, sm FRET measurements in vivo are achieved opening many exciting possibilities. The FRET based TF sensor is then internalised as a step towards real-time in vivo monitoring of TF concentrations. Finally a system based on DNA nanotechnology is presented for the non-covalent encapsulation and release of TFs. Such a system could be delivered into a cell to alter levels of gene expression using external stimuli as inputs. We believe these tools will generate valuable information in the study of prokaryotic gene expression as well as providing a potential commercial avenue towards diagnostics.

This work investigates the dynamics of gene regulatory network formed by Oct4, Sox2 and Nanog in embryonic stem cells (ESCs). Despite a large number of existing studies on stem cells, current technologies used often force a compromise between quantification of gene expression via bulk measurements and qualitative imaging of cell heterogeneity. There are few options that allow for accurate and quantitative single-cell analysis that is robust yet not associated with a high degree of technical difficulty or obscured by amplification. Here, we adapted a high resolution, single-molecule RNA fluorescent in situ hybridization technique (smFISH) to study gene expression of the core pluripotency circuit upon various types of perturbations such as differentiation, induction or knockdown of one of the three pluripotent factors. We used previously-published smFISH procedures as our initial template for investigating gene regulatory dynamics of the core pluripotency circuit during those perturbation assays. To obtain a more comprehensive picture of the regulatory circuit, we developed a modified smFISH strategy to measure mRNA and protein expression simultaneously in single ESCs. By incorporating a novel modification into the smFISH technique which allows accurate quantification of transcripts that differ by short sequences, we managed to identify a few interesting features of the core pluripotency circuit. Taken together, we demonstrated our ability to perform single-cell, single-molecule assays that reveal highly quantitative information in unprecedented detail.

ATP synthase is a ubiquitous transmembrane protein that utilises the free energy available from ion gradients across lipid membranes to synthesise adenosine triphosphate (ATP). It may be separated into two parts - the membrane-embedded (i.e. hydrophobic) FO and the hydrophilic F₁. Each undergoes a rotary motion. Single-molecule studies on the rotation of the isolated hydrophilic F₁ have been performed for many years; attempts to construct an experiment in which to view the rotation of the membrane-embedded F₁F_O complex under high space- and time- resolution (such as by attachment of a rotational probe) have not yet seen a satisfactory method emerge in the literature. Most particularly, a clear ability to generate and control a proton-motive force across the membrane in which the F₁F_O is sited is needed to probe ATP synthesis. This thesis presents the development of a candidate method for such single-molecule studies. By the use of a water-in-oil emulsion, giant unilamellar lipid vesicles are formed which entrap arbitrary components - including functionalised gold nanospheres of 60-100 nm diameter, which move freely in the internal space. A charge-based lipid fusion is developed, using mixtures of natural lipid extracts with anionic and cationic lipids. It is demonstrated that anionic giant vesicles fuse with cationic small vesicles with full content mixing and transfer of bilayer leaflets. It is shown that F₁F_O is functional in the cationic lipid mixture. Methods are shown to bind such a cationic proteoliposome to a surface and for it to fuse with an anionic giant vesicle containing functionalised gold nanospheres. Backscatter laser darkfield is used to search for rotation of the gold nanospheres under ATP hydrolysis conditions of the F₁F_O; unidirectional rotation is seen in one instance and other suggestive traces are shown with speculative analysis. Further work is proposed.

The miniaturization afforded by the integration of microfluidic technologies within lab-on-a-chip devices has greatly enhanced analytical capabilities in several key applications. Microfluidics has been utilized in a wide range of areas including sample preparation and analysis, DNA microarrays, cell detection, as well as environmental monitoring. The use of microfluidics in these applications offer many unique advantages: reduction in the required sample size, reduction in analysis time, lowered cost through batch fabrication, potentially higher throughput and the vision of having such devices used in portable systems. Nanopore sensors are a relatively new technology capable of detection and analysis with single-molecule sensitivity, and show promise in many applications related to the diagnosis and treatment of many diseases. Recently, some research groups demonstrated the integration of nanopores within microfluidic devices to increase analytical throughput. This thesis describes a methodology for integrating nanopore sensors within microfluidic devices with the aim of enhancing the analytical capabilities required to analyze biomolecular samples. In this work, the first generation of an integrated nanopore-microfluidic device contained multiple independently addressable microfluidic channels to fabricate an array of nanopore sensors using controlled breakdown (CBD). Next, for the second generation, we added pneumatic microvalves to manipulate electrical and fluidic access through connected microfluidic channels. As a proof-of-concept, single molecules (single- and double-stranded DNA, proteins) were successfully detected in the devices. It is also demonstrated that inclusion of the microfluidic via (microvia) limited the exposed area of the embedded silicon nitride membrane to the solution. This helped in localizing nanopore formation by confining the electric field to specific regions of the insulating membrane while significantly reducing high frequency noise in the ionic current signal through the reduction of chip capacitance. The devices highlighted in this thesis were designed and fabricated using soft lithography techniques which are available in most biotechnology laboratories. The core of this thesis is based on two scientific articles (Chapters 3 and 4), which are published in peer-reviewed scientific journals. These chapters are preceded by an introductory chapter and another chapter detailing the experimental setup and the methods used during the course of this study.

