Expanding the range of graphene energy transfer with multilayer graphene.

The interaction between single emitters and graphene in the context of energy transfer has attracted significant attention due to its potential applications in fields such as biophysics and super-resolution microscopy. In this study, we investigate the influence of the number of graphene layers on graphene energy transfer (GET) by placing single dye molecules at defined distances from monolayer, bilayer, and trilayer graphene substrates. We employ DNA origami nanostructures as chemical adapters to position the dye molecules precisely. Fluorescence lifetime measurements and analysis reveal an additive effect of graphene layers on the energy transfer rate extending the working range of GET up to distances of approximately 50-60 nm. Moreover, we show that switching a DNA pointer strand between two positions on a DNA origami nanostructure at a height of >28 nm above graphene is substantially better visualized with multilayer graphene substrates suggesting enhanced capabilities for applications such as biosensing and super-resolution microscopy for larger systems and distances. This study provides insights into the influence of graphene layers on energy transfer dynamics and offers new possibilities for exploiting graphene's unique properties in various nanotechnological applications.

Deep-LASI, single-molecule data analysis software.

By avoiding ensemble averaging, single-molecule methods provide novel means of extracting mechanistic insights into function of material and molecules at the nanoscale. However, one of the big limitations is the vast amount of data required for analyzing and extracting the desired information, which is time-consuming and user dependent. Here, we introduce Deep-LASI, a software suite for the manual and automatic analysis of single-molecule traces, interactions, and the underlying kinetics. The software can handle data from one-, two- and three-color fluorescence data, and was particularly designed for the analysis of two- and three-color single-molecule fluorescence resonance energy transfer experiments. The functionalities of the software include: the registration of multiple-channels, trace sorting and categorization, determination of the photobleaching steps, calculation of fluorescence resonance energy transfer correction factors, and kinetic analyses based on hidden Markov modeling or deep neural networks. After a kinetic analysis, the ensuing transition density plots are generated, which can be used for further quantification of the kinetic parameters of the system. Each step in the workflow can be performed manually or with the support of machine learning algorithms. Upon reading in the initial data set, it is also possible to perform the remaining analysis steps automatically without additional supervision. Hence, the time dedicated to the analysis of single-molecule experiments can be reduced from days/weeks to minutes. After a thorough description of the functionalities of the software, we also demonstrate the capabilities of the software via the analysis of a previously published dynamic three-color DNA origami structure fluctuating between three states. With the drastic time reduction in data analysis, new types of experiments become realistically possible that complement our currently available palette of methodologies for investigating the nanoworld.

Deep-LASI: deep-learning assisted, single-molecule imaging analysis of multi-color DNA origami structures.

Single-molecule experiments have changed the way we explore the physical world, yet data analysis remains time-consuming and prone to human bias. Here, we introduce Deep-LASI (Deep-Learning Assisted Single-molecule Imaging analysis), a software suite powered by deep neural networks to rapidly analyze single-, two- and three-color single-molecule data, especially from single-molecule Förster Resonance Energy Transfer (smFRET) experiments. Deep-LASI automatically sorts recorded traces, determines FRET correction factors and classifies the state transitions of dynamic traces all in ~20-100 ms per trajectory. We benchmarked Deep-LASI using ground truth simulations as well as experimental data analyzed manually by an expert user and compared the results with a conventional Hidden Markov Model analysis. We illustrate the capabilities of the technique using a highly tunable L-shaped DNA origami structure and use Deep-LASI to perform titrations, analyze protein conformational dynamics and demonstrate its versatility for analyzing both total internal reflection fluorescence microscopy and confocal smFRET data.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate ofcis/transphotoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule. In this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turning PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Water Harvesting at the Single-Crystal Level.

