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Robust, high-yield integration of nanoscale components such as graphene nanoribbons, nanoparticles, or single-molecules with conventional electronic circuits has proven to be challenging. This difficulty arises because the contacts to these nanoscale devices must be precisely fabricated with angstrom-level resolution to make reliable connections, and at manufacturing scales this cannot be achieved with even the highest-resolution lithographic tools. Here we introduce an approach that circumvents this issue by precisely creating nanometer-scale gaps between metallic carbon electrodes by using a self-aligning, solution-phase process, which allows facile integration with conventional electronic systems with yields approaching 50%. The electrode separation is controlled by covalently binding metallic single-walled carbon nanotube (mCNT) electrodes to individual DNA duplexes to create mCNT-DNA-mCNT nanojunctions, where the gap is precisely matched to the DNA length. These junctions are then integrated with top-down lithographic techniques to create single-molecule circuits that have electronic properties dominated by the DNA in the junction, have reproducible conductance values with low dispersion, and are stable and robust enough to be utilized as active, high-specificity electronic biosensors for dynamic single-molecule detection of specific oligonucleotides, such as those related to the SARS-CoV-2 genome. This scalable approach for high-yield integration of nanometer-scale devices will enable opportunities for manufacturing of hybrid electronic systems for a wide range of applications. © 2024 American Chemical Society., NSF-2036865/2328217; W. M. Keck Foundation, WMKF

Robust, high-yield integration of nanoscale components such as graphene nanoribbons, nanoparticles, or single-molecules with conventional electronic circuits has proven to be challenging. This difficulty arises because the contacts to these nanoscale devices must be precisely fabricated with angstrom-level resolution to make reliable connections, and at manufacturing scales this cannot be achieved with even the highest-resolution lithographic tools. Here we introduce an approach that circumvents this issue by precisely creating nanometer-scale gaps between metallic carbon electrodes by using a self-aligning, solution-phase process, which allows facile integration with conventional electronic systems with yields approaching 50%. The electrode separation is controlled by covalently binding metallic single-walled carbon nanotube (mCNT) electrodes to individual DNA duplexes to create mCNT-DNA-mCNT nanojunctions, where the gap is precisely matched to the DNA length. These junctions are then integrated with top-down lithographic techniques to create single-molecule circuits that have electronic properties dominated by the DNA in the junction, have reproducible conductance values with low dispersion, and are stable and robust enough to be utilized as active, high-specificity electronic biosensors for dynamic single-molecule detection of specific oligonucleotides, such as those related to the SARS-CoV-2 genome. This scalable approach for high-yield integration of nanometer-scale devices will enable opportunities for manufacturing of hybrid electronic systems for a wide range of applications. © 2024 American Chemical Society., NSF-2036865/2328217; W. M. Keck Foundation, WMKF

DNA methylation is a prominent epigenetic modification modulating proper access to and storage of genomic information in many eukaryotic cells. The distribution of methylated CpG dinucleotides (mCGs) throughout the genome is dynamic and proper maintenance is required throughout the life of an organism. The density of DNA methylation is often correlated with transcriptional outputs, but the underlying mechanism of how mCG density is readout by the cell is not fully understood. One feature of densely methylated regions of the genome is there ability to recruit reader proteins containing Methyl-CpG Binding Domains (MBDs), which help regulate gene expression. Here we study MBD binding to densely methylated DNA using a single molecule fluorescent imaging system called DNA curtains. These techniques are inherently limited by the diffraction of light, complicating our ability to map MBD-binding events to local regions of DNA methylation. In this study, we developed a high resolution system using dCas9 technology to map binding events on DNA curtains with high accuracy. Our novel single molecule approach confirmed that MBDs selectively bind densely methylated DNA and revealed special features within these regions that may be important to MBD recruitment throughout the genome.

Cyanine fluorophores are extensively used in fluorescence spectroscopy and imaging. Upon continuous excitation, especially at excitation conditions used in single-molecule and super-resolution experiments, photo-isomerized states of cyanines easily reach population probabilities of around 50%. Still, effects of photo-isomerization are largely ignored in such experiments. Here, we studied the photo-isomerization of the pentamethine cyanine 5 (Cy5) by two similar, yet complementary means to follow fluorophore blinking dynamics: fluorescence correlation spectroscopy (FCS) and transient-state (TRAST) excitation-modulation spectroscopy. Additionally, we combined TRAST and spectrofluorimetry (spectral-TRAST), whereby the emission spectra of Cy5 were recorded upon different rectangular pulse-train excitations. We also developed a framework for analyzing transitions between multiple emissive states in FCS and TRAST experiments, how the brightness of the different states is weighted, and what initial conditions that apply. Our FCS, TRAST, and spectral-TRAST experiments showed significant differences in dark-state relaxation amplitudes for different spectral detection ranges, which we attribute to an additional red-shifted, emissive photo-isomerized state of Cy5, not previously considered in FCS and single-molecule experiments. The photo-isomerization kinetics of this state indicate that it is formed under moderate excitation conditions, and its population and emission may thus deserve also more general consideration in fluorescence imaging and spectroscopy experiments., QC 20230307

Spectroscopy is a key analytical tool that provides valuable insight into molecular structure and is widely used to identify chemical samples. Action spectroscopy combines spectroscopic methods with mass spectrometry, enabling the analysis of low-density gas phase molecular ions. So far, commonly reported action spectroscopy techniques destructively record spectra of ensembles of molecular ions, where the presence of multiple chemical and isomeric species greatly complicates the interpretation of the spectra. Here, we extend the action spectroscopy to the purest possible sample: a single gas phase molecule. The resulting single molecule vibrational spectroscopy is initially demonstrated by the first recorded spectrum of a single gas phase polyatomic molecule to our knowledge. This thesis presents the experimental setup, method, and results of the achieved single-molecule vibrational spectroscopy. The apparatus centers around a cryogenic ion trap, where a single molecular ion is confined after being mass-selected by a quadrupole mass filter. A laser-cooled 88Sr+ ion is co-trapped with the molecular ion. The two ions are sympathetically cooled to a Coulomb crystal at milliKelvin temperatures. We developed nondestructive mass spectrometry based on the crystal's secular motion frequencies. This technique enables ``tagging'' spectroscopy with a single molecular ion, where vibrational transitions are monitored by a nitrogen molecule tag. The single molecule vibrational spectra are deduced based on the vibration-induced de-tagging rate. We showed this single molecule vibrational spectroscopy is generalizable to a broad class of molecular ions and is a powerful analytical tool for the nondestructive identification of single molecules.