Telomerase and telomere play crucial roles in the maintenance of genomic stability. Through its ability to extend chromosome ends with G-rich telomeric sequence, telomerase solves the end-replication problem of linear chromosomes and allows complete replication of the genetic information. Telomere along with its protein partners solves the end-protection problem and guards the chromosome ends against aberrant DNA damage response. In this thesis, I present two single-molecule fluorescence-based studies that determined the functional structure of telomerase RNA within active telomerase holoenzyme and probed the structure of telomere and its dependence on telomere binding proteins. In the first study, we developed a single-molecule Förster resonance energy transfer (FRET) assay to interrogate the structure of telomerase RNA within active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity. FRET signals from individual enzyme molecules during active binding events were then used to determine the organization of telomerase RNA within active telomerase. Using this assay, we have identified an active conformation of telomerase in which the conserved telomerase RNA pseudoknot is properly folded. In the second study, we used super-resolution fluorescence technique STochastic Optical Reconstruction Microscopy (STORM) to probe the structure of mammalian telomere. We showed that previously described telomere loop structures are detected by STORM imaging. Removal of telomere-binding protein TRF2 significantly reduces the fraction of telomeres found in loops. Furthermore, this reduction of telomere loops occurs in the absence of ATM-dependent DNA damage signaling and non-homologous end joining mediated chromosome fusion, suggesting a direct role of TRF2 in the formation or maintenance of telomere loops.

This thesis explores stochastic modeling and Bayesian inference strategies in the context of the following three problems: 1) Modeling the complex interactions between and within molecules; 2) Extracting information from stepwise signals that are commonly found in biophysical experiments; 3) Improving the computational efficiency of a non-parametric Bayesian inference algorithm. Chapter 1 studies the data from a recent single-molecule biophysical experiment on enzyme kinetics. Using a stochastic network model, we analyze the autocorrelation of experimental fluorescence intensity and the autocorrelation of enzymatic reaction times. This chapter shows that the stochastic network model is capable of explaining the experimental data in depth and further explains why the enzyme molecules behave fundamentally differently from what the classical model predicts. The modern knowledge on the molecular kinetics is often learned through the information extracted from stepwise signals in experiments utilizing fluorescence spectroscopy. Chapter 2 proposes a new Bayesian method to estimate the change-points in stepwise signals. This approach utilizes marginal likelihood as the tool of inference. This chapter illustrates the impact of the choice of prior on the estimator and provides guidelines for setting the prior. Based on the results of simulation study, this method outperforms several existing change-points estimators under certain settings. Furthermore, DNA array CGH data and single molecule data are analyzed with this approach. Chapter 3 focuses on the optional Polya tree, a newly established non-parametric Bayesian approach (Wong and Li 2010). While the existing study shows that the optional Polya tree is promising in analyzing high dimensional data, its applications are hindered by the high computational costs. A heuristic algorithm is proposed in this chapter, with an attempt to speed up the optional Polya tree inference. This study demonstrates that the new algorithm can reduce the running time significantly with a negligible loss of precision.
Statistics

F₁-ATPase, the sector of ATP synthase where the synthesis of cellular ATP occurs, is a rotary molecular motor in its own right. Driven by ATP hydrolysis, direct observation of the rotation of the central axis within single molecules of F₁ is possible. Operating at close to 100% efficiency, F₁ from thermophilic Bacillus has been shown to produce ~40pN˙nm of torque during rotation. This thesis details the groundwork required for the direct measurement of the torque produced by F₁ using a rotary angle clamp, an optical trapping system specifically designed for application to rotary molecular motors. Proof-of-concept experiments will be presented thereby demonstrating the ability to directly manipulate single F₁ molecules from Escherichia coli and yeast mitochondria (Saccharomyces cerevisiae), along with activation of F₁ out of its inhibited state by the application of external torque. Despite in-depth knowledge of the rotary mechanism of F₁ from thermophilic Bacillus, the rotation of F₁ from Escherichia coli is relatively poorly understood. A detailed mechanical characterization of E.coli F₁ will be presented here, with particular attention to the ground states within the catalytic cycle, notably the ATP-binding state, the catalytic state and the inhibited state. The fundamental mechanism of E.coli F₁ appears to depart little from that of F₁ from thermophilic Bacillus, although, at room temperature, chemical processes occur faster within the E.coli enzyme, in line with considerations regarding the physiological conditions of the different species. Also presented here is the verification of the rotary nature of yeast mitochondrial F₁. The torque produced by F₁ from thermophilic Bacillus, E.coli and yeast mitochondria is the same, within experimental error, despite their diverse evolutionary and environmental origins.

Faithful replication of genomic DNA is crucial for the survival of a cell. In order to achieve high-level accuracy in copying its genome, all cells employ replicative DNA polymerases that have intrinsic high fidelity. When an error occurs on the template DNA strand, in the form of lesions caused by diverse chemicals, reactive oxygen species, or UV light, the high-fidelity replicative DNA polymerases are stalled. To bypass these replication blocks, cells harbor multiple specialized translesion DNA polymerases that are error-prone and therefore able to accommodate the lesions and continue DNA synthesis. As a result of their low fidelity, the translesion polymerases are associated with increased mutagenesis, drug resistance, and cancer. Therefore, the access of the translesion polymerases to DNA needs to be tightly controlled, but how this is achieved has been the subject of debate. This Thesis presents the development of a co-localization single-molecule spectroscopy (CoSMoS) method to directly visualize the loading of the Escherichia coli replicative polymerase on DNA, as well as the exchange between the replicative polymerase and the translesion polymerases Pol II and Pol IV. In contrast to the toolbelt model for the exchange between the polymerases, this work shows that the translesion polymerases Pol II and Pol IV do not form a stable complex with the replicative polymerase Pol IIIα on the β-clamp. Furthermore, we find that the sequential activities of the replication proteins: clamp loader, clamp, and Pol IIIα, are highly organized while the exchange with the translesion polymerases is disordered. This exchange is not determined by lesion-recognition but instead a concentration-dependent competition between the replicative and translesion polymerases for the hydrophobic groove on the surface of the β-clamp. Hence, our results provide a unique insight into the temporal organization of events in DNA replication and translesion synthesis.

In the field of biophysics, the study of the thermodynamic characteristics of biomolecules, such as DNA, RNA or proteins, allows us to understand more about the building blocks of life. The thermodynamic characterization of the biomolecule gives us clues as to their functions and capabilities inside every living organism. The thermodynamic characterization of nucleic acids describes how temperature affects the stability and the structure of double stranded DNA. The melting temperature of DNA (T(M)) is defined as the temperature at which half of the DNA strands in a bulk solution experiment are in the double stranded DNA (dsDNA) or random coil configuration and half of the DNA are in the single-stranded DNA (ssDNA) configuration. Using T(M), it has been possible to experimentally determine the thermodynamic parameters of Delta-G, Delta-H and Delta-S. Viceversa, when the thermodynamic parameters of a given nucleic acid sequence are known, the TM can be predicted. This effect has important applications for biomolecule techniques such as PCR (Polymerase chain reaction) or sequencing. Traditionally thermodynamic properties of DNA have been determined using bulk techniques such as calorimetry or UV absorbance. In both cases the melting temperature has been determined by changing the temperature or pH of the entire sample. Over the past two decades single molecule force spectroscopy has been established as a powerful, accurate and bulk-complementary method of characterizing the thermodynamics of nucleic acids. Optical trapping is an experimental technique which allows force to be exerted on a micrometric particle by using the radiation pressure of light. The miniTweezers (mT) is the newest generation of optical tweezers instruments. This instrument can be used to exert and measure forces in a range between 1-200 pN and has unprecedented resolution (0.1 pN in force an around 1 nm in distance) with very high thermal and noise stability. Optical trapping is very useful in the field of molecular biology because it allows forces to be exerted on single biomolecules bonded to the micrometric particle. This technique is used to carry out pulling experiments on single molecules allowing us to study the mechanical, thermodynamic and kinetic properties of the molecule. Mechanical melting or unzipping is a process that consists of pulling apart the two strands of the dsDNA until the base pairs are disrupted and the molecule converts into ssDNA. In this case, and in contrast to other techniques, force, rather than temperature or pH, is used to open the molecule. Past experiments have shown that better resolution can be obtained using single molecule techniques than can be obtained using bulk experiments. Although force unzipping provides a direct estimation of Delta-G at room temperature, extracting the value of TM always requires the determination of the Delta-H and Delta-S contributions and until now has not been reliable accomplished.
En el campo de la biofísica, el estudio de las características termodinámicas de las biomoléculas, como ADN, ARN o proteínas, permite conocer más sobre los componentes básicos de la vida. La caracterización termodinámica de las biomoléculas nos proporciona pistas sobre sus funciones y capacidades dentro de un organismo vivo. La caracterización termodinámica de los ácidos nucleicos describe como la temperatura afecta la estabilidad y la estructura de la doble cadena de ADN. La temperatura de melting del ADN (TM) se define como la temperatura a la cual la mitad de las moléculas de ADN disueltas en una solución se encuentran en configuración de doble cadena (dsDNA) y la otra mitad se encuentra en la configuración de cadena individual (ssDNA). Conociendo el valor de la TM es posible determinar experimentalmente los parámetros termodinámicos: Delta-G, Delta-H y Delta-S. Viceversa, cuando los parámetros termodinámicos de la secuencia de un ácido nucleico es conocido, la TM puede ser predecida. Este efecto tiene importantes aplicaciones en técnicas de biología molecular como PCR (en inglés Polymerase chain reaction) o secuenciación. Tradicionalmente las propiedades termodinámicas del ADN han sido medidas utilizando técnicas de volumen como calorimetría o absorbancia de UV. En ambos casos la TM ha sido calculada modificando la temperatura o el pH de toda la muestra. En las pasadas dos décadas, las técnicas de espectroscopía de fuerzas sobre moléculas individuales, han sido reconocidas como técnicas de un gran valor y precisión cuyos resultados en el estudio de la caracterización termodinámica pueden ser considerados perfectamente complementarios a los medidos en técnicas de volumen. La técnica de atrapamiento óptico es una técnica experimental la cual permite ejercer fuerza sobre una partícula micrométrica utilizando la presión de radiación de la luz. Las minipinzas (en inglés minitweezers) es una nueva generación a los instrumentos de pinzas ópticas. Este instrumento puede ser usado para ejercer y medir fuerzas en un rango de entre 1-200pN y con una resolución en fuerza y distancia sin precedentes. El atrapamiento óptico es muy útil en el campo de la biología molecular permitiendo ejercer fuerzas sobre biomoléculas individuales enganchadas. Esta técnica es usada para llevar a cabo experimentos de estiramiento sobre moléculas individuales permitiendo el estudio de las propiedades mecánicas, termodinámicas y cinéticas de la molécula bajo estudio. El experimento de unzipping o melting mecánico es un proceso que consiste en separar las dos hebras de la dsDNA hasta que los enlaces entre los pares de bases complementarios son deshechos y la molécula se convierte en ssDNA. En este caso la fuerza es usada como medio para abrir la molécula, en vez de la temperatura o el pH como en otras técnicas. Pasados experimentos han mostrado que podemos obtener mejor resolución utilizando técnicas de moléculas individuales que utilizando técnicas en volumen. Aunque la fuerza de unzipping nos proporciona una estimación directa de Delta-G a temperatura ambiente, para poder extraer el valor de TM requiere conocer las contribuciones de Delta-H y Delta-S y hasta ahora no ha sido posible. Para llevar a cabo una completa caracterización termodinámica de ácidos nucleicos es importante conocer ambas magnitudes (Fuerza y Temperatura). El mejor camino para hacer este análisis es llevar a cabo experimentos de unzipping sobre moléculas individuales de ADN a diferentes temperaturas. Por ello hemos desarrollado un novedoso instrumento de pinzas ópticas con un controlador de temperatura que nos permite modificar y cambiar la temperatura de manera local y rápida. Se ha usado un específico láser calentador con una longitud de onda con una alta absorción en agua que permite cubrir un amplio rango de temperaturas. Este instrumento nos permite grabar diversas curvas de fuerza/extensión para una molécula individual a varias temperaturas con una buena estabilidad térmica y mecánica. Este diseño tiene ciertas mejoras para reducir la convección, el cual ha sido un grave problema en previos equipos calentados a través de un láser. Este equipo ha sido usado para hacer experimentos de ADN, lo que nos ha permitido hacer un análisis promediado de Delta-G, Delta-S y Delta-H entre pares de bases en un rango de temperatura entre 5ºC y 50ºC.

The influenza virus hemagglutinin (HA) surface protein is a primary antigenic target for neutralization of viral infection. HA also mediates membrane fusion between the virus and a cell, which is the first critical step during infection. Traditional techniques to study infection neutralization by antibodies or the membrane fusion process rely on ensemble measurements, confounding the precise mechanism of infection neutralization and obscuring transient conformational intermediates. This dissertation describes advances made in a fluorescence microscopy-based single-particle fusion assay to overcome the limitations of ensemble measurements in these types of studies. Virus particles are labeled to visualize lipid mixing between a virus and a target membrane formed upon a glass or polymer support. Optionally, the viral lumen can be labeled to visualize the subsequent release of viral contents.

Protein-DNA interactions govern the fundamental cellular processes of DNA replication, transcription, repair, and chromosome organisation. Despite their importance, the detailed molecular mechanisms of protein-DNA interactions and their organisation in the cell remain elusive. The complexity of molecular biology demands new experimental concepts that resolve the structural and functional diversity of biomolecules. In this thesis, I describe fluorescence methods that give a direct view on protein-DNA interactions at the single-molecule level. These methods employ photoswitching to control the number of active fluorophores in the sample. Forster Resonance Energy Transfer (FRET) measures the distance between a donor and an acceptor fluorophore to report on biomolecular structure and dynamics in vitro. Because a single distance gives only limited structural information, I developed "switchable FRET" that employs photoswitching to sequentially probe multiple FRET pairs per molecule. Switchable FRET resolved two distances within static and dynamic DNA constructs and protein-DNA complexes. Towards application of switchable FRET, I investigated aspects of the nucleotide selection mechanism of DNA polymerase. I further explored application of single-molecule imaging in the complex environment of the living cell. Photoswitching was used to resolve the precise localisations of individual fluorophores. I constructed a super-resolution fluorescence microscope to image fixed cellular structures and track the movement of individual fluorescent fusion proteins in live bacteria. I applied the method to directly visualise DNA repair processes by DNA polymerase I and ligase, generating a quantitative account of their repair rates, search times, copy numbers, and spatial distribution in the cell. I validated the approach by tracking diffusion of replisome components and their association with the replication fork. Finally, super-resolution microscopy showed dense clusters of SMC (Structural Maintenance of Chromosomes) protein complexes in vivo that have previously been hidden by the limited resolution of conventional microscopy.

DNA polymerases are a family of molecular machines involved in high-fidelity DNA replication and repair, of which DNA polymerase I (Pol) is one the best-characterized members. Pol is a strand-displacing polymerase responsible for Okazaki fragment synthesis and base-excision repair in bacteria; it consists of three protein domains, which harbour its 5’-3' polymerase, 3’-5’ exonuclease and 5’ endonuclease activities. In the first part of the thesis, we use a combination of single-molecule Förster resonance energy transfer (smFRET) and rigid-body docking to probe the structure of Pol bound to its gapped-DNA substrate. We show that the DNA substrate is highly bent in the complex, and that the downstream portion of the DNA is partly unwound. Using all-atom molecular dynamics (MD) simulations, we identify residues in the polymerase important for strand displacement and for downstream DNA binding. Moreover, we use coarse-grained simulations to investigate the dynamics of the gapped-DNA substrate alone, allowing us to propose a model for specific recognition and binding of gapped DNA by Pol. In the second part of the thesis, we focus on the catalytically important conformational change in Pol that involves the closing of the ‘fingers’ subdomain of the protein around an incoming nucleotide. We make use of the energy decomposition method (EDM) to predict the stability-determining residues for the closed and open conformations of Pol, and test their relevance by site-directed mutagenesis. We apply the unnatural amino acid approach and a single-molecule FRET assay of Pol fingers-closing, to show that substitutions in the stability-determining residues significantly affect the conformational equilibrium of Pol. In the final part of the thesis, we attempt to study Pol in its native environment of the living cell. We make use of the recently developed method of internalization by electroporation, and optimize it for organically labelled proteins. We demonstrate the internalization and single-molecule tracking of Pol, and provide preliminary data of intra-molecular FRET in Pol, both at the single-cell and single-molecule levels. Finally, by measuring smFRET within an internalized gapped-DNA construct, we observe DNA binding and bending by endogenous Pol, confirming the physiological relevance of our in vitro Pol-DNA structure.