Metal-organic frameworks (MOFs) have emerged as a class of porous materials with facile uptake and release of water, turning them into excellent substrates for real-world atmospheric water harvesting applications. The performance of different MOF systems was experimentally characterized at the bulk level by assessing the total amount of water taken up and the release kinetics, leaving the question behind of what the upper limit of the pristine materials actually is. Moreover, recent devices rely on fluidized bed reactors that exploit the harvesting capacities of MOFs at the single-crystal (SC) level. In this publication, we present a novel methodology based on Raman spectroscopy, for acquiring water adsorption isotherms and kinetic curves with a sub-micrometer resolution that provides valuable insights into the material behavior probing the pristine MOF at the SC level. We investigated isolated MOF-801 particles in situ and could dissect contributions of intra- and inter-particle effects on the water harvesting performance of MOF-801 via adsorption-desorption isotherms and kinetic curves. Using spontaneous Raman spectroscopy, we found an almost 20-fold faster uptake for the undisturbed crystalline material. Correlative imaging based on four-wave mixing and coherent anti-Stokes Raman scattering further localized the uptaken water inside MOF-801 and identified inter-particle condensation as the main source for the discrepancies between the performance at the bulk and SC level. Our studies determined an upper limit of around 91.9 L/kgMOF/day for MOF-801.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Multicolor 3D Orbital Tracking.

Feedback-based single-particle tracking (SPT) is a powerful technique for investigating particle behavior with very high spatiotemporal resolution. The ability to follow different species and their interactions independently adds a new dimension to the information available from SPT. However, only a few approaches have been expanded to multiple colors and no method is currently available that can follow two differently labeled biomolecules in 4 dimensions independently. In this proof-of-concept paper, the new modalities available when performing 3D orbital tracking with a second detection channel are demonstrated. First, dual-color tracking experiments are described studying independently diffusing particles of different types. For interacting particles where their motion is correlated, a second modality is implemented where a particle is tracked in one channel and the position of the second fluorescence species is monitored in the other channel. As a third modality, 3D orbital tracking is performed in one channel while monitoring its spectral signature in a second channel. This last modality is used to successfully readout accurate Förster Resonance Energy Transfer (FRET) values over time while tracking a mobile particle.

Reticular Nanoscience: Bottom-Up Assembly Nanotechnology.

The chemistry of metal-organic and covalent organic frameworks (MOFs and COFs) is perhaps the most diverse and inclusive among the chemical sciences, and yet it can be radically expanded by blending it with nanotechnology. The result is reticular nanoscience, an area of reticular chemistry that has an immense potential in virtually any technological field. In this perspective, we explore the extension of such an interdisciplinary reach by surveying the explored and unexplored possibilities that framework nanoparticles can offer. We localize these unique nanosized reticular materials at the juncture between the molecular and the macroscopic worlds, and describe the resulting synthetic and analytical chemistry, which is fundamentally different from conventional frameworks. Such differences are mirrored in the properties that reticular nanoparticles exhibit, which we described while referring to the present state-of-the-art and future promising applications in medicine, catalysis, energy-related applications, and sensors. Finally, the bottom-up approach of reticular nanoscience, inspired by nature, is brought to its full extension by introducing the concept of augmented reticular chemistry. Its approach departs from a single-particle scale to reach higher mesoscopic and even macroscopic dimensions, where framework nanoparticles become building units themselves and the resulting supermaterials approach new levels of sophistication of structures and properties.

Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides cth GAMP.

The cGAS-STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the cyclic GMP-AMP synthase (cGAS) catalyzing the production of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP) in mammals. This cyclic dinucleotide acts as a second messenger, activating the stimulator of interferon genes (STING) that finally triggers the transcription of interferon genes and inflammatory cytokines. Due to the therapeutic potential of this pathway, both the production and the detection of cGAMP via fluorescent moieties for assay development is of great importance. Here, we introduce the paralleled synthetic access to the intrinsically fluorescent, cyclic dinucleotides 2'3'-cth GAMP and 3'3'-cth GAMP based on phosphoramidite and phosphate chemistry, adaptable for large scale synthesis. We examine their binding properties to murine and human STING and confirm biological activity including interferon induction by 2'3'-cth GAMP in THP-1 monocytes. Two-photon imaging revealed successful cellular uptake of 2'3'-cth GAMP in THP-1 cells.

Single Crystals Heterogeneity Impacts the Intrinsic and Extrinsic Properties of Metal-Organic Frameworks.

At present, an enormous characterization gap exists between the study of the crystal structure of a material and its bulk properties. Individual particles falling within this gap cannot be fully characterized in a correlative manner by current methods. The authors address this problem by exploiting the noninvasive nature of optical microscopy and spectroscopy for the correlative analysis of metal-organic framework particles in situ. They probe the intrinsic as well as extrinsic properties in a correlated manner. The authors show that the crystal shape of MIL-88A strongly impacts its optical absorption. Furthermore, the question of how homogeneously water is distributed and adsorbed within one of the most promising materials for harvesting water from humid air, MOF-801, is addressed. The results demonstrate the considerable importance of the particle level and how it can affect the property of the material.