Antifreeze proteins (AFPs) bind ice to reduce freezing temperatures and arrest ice crystal ripening, making AFPs essential for the survival of many organisms in ice-laden environments and attractive as biocompatible antifreezes in many applications. While their activity was identified over 50 years ago, the physical mechanisms through which they function are still debated because experimental insights at the molecular scale remain elusive. Here, we introduce subzero nanoscopy by the design and incorporation of a freezing stage on a commercial super-resolution setup to resolve the interfacial dynamics of single AFPs with ice crystal surfaces. Using this method, we demonstrate irreversible binding and immobilization (i.e., pinning) of individual proteins to the ice/water interface. Surprisingly, pinning is lost and adsorption becomes reversible when freezing point depression activity, but not ice recrystallization inhibition, is eliminated by a single mutation in the ice-binding site of the AFP. Our results provide direct experimental evidence for the adsorption-inhibition paradigm, pivotal to all theoretical descriptions of freezing point depression activity, but also reveal that reversible binding to ice must be accounted for in an all-inclusive model for AFP activity. These mechanistic insights into the relation between interfacial interactions and activity further our understanding and may serve as leading principles in the future design of highly potent, biocompatible antifreezes with tunable affinity.

The main goal of the studies presented in this dissertation is to continue the endeavor in improving of energy-probing capability of STM for single molecule spectroscopy. Here, we extend energy spectrum into THz region for STM vibrational spectroscopy by combining an 8-Kelvin scanning tunneling STM with a continuous wave THz source. Development of THz rectification technique allowed probing of vibrational excitations with simultaneous chemical sensitivity, THz field sensitivity and <10 MHz energy resolution. On the other hand, by incorporating tunable NIR lasers into an STM junction, we demonstrate the energy-resolved probing of vibrational overtone resonant excitation through the vibration-mediated conformational transition of single pyrrolidine molecules. Photon equilibrium action spectroscopy was performed to observe wavelength dependent resonance excitation features with sub-meV energy resolution. These features were shown to be sensitive to the molecular local adsorption environment while being insensitive to the junction near field profile induced by junction plasmon excitation or the optical setup. Moreover, the light induced equilibrium change in the reversible conformational transition of pyrrolidine leads to generation of photocurrent. Using optical rectification spectroscopy, we investigated the influence of laser power and tunneling setpoint on the photocurrent. The photocurrent was shown to be governed by the competition between photon and electron contribution to the molecular reversible transition, which can be tuned through laser power, bias and tip-molecular interaction. Through the optical rectification study of pyrrolidine, we demonstrate its potential application as a tunable single-molecule photo-rectifier device when trapped between two electrodes.

Cells sense and respond to mechanical stimuli for activation, proliferation, migration, and differentiation. The associated mechanosensing and biomechanical properties of cells and tissues are significantly implicated in the context of cancer, fibrosis, dementia, and cardiovascular diseases. To gain more mechanobiology insights, dynamic force spectroscopies (DFSs), particularly optical tweezers (OT), have been further advanced to enable in situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside of a cell. In this review, we first explain the classic OT-DFS rationales and discuss their applications to protein biophysics, extracellular biomechanics, and receptor-mediated cell mechanosensing. As a non-invasive technique, optical tweezers’s unique advantages in probing cytoplasmic protein behaviors and manipulating organelles inside living cells have been increasingly explored in recent years. Hereby, we then introduce and highlight the emerging OT rationales for intracellular force measurement including refractive index matching, active–passive calibration, and change of light momentum. These new approaches enable intracellular OT-DFS and mechanical measurements with respect to intracellular motor stepping, cytosolic micro-rheology, and biomechanics of irregularly shaped nuclei and vesicles. Last but not least, we foresee future OT upgrades with respect to overcoming phototoxicity and system drifting for longer duration live-cell measurements; multimodal integration with advanced imaging and nanotechnology to obtain higher spatiotemporal resolution; and developing simultaneous, automated, and artificial intelligence–inspired multi-trap systems to achieve high throughput. These further developments will grant unprecedented accessibility of OT-DFS and force measurement nanotools to a wider biomedical research community, ultimately opening the floodgates for exciting live-cell mechanobiology and novel therapeutic discoveri

Pausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our mechanistic understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high-throughput magnetic tweezers, we examine the Escherichia coli RNAp transcription dynamics over a wide range of forces and NTP concentrations. Dwell-time analysis and stochastic modeling identify, in addition to a short-lived elemental pause, two distinct long-lived backtrack pause states differing in recovery rates. We identify two stochastic sources of transcription heterogeneity: alterations in short-pause frequency that underlies elongation-rate switching, and variations in RNA cleavage rates in long-lived backtrack states. Together with effects of force and Gre factors, we demonstrate that recovery from deep backtracks is governed by intrinsic RNA cleavage rather than diffusional Brownian dynamics. We introduce a consensus mechanistic model that unifies our findings with prior models., BN/Cees Dekker Lab, BN/Martin Depken Lab, BN/Bionanoscience, BN/Nynke Dekker Lab

[EN] CONSPECTUS: Molecular vibrations constitute one of the smallest mechanical oscillators available for micro-/nanoengineering. The energy and strength of molecular oscillations depend delicately on the attached specific functional groups as well as on the chemical and physical environments. By exploiting the inelastic interaction of molecules with optical photons, Raman scattering can access the information contained in molecular vibrations. However, the low efficiency of the Raman process typically allows only for characterizing large numbers of molecules. To circumvent this limitation, plasmonic resonances supported by metallic nanostructures and nanocavities can be used because they localize and enhance light at optical frequencies, enabling surface-enhanced Raman scattering (SERS), where the Raman signal is increased by many orders of magnitude. This enhancement enables few- or even single-molecule characterization. The coupling between a single molecular vibration and a plasmonic mode constitutes an example of an optomechanical interaction, analogous to that existing between cavity photons and mechanical vibrations. Optomechanical systems have been intensely studied because of their fundamental interest as well as their application in practical implementations of quantum technology and sensing. In this context, SERS brings cavity optomechanics down to the molecular scale and gives access to larger vibrational frequencies associated with molecular motion, offering new possibilities for novel optomechanical nanodevices. The molecular optomechanics description of SERS is recent, and its implications have only started to be explored. In this Account, we describe the current understanding and progress of this new description of SERS, focusing on our own contributions to the field. We first show that the quantum description of molecular optomechanics is fully consistent with standard classical and semiclassical models often used to describe SERS. Furthermore, we n

The main goal of the studies presented in this dissertation is to continue the endeavor in improving of energy-probing capability of STM for single molecule spectroscopy. Here, we extend energy spectrum into THz region for STM vibrational spectroscopy by combining an 8-Kelvin scanning tunneling STM with a continuous wave THz source. Development of THz rectification technique allowed probing of vibrational excitations with simultaneous chemical sensitivity, THz field sensitivity and <10 MHz energy resolution. On the other hand, by incorporating tunable NIR lasers into an STM junction, we demonstrate the energy-resolved probing of vibrational overtone resonant excitation through the vibration-mediated conformational transition of single pyrrolidine molecules. Photon equilibrium action spectroscopy was performed to observe wavelength dependent resonance excitation features with sub-meV energy resolution. These features were shown to be sensitive to the molecular local adsorption environment while being insensitive to the junction near field profile induced by junction plasmon excitation or the optical setup. Moreover, the light induced equilibrium change in the reversible conformational transition of pyrrolidine leads to generation of photocurrent. Using optical rectification spectroscopy, we investigated the influence of laser power and tunneling setpoint on the photocurrent. The photocurrent was shown to be governed by the competition between photon and electron contribution to the molecular reversible transition, which can be tuned through laser power, bias and tip-molecular interaction. Through the optical rectification study of pyrrolidine, we demonstrate its potential application as a tunable single-molecule photo-rectifier device when trapped between two electrodes.

The main goal of the studies presented in this dissertation is to demonstrate the measurement and manipulation of ultrafast quantum coherence in single molecules. Low temperature scanning tunneling microscopy (STM) with inelastic electron tunneling spectroscopy (IETS) allows determination of molecular fingerprints with atomic-scale spatial resolution. Femtosecond (fs) lasers in the terahertz (THz) range can be used to monitor the quantum coherence of molecules and atoms. Here we combine STM-IETS with fs THz pulses into a fs THz-STM and perform the time-resolved THz rectification spectroscopy (t-TRS) in the STM junction.When THz pulses are introduced into the STM junction, an effective voltage will be developed by the THz electric field which behaves as a modulation of the static sample bias. A non-zero rectification current can then be monitored due to the nonlinearity originated from the molecular excitation in the STM junction. THz photon absorption can also occur when the energy separation of the ground and excited states in the molecule, such as the hydrogen molecule (H2), matches the frequency range of the THz pulses. It thus becomes possible to prepare the superposition state of the H2 two-level system and track its temporal evolution from time domain pump-probe spectroscopy. The coherence measurement is successfully demonstrated on a single H2 molecule by our fs THz-STM. The extreme sensitivity of the H2 TLS coherent oscillation to the underlying surface chemical environment is discovered by scanning across the Cu2N surface while performing pump-probe spectroscopy, which shows promising applications in quantum sensing. Both the oscillation frequency and the dephasing time of the initial wave packet fo the H2 TLS are highly dependent on the applied external electric field under the giant Stark effect. By sweeping the sample bias and the corresponding electric field across the STM junction, we realized the electric manipulation of the quantum coherence of the H

Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR–HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR : EGF stoichiometry of approximately 4 : 1 for plasma membrane-colocalized EGFR–EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.

Tuberculosis (TB) is an airborne disease that kills 1.5 million people and affects ~ 9 million worldwide. Its etiological agent, Mycobacterium tuberculosis (Mtb), persists in a third of the general population. There is also an emergent problem of drug and multidrug resistance in Mtb that creates the need for novel drugs. In the treatment against TB, one target of antibiotics is the prokaryotic RNA polymerase (RNAP), an essential multitask enzyme that catalyzes RNA synthesis during transcription. Rifampicin, the first-line antibiotic anti-TB, binds and inhibits the MtbRNAP.Consequently, combatting TB requires a better understanding of the primary mechanism of transcription by MtbRNAP. In fact, during Mtb transcription, unique and essential factors might affect the MtbRNAP dynamics and regulate its activity. That regulation plays a significant role in the adaptation and pathogenesis of the parasite. Therefore, understanding how these factors and new antibiotics modulate together the MtbRNAP activity will contribute valid information to address TB. To this end, we use a single-molecule approach based on high-resolution optical tweezers to study transcription elongation by MtbRNAP in the context of its DNA elements, transcriptional factors, and antibiotics.

Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity and kinetics measurements enabled the construction of energy landscapes in sequence space, uncovering relationships between sequence, structure, and function. Here, we review the approaches to perform ensemble fluorescence experiments on next-generation sequencing chips for variations of DNA, RNA, and protein sequences. As the next step, we anticipate that these fluorescence experiments will be pushed to the single-molecule level, which can directly uncover kinetics and molecular heterogeneity in an unprecedented high-throughput fashion. Molecular biophysics in sequence space, both at the ensemble and single-molecule level, leads to new mechanistic insights. The wide spectrum of applications in biology and medicine ranges from the fundamental understanding of evolutionary pathways to the development of new therapeutics., Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., BN/Bionanoscience, BN/Chirlmin Joo Lab

[EN] CONSPECTUS: Molecular vibrations constitute one of the smallest mechanical oscillators available for micro-/nanoengineering. The energy and strength of molecular oscillations depend delicately on the attached specific functional groups as well as on the chemical and physical environments. By exploiting the inelastic interaction of molecules with optical photons, Raman scattering can access the information contained in molecular vibrations. However, the low efficiency of the Raman process typically allows only for characterizing large numbers of molecules. To circumvent this limitation, plasmonic resonances supported by metallic nanostructures and nanocavities can be used because they localize and enhance light at optical frequencies, enabling surface-enhanced Raman scattering (SERS), where the Raman signal is increased by many orders of magnitude. This enhancement enables few- or even single-molecule characterization. The coupling between a single molecular vibration and a plasmonic mode constitutes an example of an optomechanical interaction, analogous to that existing between cavity photons and mechanical vibrations. Optomechanical systems have been intensely studied because of their fundamental interest as well as their application in practical implementations of quantum technology and sensing. In this context, SERS brings cavity optomechanics down to the molecular scale and gives access to larger vibrational frequencies associated with molecular motion, offering new possibilities for novel optomechanical nanodevices. The molecular optomechanics description of SERS is recent, and its implications have only started to be explored. In this Account, we describe the current understanding and progress of this new description of SERS, focusing on our own contributions to the field. We first show that the quantum description of molecular optomechanics is fully consistent with standard classical and semiclassical models often used to describe SERS. Furthermore, we n

Cells sense and respond to mechanical stimuli for activation, proliferation, migration, and differentiation. The associated mechanosensing and biomechanical properties of cells and tissues are significantly implicated in the context of cancer, fibrosis, dementia, and cardiovascular diseases. To gain more mechanobiology insights, dynamic force spectroscopies (DFSs), particularly optical tweezers (OT), have been further advanced to enable in situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside of a cell. In this review, we first explain the classic OT-DFS rationales and discuss their applications to protein biophysics, extracellular biomechanics, and receptor-mediated cell mechanosensing. As a non-invasive technique, optical tweezers’s unique advantages in probing cytoplasmic protein behaviors and manipulating organelles inside living cells have been increasingly explored in recent years. Hereby, we then introduce and highlight the emerging OT rationales for intracellular force measurement including refractive index matching, active–passive calibration, and change of light momentum. These new approaches enable intracellular OT-DFS and mechanical measurements with respect to intracellular motor stepping, cytosolic micro-rheology, and biomechanics of irregularly shaped nuclei and vesicles. Last but not least, we foresee future OT upgrades with respect to overcoming phototoxicity and system drifting for longer duration live-cell measurements; multimodal integration with advanced imaging and nanotechnology to obtain higher spatiotemporal resolution; and developing simultaneous, automated, and artificial intelligence–inspired multi-trap systems to achieve high throughput. These further developments will grant unprecedented accessibility of OT-DFS and force measurement nanotools to a wider biomedical research community, ultimately opening the floodgates for exciting live-cell mechanobiology and novel therapeutic discoveri

Pausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our mechanistic understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high-throughput magnetic tweezers, we examine the Escherichia coli RNAp transcription dynamics over a wide range of forces and NTP concentrations. Dwell-time analysis and stochastic modeling identify, in addition to a short-lived elemental pause, two distinct long-lived backtrack pause states differing in recovery rates. We identify two stochastic sources of transcription heterogeneity: alterations in short-pause frequency that underlies elongation-rate switching, and variations in RNA cleavage rates in long-lived backtrack states. Together with effects of force and Gre factors, we demonstrate that recovery from deep backtracks is governed by intrinsic RNA cleavage rather than diffusional Brownian dynamics. We introduce a consensus mechanistic model that unifies our findings with prior models., BN/Cees Dekker Lab, BN/Martin Depken Lab, BN/Bionanoscience, BN/Nynke Dekker Lab

Intermittent jumping force is an operational atomic-force microscopy mode that produces simultaneous topography and tip-sample maximum-adhesion images based on force spectroscopy. In this work, the operation conditions have been implemented scanning in a repulsive regime and applying very low forces, thus avoiding unspecific tip-sample forces. Remarkably, adhesion images give only specific rupture events, becoming qualitative and quantitative molecular recognition maps obtained at reasonably fast rates, which is a great advantage compared to the force–volume modes. This procedure has been used to go further in discriminating between two similar protein molecules, avidin and streptavidin, in hybrid samples. The adhesion maps generated scanning with biotinylated probes showed features identified as avidin molecules, in the range of 40–80 pN; meanwhile, streptavidin molecules rendered 120–170 pN at the selected working conditions. The gathered results evidence that repulsive jumping force mode applying very small forces allows the identification of biomolecules through the specific rupture forces of the complexes and could serve to identify receptors on membranes or samples or be applied to design ultrasensitive detection technologies.

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

[EN] Statistical mechanics is a physics theory that deals with ensembles of microstates of a system compatible with environmental constraints and that on average define a thermodynamic state. The evolution of a small system is normally subjected to changing constraints, as set by a protocol, and involves a stochastic dependence on previous events. Here, we generalize the dynamic trajectories described by a realization of a physical system without dissipation to include those in which the history of previous events is necessary to understand its future. This framework is then used to characterize the processes experienced by the stochastic system, as derived from ensemble averages over the available pathways. We find that the pathways that the system traces in the presence of a protocol entail different statistics from those in its absence and prove that both types of pathways are equivalent in the limit of independent events. Such equivalence implies that a thermodynamic system cannot evolve away from equilibrium in the absence of memory. These results are useful to interpret single-molecule experiments in biophysics and other fields in nanoscience, as well as an adequate platform to describe non-equilibrium processes.

[EN] Statistical mechanics is a physics theory that deals with ensembles of microstates of a system compatible with environmental constraints and that on average define a thermodynamic state. The evolution of a small system is normally subjected to changing constraints, as set by a protocol, and involves a stochastic dependence on previous events. Here, we generalize the dynamic trajectories described by a realization of a physical system without dissipation to include those in which the history of previous events is necessary to understand its future. This framework is then used to characterize the processes experienced by the stochastic system, as derived from ensemble averages over the available pathways. We find that the pathways that the system traces in the presence of a protocol entail different statistics from those in its absence and prove that both types of pathways are equivalent in the limit of independent events. Such equivalence implies that a thermodynamic system cannot evolve away from equilibrium in the absence of memory. These results are useful to interpret single-molecule experiments in biophysics and other fields in nanoscience, as well as an adequate platform to describe non-equilibrium processes.

[EN] Statistical mechanics is a physics theory that deals with ensembles of microstates of a system compatible with environmental constraints and that on average define a thermodynamic state. The evolution of a small system is normally subjected to changing constraints, as set by a protocol, and involves a stochastic dependence on previous events. Here, we generalize the dynamic trajectories described by a realization of a physical system without dissipation to include those in which the history of previous events is necessary to understand its future. This framework is then used to characterize the processes experienced by the stochastic system, as derived from ensemble averages over the available pathways. We find that the pathways that the system traces in the presence of a protocol entail different statistics from those in its absence and prove that both types of pathways are equivalent in the limit of independent events. Such equivalence implies that a thermodynamic system cannot evolve away from equilibrium in the absence of memory. These results are useful to interpret single-molecule experiments in biophysics and other fields in nanoscience, as well as an adequate platform to describe non-equilibrium processes.

Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.

Single-molecule techniques allow the visualization of the molecular dynamics of nucleic acids and proteins with high spatiotemporal resolution. Valuable kinetic information of biomolecules can be obtained when the discrete states within single-molecule time trajectories are determined. Here, we present a fast, automated, and bias-free step detection method, AutoStepfinder, that determines steps in large datasets without requiring prior knowledge on the noise contributions and location of steps. The analysis is based on a series of partition events that minimize the difference between the data and the fit. A dual-pass strategy determines the optimal fit and allows AutoStepfinder to detect steps of a wide variety of sizes. We demonstrate step detection for a broad variety of experimental traces. The user-friendly interface and the automated detection of AutoStepfinder provides a robust analysis procedure that enables anyone without programming knowledge to generate step fits and informative plots in less than an hour., BN/Chirlmin Joo Lab, BN/Technici en Analisten, BN/Cees Dekker Lab

Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

Single-molecule techniques allow the visualization of the molecular dynamics of nucleic acids and proteins with high spatiotemporal resolution. Valuable kinetic information of biomolecules can be obtained when the discrete states within single-molecule time trajectories are determined. Here, we present a fast, automated, and bias-free step detection method, AutoStepfinder, that determines steps in large datasets without requiring prior knowledge on the noise contributions and location of steps. The analysis is based on a series of partition events that minimize the difference between the data and the fit. A dual-pass strategy determines the optimal fit and allows AutoStepfinder to detect steps of a wide variety of sizes. We demonstrate step detection for a broad variety of experimental traces. The user-friendly interface and the automated detection of AutoStepfinder provides a robust analysis procedure that enables anyone without programming knowledge to generate step fits and informative plots in less than an hour., BN/Chirlmin Joo Lab, BN/Technici en Analisten, BN/Cees Dekker Lab

Nonostante gli enormi passi avanti sulla comprensione dei meccanismi d’azione del cancro e nello sviluppo di farmaci antitumorali, la maggior parte delle forme di cancro è ancora incurabile. La ragione di tale insuccesso è radicata nella complessità di questa malattia, che è ancora poco conosciuta sia in fase di insorgenza, dovuta alla misregolazione di oncogeni, sia in fase di metastatizzazione delle cellule cancerose. Lo studio di tali caratteristiche da un punto di vista meccanico può fornire una visione differente dei meccanismi di azione del cancro ed aiutare a contestualizzarli all’interno di processi estremamente complessi. L’insorgenza del cancro è dovuta ad una mancata regolazione del ciclo cellulare, che a sua volta dipende spesso da un’alterata espressione dei cosiddetti oncogeni. Ne è un esempio il proto-oncogene c-KIT, che codifica per un fattore di crescita delle cellule staminali. La regolazione della sua espressione è controllata dal suo promotore prossimale, la cui struttura è caratterizzata dalla presenza di 3 superstrutture tridimensionali complesse che si formano su uno dei due filamenti della sua sequenza, chiamate G-quadruplexes. Tali strutture sembrerebbero avere un ruolo nel riconoscimento del promotore da parte della polimerasi e nell’assemblaggio della macchina trascrizionale, ma la loro funzione non è ancora chiara. Lo studio della nanomeccanica di tali strutture a livello di singola molecola (grazie al Magnetic Tweezers) può fornire importanti informazioni sul loro ruolo e sulle modalità con cui regolano l’espressione dell’oncogene. In seguito all’insorgenza del cancro, le cellule coinvolte subiscono forti cambiamenti strutturali: iniziano a dividersi velocemente e a migrare incontrollatamente. Entrambi questi processi richiedono un grande cambiamento nella struttura cellulare ed in particolare nella rigidità della cellula. La leucemia linfoide cronica (CLL) rappresenta un caso esemplare di tali capacità migratorie, non solo, With the term cancer are intended many species of diseases having quite different properties from each other. Despite such vast differences, the mechanisms beyond the onset of any kind of cancer are very similar and can be classified in two main groups depending on their stage. The first is related to the dysregulation of particular genes (oncogenes), that results in an impairment of the cell cycle. The second concerns the ability of cancer cells to continuously divide and migrate through tissues, that results in a highly invasive potential. From a mechanical point of view, the investigation of such features can be crucial for a deeper understanding of cancer onset and progression as well as for the study of novel pharmacological treatments. The outbreak of cancer is caused by a deficiency in the regulation of the cell cycle which, in turn, often depends on an abnormal expression of oncogenes. It is the case of the proto-oncogene c-KIT, that encodes for a mast/stem cell growth factor receptor. Its regulation relies mainly on its promoter, which is constituted by 3 distinct three-dimensional DNA structures called G-quadruplexes (G4s). Those structures can be studied by means of nanomechanical tools such as Magnetic Tweezers, which can recognize folded G4s at single-molecule level, thus enabling to study their role in the regulation of the oncogene. After the onset of cancer, a generic cell undergoes mechanical changes: it divides quickly, and it starts migrating. Both phenomena require a modification in the cell structural phenotype, eventually modifying its rigidity. Chronic lymphocytic leukemia is a case in point: malignant B lymphocytes continuously traffic between peripheral blood and lymphoid tissues. Such frequent migrations require a change in the rigidity of cells. In this case, Atomic Force Microscopy can provide a nanomechanical approach allowing to measure the stiffness of single cells from patients with leukemia, which is slightly decreased if compared to

A detailed study on the single-molecule ferroelectric property of Preyssler-type polyoxometalates (POMs), [M3+P5W30O110](12-) (M = La, Gd, and Lu), is performed by density functional theory calculations. Linked to one H2O molecule, the cation (M3+) encapsulated in the cavity of the Preyssler framework is off-centered, and it generates a permanent dipole, which is essential for a ferroelectric ground state. Accompanied with a 180 degrees rotation of H2O, the switching of M3+ between two isoenergetic sites on both sides of the cavity results in a calculated barrier of 1.15 eV for Gd3+, leading to the inversion of electric polarization. The height of the barrier is in good agreement with the experimentally measured barrier for the Tb3+ ion, whose ionic radius is similar to Gd3+. The total polarization value of the crystal is estimated to be 4.7 mu C/cm(2) as calculated by the modern theory of polarization, which is quite close to the experimental value. Considering that the order of contributions to the polarization is M3+-H2O > counter-cations (K+) > [P5W30O110](15-), the interconversion of M3+-H2O between the two isoenergetic sites is predicted to be the main origin of ferroelectricity with a polarization contribution of 3.4 mu C/cm(2); the K+ counter-cations contribute by 1.2 mu C/cm(2) and it cannot be disregarded, while the framework appears to contribute negligibly to the total polarization. Our study suggests that a suitable choice of M3+-H2O could be used to tune the single-molecule ferroelectricity in Preyssler-type polyoxometalates.

Single-molecule enzymology has proven to be a powerful technique for understanding the driving forces behind protein kinetics and function. Carbon nanotube field-effect transistors (CNTFETs) have been implemented as a novel platform to study the behavior of enzymes. This solid-state technique has contributed to the single-molecule field by providing both microsecond resolution and long-duration recordings. Bandwidths extending to 1 MHz have identified enzymatic conformations as short as 5 µs. Ten-minute recordings have shown enzymatic rates fluctuating over two orders of magnitude from minute to minute.This dissertation documents multiple advances expanding the scope of this CNTFET technique. Progress was made on three fronts: incorporating variable temperature control, combining this solid-state electronic technique with TIRF to acquire simultaneous electronic and fluorescent recordings from a polymerase, and expanding experiments to new molecular catalysts.Taq DNA polymerase was used as a model system to demonstrate variable temperature single-molecule enzymology. No other single-molecule technique can access high enough temperatures to study Taq polymerase, the industry standard for the billion dollar polymerase chain reaction (PCR) industry. Successful single-molecule measurements of Taq polymerase in four different orientations up to 85 °C are reported. Each attachment site transduced unique signals. One attachment site generated events with rates that increased with temperature and match ensemble catalytic rates. Taq’s catalytic activity ranged from 4 s-1 at 22 °C to up to 96 s-1 at 85 °C.Other results from Taq polymerase focused on the importance of the attachment site and molecular orientation. Another attachment site transduced both catalytic turnovers, as well as another noncatalytic fluttering motion. In this signal, two distinct populations of events were detected: one population with 20 µs event durations and another with 125 µs event durations. The lon

The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.

The HIV-1 capsid is a protein shell utilised by the virus as a binding platform to hijack host machineries to promote infection. Characterisation of novel cofactors binding to capsid demands sensitive and quantitative assays to better understand their underlying binding mechanisms. Here, I have developed three complementary fluorescence microscopy-based platforms for quantitative characterisation of capsid binding analytes using tubular, spherical or conical lattices self-assembled from recombinant capsid protein (CA) with engineered cysteine residues for cross-linking as surrogates for the authentic capsid. The first approach focused on the development of a TIRFM biosensor using CA tubes immobilised on a glass surface as the biorecognition substrate for sensitive single-molecule binding studies. The biosensor was assessed in its ability to measure binding affinity, stoichiometry and kinetics using known capsid-binding cofactors as part of its validation process. Biosensor measurements confirmed that binding of the host cell protein cyclophilin A (CypA) to the tubular lattice is substoichiometric due to steric hindrance between CypA molecules. Binding analysis of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) expressed as a full-length protein in a cell-free expression system suggested a role of oligomerisation to enhance binding via avidity. In the second approach, the biosensor surface was modified with cross-linked CA spheres as an alternative substrate containing pentameric defects and highly curved regions that are lacking in CA tubes. Binding of CypA to spheres revealed a significantly higher CypA to CA binding ratio at saturation suggesting a role of curvature in accommodating specific host cofactors. The third approach utilises confocal microscopy-based single molecule spectroscopy and was developed as a medium-throughput capsid interactor screening platform that uses conical CA self-assemblies as the substrate. Fluorescence-tagged analyte

CASSINA, VALERIA, open, In the last few decades, the constant development of novel single molecule techniques has created the basis for a new paradigm in the field of biophysics. Among all, the nanomanipulation of individual biomolecules revealed new insights into the mechanics of biological molecules, in particular proteins and DNA, improving the understanding of the fundamental relation between structural properties and biological functions. Therefore, several single-molecule nanomanipulation methods have been developed, including Atomic Force Microscopy (AFM), Magnetic Tweezers (MT) and Flow Stretching (F-S) coupled with fluorescence. All these technique were employed in this Thesis for the characterisation of biological macromolecules by single molecule force spectroscopy (SMFS). In this Thesis I focus mainly on several aspects of a few different proteins trying to depict a frame in which the strong link between proteins function and their structure can be clarified. With this aim, I study the conformational states of an intrinsically disordered protein (IDP) involved in Parkinson's Disease, the a-synuclein (AS) and the structural change driving the DNA compaction mediated by structural maintenance protein, the condensin. Finally, I present a structural study of a DNA-analogue by thermal shifting essays and single molecule experiments. I included also a technical implementation of a (F-S) combined with TIRF set up to promote the high-speed exchanging buffer for study protein DNA interactions. In the AS single molecule force spectroscopy (SMFS) study, I afford the problem of AS lacking of well defined structure by stretching and unfolding a single polyprotein containing the human AS by employing a SMFS approach. The analysis of the different unfolding pathways gives information about the structural conformation of the protein before the mechanical denaturation. The AS was found to assume three distinct conformational states ranging from a random coil to a highly structured conformation., Negli ultimi decenni, lo sviluppo di nuove tecniche di singola molecola ha creato le basi per nuovi paradigmi nel campo della biofisica. In particolare, la nanomanipolazione di singole biomacromolecole ha permesso la caratterizzazione meccanica di proteine e DNA, cercando di evidenziare la relazione fondamentale tra struttura e funzione biologica. Nel corso degli anni sono stati sviluppati diversi metodi di nanomanipolazione: tra questi, il Microscopio a Forza Atomica (AFM), il Magnetic Tweezers (MT) e il Flow-Stretching (F-S) accoppiato a fluorescenza. Queste tre tecniche sono state utilizzate per la caratterizzazione di singole biomacromolecole con tecniche di spettroscopia di forza a livello di singola molecola (SMFS) in quattro diversi progetti. In questa Tesi mi sono occupata principalmente di caratterizzare strutturalmente diverse proteine e correlarne la conformazione alla funzione biologica. In questa ottica, sono state studiate le diverse conformazioni strutturali di una proteina intrinsecamente disordinata (IDP) coinvolta nella malattia del Parkison’s (PD), chiamata alfa-synucleina (AS) e il riarrangiamento strutturale di una proteina coinvolta nel mantenimento strutturale cromosomico, la condensina, durante la condensazione di singoli filamenti di DNA. Inoltre, è stata studiata la struttura di un DNA-analogo sia caratterizzandone la stabilità termica sia attraverso studi di nanomeccanica in SMFS. Infine, è stata descritta una implementazione tecnica di un apparato di F-S accoppiato con illuminazione TIRF, per ottenere una rapida sostituzione di soluzioni durante esperimenti di microfluidica, ottenendo un setup particolarmente indicato per studi di interazione tra DNA e proteine. Durante lo studio della proteina AS in SMFS, il problema della mancanza di una struttura secondaria ben definita è stato risolto con l’impego di una singola poliproteina, contenente un modulo di AS umana. Sono state caratterizzati tre diversi stati conformazionali per AS, da uno s, No, open, Corti, R

Single-molecule enzymology has proven to be a powerful technique for understanding the driving forces behind protein kinetics and function. Carbon nanotube field-effect transistors (CNTFETs) have been implemented as a novel platform to study the behavior of enzymes. This solid-state technique has contributed to the single-molecule field by providing both microsecond resolution and long-duration recordings. Bandwidths extending to 1 MHz have identified enzymatic conformations as short as 5 µs. Ten-minute recordings have shown enzymatic rates fluctuating over two orders of magnitude from minute to minute.This dissertation documents multiple advances expanding the scope of this CNTFET technique. Progress was made on three fronts: incorporating variable temperature control, combining this solid-state electronic technique with TIRF to acquire simultaneous electronic and fluorescent recordings from a polymerase, and expanding experiments to new molecular catalysts.Taq DNA polymerase was used as a model system to demonstrate variable temperature single-molecule enzymology. No other single-molecule technique can access high enough temperatures to study Taq polymerase, the industry standard for the billion dollar polymerase chain reaction (PCR) industry. Successful single-molecule measurements of Taq polymerase in four different orientations up to 85 °C are reported. Each attachment site transduced unique signals. One attachment site generated events with rates that increased with temperature and match ensemble catalytic rates. Taq’s catalytic activity ranged from 4 s-1 at 22 °C to up to 96 s-1 at 85 °C.Other results from Taq polymerase focused on the importance of the attachment site and molecular orientation. Another attachment site transduced both catalytic turnovers, as well as another noncatalytic fluttering motion. In this signal, two distinct populations of events were detected: one population with 20 µs event durations and another with 125 µs event durations. The lon

Sounds of Science is the first movement of a symphony for many (scientific) instruments and voices, united in celebration of the frontiers of science and intended for a general audience. John Goodenough, the maestro who transformed energy usage and technology through the invention of the lithium-ion battery, opens the programme, reflecting on the ultimate limits of battery technology. This applied theme continues through the subsequent pieces on energy-related topics - the sodium-ion battery and artificial fuels, by Martin Månsson - and the ultimate challenge for 3D printing, the eventual production of life, by Anthony Atala. A passage by Gerianne Alexander follows, contemplating a related issue: How might an artificially produced human being behave? Next comes a consideration of consciousness and free will by Roland Allen and Suzy Lidström. Further voices and new instruments enter as Warwick Bowen, Nicolas Mauranyapin and Lars Madsen discuss whether dynamical processes of single molecules might be observed in their native state. The exploitation of chaos in science and technology, applications of Bose-Einstein condensates and the significance of entropy follow in pieces by Linda Reichl, Ernst Rasel and Roland Allen, respectively. Mikhail Katsnelson and Eugene Koonin then discuss the potential generalisation of thermodynamic concepts in the context of biological evolution. Entering with the music of the cosmos, Philip Yasskin discusses whether we might be able to observe torsion in the geometry of the Universe. The crescendo comes with the crisis of singularities, their nature and whether they can be resolved through quantum effects, in the composition of Alan Coley. The climax is Mario Krenn, Art Melvin and Anton Zeilinger's consideration of how computer code can be autonomously surprising and creative. In a harmonious counterpoint, his 'Guidelines for considering AIs as coauthors', Roman Yampolskiy concludes that code is not yet able to take responsibility for coa

Negli ultimi decenni, lo sviluppo di nuove tecniche di singola molecola ha creato le basi per nuovi paradigmi nel campo della biofisica. In particolare, la nanomanipolazione di singole biomacromolecole ha permesso la caratterizzazione meccanica di proteine e DNA, cercando di evidenziare la relazione fondamentale tra struttura e funzione biologica. Nel corso degli anni sono stati sviluppati diversi metodi di nanomanipolazione: tra questi, il Microscopio a Forza Atomica (AFM), il Magnetic Tweezers (MT) e il Flow-Stretching (F-S) accoppiato a fluorescenza. Queste tre tecniche sono state utilizzate per la caratterizzazione di singole biomacromolecole con tecniche di spettroscopia di forza a livello di singola molecola (SMFS) in quattro diversi progetti. In questa Tesi mi sono occupata principalmente di caratterizzare strutturalmente diverse proteine e correlarne la conformazione alla funzione biologica. In questa ottica, sono state studiate le diverse conformazioni strutturali di una proteina intrinsecamente disordinata (IDP) coinvolta nella malattia del Parkison’s (PD), chiamata alfa-synucleina (AS) e il riarrangiamento strutturale di una proteina coinvolta nel mantenimento strutturale cromosomico, la condensina, durante la condensazione di singoli filamenti di DNA. Inoltre, è stata studiata la struttura di un DNA-analogo sia caratterizzandone la stabilità termica sia attraverso studi di nanomeccanica in SMFS. Infine, è stata descritta una implementazione tecnica di un apparato di F-S accoppiato con illuminazione TIRF, per ottenere una rapida sostituzione di soluzioni durante esperimenti di microfluidica, ottenendo un setup particolarmente indicato per studi di interazione tra DNA e proteine. Durante lo studio della proteina AS in SMFS, il problema della mancanza di una struttura secondaria ben definita è stato risolto con l’impego di una singola poliproteina, contenente un modulo di AS umana. Sono state caratterizzati tre diversi stati conformazionali per AS, da u, In the last few decades, the constant development of novel single molecule techniques has created the basis for a new paradigm in the field of biophysics. Among all, the nanomanipulation of individual biomolecules revealed new insights into the mechanics of biological molecules, in particular proteins and DNA, improving the understanding of the fundamental relation between structural properties and biological functions. Therefore, several single-molecule nanomanipulation methods have been developed, including Atomic Force Microscopy (AFM), Magnetic Tweezers (MT) and Flow Stretching (F-S) coupled with fluorescence. All these technique were employed in this Thesis for the characterisation of biological macromolecules by single molecule force spectroscopy (SMFS). In this Thesis I focus mainly on several aspects of a few different proteins trying to depict a frame in which the strong link between proteins function and their structure can be clarified. With this aim, I study the conformational states of an intrinsically disordered protein (IDP) involved in Parkinson's Disease, the a-synuclein (AS) and the structural change driving the DNA compaction mediated by structural maintenance protein, the condensin. Finally, I present a structural study of a DNA-analogue by thermal shifting essays and single molecule experiments. I included also a technical implementation of a (F-S) combined with TIRF set up to promote the high-speed exchanging buffer for study protein DNA interactions. In the AS single molecule force spectroscopy (SMFS) study, I afford the problem of AS lacking of well defined structure by stretching and unfolding a single polyprotein containing the human AS by employing a SMFS approach. The analysis of the different unfolding pathways gives information about the structural conformation of the protein before the mechanical denaturation. The AS was found to assume three distinct conformational states ranging from a random coil to a highly structured conformation.

Background: DNA bridging promoted by the H-NS protein, combined with the compaction induced by cellular crowding, plays a major role in the structuring of the E. coli genome. However, only few studies consider the effects of the physical interplay of these two factors in a controlled environment. Methods: We apply a single molecule technique (Magnetic Tweezers) to study the nanomechanics of compaction and folding kinetics of a 6 kb DNA fragment, induced by H-NS bridging and/or PEG crowding. Results: In the presence of H-NS alone, the DNA shows a step-wise collapse driven by the formation of multiple bridges, and little variations in the H-NS concentration-dependent unfolding force. Conversely, the DNA collapse force observed with PEG was highly dependent on the volume fraction of the crowding agent. The two limit cases were interpreted considering the models of loop formation in a pulled chain and pulling of an equilibrium globule respectively. Conclusions: We observed an evident cooperative effect between H-NS activity and the depletion of forces induced by PEG. General Significance: Our data suggest a double role for H-NS in enhancing compaction while forming specific loops, which could be crucial in vivo for defining specific mesoscale domains in chromosomal regions in response to environmental changes.

Surface-enhanced Raman spectroscopy (SERS) is a surface analytical technique, which enhances the Raman signal based on the localized surface plasmon resonance (LSPR) phenomenon. It has been successfully used for single molecule (SM) detection and has extended SERS to numerous applications in biomolecular detection. However, SM detection by SERS is still challenging especially with traditional SERS substrates and detection methods. In addition, the fundamental understanding of the SERS enhancement mechanism is still elusive. Furthermore, the application of SERS in biological field is still in the early stage. To address these challenges, there are two main aspects of SERS studied in my dissertation: (a) fundamental aspects through systematic experimental studies combined with simulations, which focus on SM detection, Raman enhancement mechanisms, and (b) the development and optimization of the SERS-based nanoprobe for biomarkers detection from fluidic devices to a single cell. In my dissertation, the following studies have been investigated. First, the sensitivity of a home-made SERS instrument was tested. SM detection was realized by utilizing a highly curved nanoelectrode (NE) to limit the number of attached nanoparticle (NP), which will allow us to have even a single NP on NE (NPoNE) junction in the SERS detection area. The molecule number in a single NPoNE junction which contributes to SERS can be hundreds or even SM. In this first study, we also conducted a correlation study between electrochemical current and SERS to monitor the dynamic formation of the plasmonic junctions. Second, we investigate electromagnetic and chemical enhancement factor tuning by the electrode potential with the assistant of Au@Ag core-shell NPs. The electrode potential induced electromagnetic enhancement (EME) tuning in the Au@Ag NPoNE structure has been confirmed by 3D Finite-difference time-domain (FDTD) simulations. Last is the design of a SERS-based nanoprobe for biomarkers detectio

Vink et al. tracked single CRISPR RNA-surveillance complexes (Cascade) in the native host cell and determined the influence of Cascade copy numbers, PAM scanning speed, and the presence of CRISPR arrays and transcription on their ability to find and clear invading mobile genetic elements from the cell.

Visualizing neural activities is an important step towards studying and understanding the human brain. Since neural signals are transmitted through modulations of their membrane potentials, a membrane potential sensor capable of translating such signals into experimental observables is essential for recording neural signals. Moreover, the capability of observing membrane potential in the functional structures of a neuron, which can be as small as femto-liters in volume, is crucial for studying important brain functions such as memory. For this reason, we developed inorganic nanosensors based on semiconductor nanorods as membrane potential sensors. These nanorods, which operate via the quantum confined Stark effect, display large voltage sensitivities by changes in fluorescence intensity, spectra and lifetime, allowing non-invasive observation of minute fluctuations in the membrane of live cells. The extreme brightness of these nanorods also allow single particle recordings, which will enable studies of membrane potentials in tiny neuronal structure such as synapses. In this thesis, we will first introduce the background, review the existing membrane potential sensors and introduce the relevant literatures regarding solid-state membrane potential sensors. For our development, we will first describe the development of a spectrally-resolved microscope for measuring the spectra of single nanoparticles in Chapter 2. Then, the characterizations of the quantum confined Stark effect in a variety of nanoparticles will be described in Chapter 3. The temporal response, long-term stability and the capability of recording electric field modulation at 1 kHz using a single nanorod will be reported and discussed. In Chapter 4, we will demonstrate a surface functionalization approach utilizing designed alpha-helical peptides and zwitterionic ligands for facilitating insertion of the nanorods into lipid membranes. These functionalized nanorods were shown to spontaneously insert into

The ability for RNA to adopt multiple structures from one sequence presents challenges when predicting RNA secondary structure. Most RNA in the cell is bound to protein, adding additional layers of complexity to the puzzle. Protein-RNA contacts are studied in this body of work within the context of telomerase, a ribonucleoprotein that adds telomeric DNA to the ends of chromosomes allowing cells preserve genomic integrity and prevent the DNA damage. Telomerase is composed of a protein and RNA component, each composed of multiple conserved domains. The contacts formed between these two units are important for understanding telomerase assembly and function. In the first study it is shown that a functionally critical domain within human telomerase RNA is structurally heterogeneous. This heterogeneity is eliminated with telomerase RNA is bound to the telomerase protein. The role of structural heterogeneity in a step-wise assembly pathway of telomerase is discussed. In the second study a previously characterized protein-RNA interaction in Tetrahymena thermophila is demonstrated to be due to a protein contaminant rather than a true telomerase protein-telomerase RNA interaction. This allows for the re-evaluation of previously proposed models for which protein-RNA interactions are important for telomerase function. Taken together these two studies shed light on the different protein-RNA interactions important for telomerase assembly and function.

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

In this thesis we used single-molecule FRET to investigate the kinetic properties of a protein called Argonaute. While traditionally one uses bulk methods to investigate the molecular properties of proteins, bulk methods do not confer information that is transient, since that is inherently averaged out. Single-molecule methods, as their name imply, allow one to observe interactions between individual molecules and their substrates. This allows one to see fast kinetics which may otherwise be missed. Furthermore, while the bulk methods give one only the average kinetics, single molecule methods also give the distribution of probabilities to access certain bound or conformational states, which in turn can give the observer information of the nature of the stochastic process. We rely in this thesis on single-molecule FRET, an abbreviation for Förster Resonance Energy Transfer: It’s a process where energy is transferred through dipole-dipole interaction from a donor fluorophore to an acceptor fluorophore. The distance between fluorophores determines the efficiency of transfer on a length scale of » 10 nm. Since many biological processes take place on this length scale, this technique is exquisitely suitable to study biological processes real-time on the smallest scale, whether it is conformational changes, protein-protein interactions, or in this case, protein target search studies., BN/Chirlmin Joo Lab

The ability for RNA to adopt multiple structures from one sequence presents challenges when predicting RNA secondary structure. Most RNA in the cell is bound to protein, adding additional layers of complexity to the puzzle. Protein-RNA contacts are studied in this body of work within the context of telomerase, a ribonucleoprotein that adds telomeric DNA to the ends of chromosomes allowing cells preserve genomic integrity and prevent the DNA damage. Telomerase is composed of a protein and RNA component, each composed of multiple conserved domains. The contacts formed between these two units are important for understanding telomerase assembly and function. In the first study it is shown that a functionally critical domain within human telomerase RNA is structurally heterogeneous. This heterogeneity is eliminated with telomerase RNA is bound to the telomerase protein. The role of structural heterogeneity in a step-wise assembly pathway of telomerase is discussed. In the second study a previously characterized protein-RNA interaction in Tetrahymena thermophila is demonstrated to be due to a protein contaminant rather than a true telomerase protein-telomerase RNA interaction. This allows for the re-evaluation of previously proposed models for which protein-RNA interactions are important for telomerase function. Taken together these two studies shed light on the different protein-RNA interactions important for telomerase assembly and function.

Visualizing neural activities is an important step towards studying and understanding the human brain. Since neural signals are transmitted through modulations of their membrane potentials, a membrane potential sensor capable of translating such signals into experimental observables is essential for recording neural signals. Moreover, the capability of observing membrane potential in the functional structures of a neuron, which can be as small as femto-liters in volume, is crucial for studying important brain functions such as memory. For this reason, we developed inorganic nanosensors based on semiconductor nanorods as membrane potential sensors. These nanorods, which operate via the quantum confined Stark effect, display large voltage sensitivities by changes in fluorescence intensity, spectra and lifetime, allowing non-invasive observation of minute fluctuations in the membrane of live cells. The extreme brightness of these nanorods also allow single particle recordings, which will enable studies of membrane potentials in tiny neuronal structure such as synapses. In this thesis, we will first introduce the background, review the existing membrane potential sensors and introduce the relevant literatures regarding solid-state membrane potential sensors. For our development, we will first describe the development of a spectrally-resolved microscope for measuring the spectra of single nanoparticles in Chapter 2. Then, the characterizations of the quantum confined Stark effect in a variety of nanoparticles will be described in Chapter 3. The temporal response, long-term stability and the capability of recording electric field modulation at 1 kHz using a single nanorod will be reported and discussed. In Chapter 4, we will demonstrate a surface functionalization approach utilizing designed alpha-helical peptides and zwitterionic ligands for facilitating insertion of the nanorods into lipid membranes. These functionalized nanorods were shown to spontaneously insert into