The Current Status of MOF and COF Applications.

The amalgamation of different disciplines is at the heart of reticular chemistry and has broadened the boundaries of chemistry by opening up an infinite space of chemical composition, structure, and material properties. Reticular design has enabled the precise prediction of crystalline framework structures, tunability of chemical composition, incorporation of various functionalities onto the framework backbone, and as a consequence, fine-tuning of metal-organic framework (MOF) and covalent organic framework (COF) properties beyond that of any other material class. Leveraging the unique properties of reticular materials has resulted in significant advances from both a fundamental and an applied perspective. Here, we wish to review the milestones in MOF and COF research and give a critical view on progress in their real-world applications. Finally, we briefly discuss the major challenges in the field that need to be addressed to pave the way for industrial applications.

Graphene-on-Glass Preparation and Cleaning Methods Characterized by Single-Molecule DNA Origami Fluorescent Probes and Raman Spectroscopy.

Graphene exhibits outstanding fluorescence quenching properties that can become useful for biophysics and biosensing applications, but it remains challenging to harness these advantages due to the complex transfer procedure of chemical vapor deposition-grown graphene to glass coverslips and the low yield of usable samples. Here, we screen 10 graphene-on-glass preparation methods and present an optimized protocol. To obtain the required quality for single-molecule and super-resolution imaging on graphene, we introduce a graphene screening method that avoids consuming the investigated sample. We apply DNA origami nanostructures to place fluorescent probes at a defined distance on top of graphene-on-glass coverslips. Subsequent fluorescence lifetime imaging directly reports on the graphene quality, as deviations from the expected fluorescence lifetime indicate imperfections. We compare the DNA origami probes with conventional techniques for graphene characterization, including light microscopy, atomic force microscopy, and Raman spectroscopy. For the latter, we observe a discrepancy between the graphene quality implied by Raman spectra in comparison to the quality probed by fluorescence lifetime quenching measured at the same position. We attribute this discrepancy to the difference in the effective area that is probed by Raman spectroscopy and fluorescence quenching. Moreover, we demonstrate the applicability of already screened and positively evaluated graphene for studying single-molecule conformational dynamics on a second DNA origami structure. Our results constitute the basis for graphene-based biophysics and super-resolution microscopy.

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ.

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE-FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE-FRET to monitor protein-protein interactions and conformational states simultaneously.

Artificial Bioaugmentation of Biomacromolecules and Living Organisms for Biomedical Applications.

The synergistic union of nanomaterials with biomaterials has revolutionized synthetic chemistry, enabling the creation of nanomaterial-based biohybrids with distinct properties for biomedical applications. This class of materials has drawn significant scientific interest from the perspective of functional extension via controllable coupling of synthetic and biomaterial components, resulting in enhancement of the chemical, physical, and biological properties of the obtained biohybrids. In this review, we highlight the forefront materials for the combination with biomacromolecules and living organisms and their advantageous properties as well as recent advances in the rational design and synthesis of artificial biohybrids. We further illustrate the incredible diversity of biomedical applications stemming from artificially bioaugmented characteristics of the nanomaterial-based biohybrids. Eventually, we aim to inspire scientists with the application horizons of the exciting field of synthetic augmented biohybrids.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.

Spatio-selective activation of nuclear translocation of YAP with light directs invasion of cancer cell spheroids.

The mechanical properties of the extracellular matrix strongly influence tumor progression and invasion. Yes-associated protein (YAP) has been shown to be a key regulator of this process translating mechanical cues from the extracellular matrix into intracellular signals. Despite its apparent role in tumor progression and metastasis, it is not clear yet, whether YAP activation can actively trigger the onset of invasion. To address this question, we designed a photo-activatable YAP (optoYAP), which allows for spatiotemporal control of its activation. The activation mechanism of optoYAP is based on optically triggered nuclear translocation of the protein. Activation of optoYAP induces downstream signaling for several hours and leads to increased proliferation in two- and three-dimensional cultures. Applied to cancer spheroids, optoYAP activation induces invasion. Site-selective activation of optoYAP in cancer spheroids strikingly directs invasion into the activated direction. Thus, nuclear translocation of YAP may be enough to trigger the onset of invasion.

Sponges as bioindicators for microparticulate pollutants?

Amongst other threats, the world's oceans are faced with man-made pollution, including an increasing number of microparticulate pollutants. Sponges, aquatic filter-feeding animals, are able to incorporate fine foreign particles, and thus may be a potential bioindicator for microparticulate pollutants. To address this question, 15 coral reef demosponges sampled around Bangka Island (North Sulawesi, Indonesia) were analyzed for the nature of their foreign particle content using traditional histological methods, advanced light microscopy, and Raman spectroscopy. Sampled sponges accumulated and embedded the very fine sediment fraction (<200 µm), absent in the surrounding sand, in the ectosome (outer epithelia) and spongin fibers (skeletal elements), which was confirmed by two-photon microscopy. A total of 34 different particle types were identified, of which degraded man-made products, i.e., polystyrene, particulate cotton, titanium dioxide and blue-pigmented particles, were incorporated by eight specimens at concentrations between 91 and 612 particle/g dry sponge tissue. As sponges can weigh several hundreds of grams, we conservatively extrapolate that sponges can incorporate on average 10,000 microparticulate pollutants in their tissue. The uptake of particles, however, appears independent of the material, which suggests that the fluctuation in material ratios is due to the spatial variation of surrounding microparticles. Therefore, particle-bearing sponges have a strong potential to biomonitor microparticulate pollutants, such as microplastics and other degraded industrial products.

Metal-Organic Framework Nanoparticles Induce Pyroptosis in Cells Controlled by the Extracellular pH.

Ion homeostasis is essential for cellular survival, and elevated concentrations of specific ions are used to start distinct forms of programmed cell death. However, investigating the influence of certain ions on cells in a controlled way has been hampered due to the tight regulation of ion import by cells. Here, it is shown that lipid-coated iron-based metal-organic framework nanoparticles are able to deliver and release high amounts of iron ions into cells. While high concentrations of iron often trigger ferroptosis, here, the released iron induces pyroptosis, a form of cell death involving the immune system. The iron release occurs only in slightly acidic extracellular environments restricting cell death to cells in acidic microenvironments and allowing for external control. The release mechanism is based on endocytosis facilitated by the lipid-coating followed by degradation of the nanoparticle in the lysosome via cysteine-mediated reduction, which is enhanced in slightly acidic extracellular environment. Thus, a new functionality of hybrid nanoparticles is demonstrated, which uses their nanoarchitecture to facilitate controlled ion delivery into cells. Based on the selectivity for acidic microenvironments, the described nanoparticles may also be used for immunotherapy: the nanoparticles may directly affect the primary tumor and the induced pyroptosis activates the immune system.

Conformational changes and catalytic inefficiency associated with Mot1-mediated TBP-DNA dissociation.

The TATA-box Binding Protein (TBP) plays a central role in regulating gene expression and is the first step in the process of pre-initiation complex (PIC) formation on promoter DNA. The lifetime of TBP at the promoter site is controlled by several cofactors including the Modifier of transcription 1 (Mot1), an essential TBP-associated ATPase. Based on ensemble measurements, Mot1 can use adenosine triphosphate (ATP) hydrolysis to displace TBP from DNA and various models for how this activity is coupled to transcriptional regulation have been proposed. However, the underlying molecular mechanism of Mot1 action is not well understood. In this work, the interaction of Mot1 with the DNA/TBP complex was investigated by single-pair Förster resonance energy transfer (spFRET). Upon Mot1 binding to the DNA/TBP complex, a transition in the DNA/TBP conformation was observed. Hydrolysis of ATP by Mot1 led to a conformational change but was not sufficient to efficiently disrupt the complex. SpFRET measurements of dual-labeled DNA suggest that Mot1's ATPase activity primes incorrectly oriented TBP for dissociation from DNA and additional Mot1 in solution is necessary for TBP unbinding. These findings provide a framework for understanding how the efficiency of Mot1's catalytic activity is tuned to establish a dynamic pool of TBP without interfering with stable and functional TBP-containing complexes.

Caging and Photoactivation in Single-Molecule Förster Resonance Energy Transfer Experiments.

Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Förster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor-acceptor pairs during solution-based smFRET. We use this "caged FRET" methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules.