RESUMEN
Most chemistry and biology occurs in solution, in which conformational dynamics and complexation underlie behaviour and function. Single-molecule techniques1 are uniquely suited to resolving molecular diversity and new label-free approaches are reshaping the power of single-molecule measurements. A label-free single-molecule method2-16 capable of revealing details of molecular conformation in solution17,18 would allow a new microscopic perspective of unprecedented detail. Here we use the enhanced light-molecule interactions in high-finesse fibre-based Fabry-Pérot microcavities19-21 to detect individual biomolecules as small as 1.2 kDa, a ten-amino-acid peptide, with signal-to-noise ratios (SNRs) >100, even as the molecules are unlabelled and freely diffusing in solution. Our method delivers 2D intensity and temporal profiles, enabling the distinction of subpopulations in mixed samples. Notably, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight and composition but different conformation can also be resolved. Detection is based on the creation of a new molecular velocity filter window and a dynamic thermal priming mechanism that make use of the interplay between optical and thermal dynamics22,23 and Pound-Drever-Hall (PDH) cavity locking24 to reveal molecular motion even while suppressing environmental noise. New in vitro ways of revealing molecular conformation, diversity and dynamics can find broad potential for applications in the life and chemical sciences.
Asunto(s)
Péptidos , Imagen Individual de Molécula , Difusión , Isomerismo , Luz , Péptidos/análisis , Péptidos/química , Péptidos/efectos de la radiación , Relación Señal-Ruido , Imagen Individual de Molécula/métodos , Soluciones , Conformación Proteica , Peso Molecular , Movimiento (Física)RESUMEN
vLUME is a virtual reality software package designed to render large three-dimensional single-molecule localization microscopy datasets. vLUME features include visualization, segmentation, bespoke analysis of complex local geometries and exporting features. vLUME can perform complex analysis on real three-dimensional biological samples that would otherwise be impossible by using regular flat-screen visualization programs.
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Procesamiento de Imagen Asistido por Computador/métodos , Imagen Individual de Molécula/métodos , Realidad Virtual , Algoritmos , Animales , Células COS , Caulobacter crescentus/química , Línea Celular , Membrana Celular/química , Chlorocebus aethiops , Clatrina/química , Humanos , Células Jurkat , Microtúbulos/química , Poro Nuclear/química , Programas InformáticosRESUMEN
Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.
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Péptidos beta-Amiloides/metabolismo , Anticuerpos/inmunología , Agregado de Proteínas , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Especificidad de Anticuerpos , Caenorhabditis elegans , Modelos Animales de Enfermedad , Epítopos , Hipocampo/metabolismo , Ratones , Unión Proteica , Conformación Proteica , Anticuerpos de Dominio ÚnicoRESUMEN
TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aß and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aß oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aß oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aß oligomerization and disaggregated preformed Aß oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aß fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aß-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal-glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aß but rather promotes Aß protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aß-induced release of sTREM2, which blocks Aß aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aß aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk.
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Péptidos beta-Amiloides/química , Amiloide/química , Glicoproteínas de Membrana/fisiología , Proteínas Mutantes/metabolismo , Mutación , Neuronas/patología , Síndromes de Neurotoxicidad/patología , Receptores Inmunológicos/fisiología , Enfermedad de Alzheimer , Amiloide/metabolismo , Animales , Ratones , Ratones Noqueados , Proteínas Mutantes/genética , Neuronas/metabolismo , Síndromes de Neurotoxicidad/etiologíaRESUMEN
Pure rotational spectra of the ground electronic states of lead monoiodide and tin monoiodide have been measured using a chirped pulsed Fourier transform microwave spectrometer over the 7-18.5 GHz region for the first time. Each of PbI and SnI has a X (2)Π1/2 ground electronic state and may have a hyperfine structure that aids the determination of the electron electric dipole moment. For each species, pure rotational transitions of a number of different isotopologues and their excited vibrational states have been assigned and fitted. A multi-isotopologue Dunham-type analysis was carried out on both species producing values for Y01, Y02, Y11, and Y21, along with Λ-doubling constants, magnetic hyperfine constants and nuclear quadrupole coupling constants. The Born-Oppenheimer breakdown parameters for Pb have been evaluated and the parameter rationalized in terms of finite nuclear field effects. Analysis of the bond lengths and hyperfine interaction indicates that the bonding in both PbI and SnI is ionic in nature. Equilibrium bond lengths have been evaluated for both species.
RESUMEN
The vast majority of chemistry and biology occurs in solution, and new label-free analytical techniques that can help resolve solution-phase complexity at the single-molecule level can provide new microscopic perspectives of unprecedented detail. Here, we use the increased light-molecule interactions in high-finesse fiber Fabry-Pérot microcavities to detect individual biomolecules as small as 1.2 kDa with signal-to-noise ratios >100, even as the molecules are freely diffusing in solution. Our method delivers 2D intensity and temporal profiles, enabling the distinction of sub-populations in mixed samples. Strikingly, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight can also be resolved. Detection is based on a novel molecular velocity filtering and dynamic thermal priming mechanism leveraging both photo-thermal bistability and Pound-Drever-Hall cavity locking. This technology holds broad potential for applications in life and chemical sciences and represents a major advancement in label-free in vitro single-molecule techniques.
RESUMEN
Optical imaging of protein aggregates in living and post-mortem tissue can often be impeded by unwanted fluorescence, prompting the need for novel methods to extract meaningful signal in complex biological environments. Historically, benzothiazolium derivatives, prominently Thioflavin T, have been the state-of-the-art fluorescent probes for amyloid aggregates, but their optical, structural, and binding properties typically limit them to in vitro applications. This study compares the use of novel uncharged derivative, PAP_1, with parent Thioflavin T as a fluorescence lifetime imaging probe. This is applied specifically to imaging recombinant α-synuclein aggregates doped into brain tissue. Despite the 100-fold lower brightness of PAP_1 compared to that of Thioflavin T, PAP_1 binds to α-synuclein aggregates with an affinity several orders of magnitude greater than Thioflavin T; thus, we observe a specific decrease in the fluorescence lifetime of PAP_1 bound to α-synuclein aggregates, resulting in a separation of >1.4 standard deviations between PAP_1-stained brain tissue background and α-synuclein aggregates that is not observed with Thioflavin T. This enables contrast between highly fluorescent background tissue and amyloid fibrils that is attributed to the greater affinity of PAP_1 for α-synuclein aggregates, avoiding the substantial off-target staining observed with Thioflavin T.
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Amiloide , alfa-Sinucleína , Péptidos beta-Amiloides , Proteínas Amiloidogénicas , Benzotiazoles , Colorantes Fluorescentes , Imagen Óptica , Espectrometría de FluorescenciaRESUMEN
Fluorescent nucleobase surrogates capable of Watson-Crick hydrogen bonding are essential probes of nucleic acid structure and dynamics, but their limited brightness and short absorption and emission wavelengths have rendered them unsuitable for single-molecule detection. Aiming to improve on these properties, we designed a new tricyclic pyrimidine nucleoside analogue with a push-pull conjugated system and synthesized it in seven sequential steps. The resulting C-linked 8-(diethylamino)benzo[b][1,8]naphthyridin-2(1H)-one nucleoside, which we name ABN, exhibits ε 442 = 20 000 M-1 cm-1 and Φ em,540 = 0.39 in water, increasing to Φ em = 0.50-0.53 when base paired with adenine in duplex DNA oligonucleotides. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to > 95% in a bright state with count rates of at least 15 kHz per molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is the first to offer robust and accessible single-molecule fluorescence detection capabilities.
RESUMEN
The benzothiazolium salt, Thioflavin T (ThT), has been widely adopted as the "gold-standard" fluorescent reporter of amyloid in vitro. Its properties as a molecular rotor result in a large-scale (â¼1000-fold) fluorescence turn-on upon binding to ß-sheets in amyloidogenic proteins. However, the complex photophysics of ThT combined with the intricate and varied nature of the amyloid binding motif means these interactions are poorly understood. To study this important class of fluorophores, we present a detailed photophysical characterization and comparison of a novel library of 12 ThT-inspired fluorescent probes for amyloid protein (PAPs), where both the charge and donor capacity of the heterocyclic and aminobenzene components have been interrogated, respectively. This enables direct photophysical juxtaposition of two structural groups: the neutral "PAP" (class 1) and the charged "mPAP" fluorophores (class 2). We quantify binding and optical properties at both the bulk and single-aggregate levels with some derivatives showing higher aggregate affinity and brightness than ThT. Finally, we demonstrate their abilities to perform super-resolution imaging of α-synuclein fibrils with localization precisions of â¼16 nm. The properties of the derivatives provide new insights into the relationship between chemical structure and function of benzothiazole probes.
RESUMEN
Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.
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G-Cuádruplex , Imagen Individual de Molécula/métodos , Línea Celular Tumoral , Colorantes Fluorescentes/química , Fase G1 , Humanos , Microscopía Fluorescente , Fase S , Imagen de Lapso de TiempoRESUMEN
Neurodegenerative diseases such as Alzheimer's and Parkinson's are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, the improved optical properties (extinction coefficient, quantum yield and brightness) of ThX can be used to monitor structural differences in oligomeric species, not observed via traditional ThT imaging.
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Hexagonal boron nitride (h-BN) is a 2D, wide band gap semiconductor that has recently been shown to display bright room-temperature emission in the visible region, sparking immense interest in the material for use in quantum applications. In this work, we study highly crystalline, single atomic layers of chemical vapor deposition grown h-BN and find predominantly one type of emissive state. Using a multidimensional super-resolution fluorescence microscopy technique we simultaneously measure spatial position, intensity, and spectral properties of the emitters, as they are exposed to continuous wave illumination over minutes. As well as low emitter heterogeneity, we observe inhomogeneous broadening of emitter line-widths and power law dependency in fluorescence intermittency; this is strikingly similar to previous work on quantum dots. These results show that high control over h-BN growth and treatment can produce a narrow distribution of emitter type and that surface interactions heavily influence the photodynamics. Furthermore, we highlight the utility of spectrally resolved wide-field microscopy in the study of optically active excitations in atomically thin two-dimensional materials.
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Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification.
Asunto(s)
Biomineralización , Daño del ADN , Poli Adenosina Difosfato Ribosa/metabolismo , Calcificación Vascular/metabolismo , Adolescente , Adulto , Anciano , Animales , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Bovinos , Línea Celular , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Osteoblastos/metabolismo , Osteoblastos/patología , Estrés Oxidativo , Ratas , Ratas Wistar , OvinosRESUMEN
[This corrects the article DOI: 10.1098/rsos.171399.].
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Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H2O2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H2O2. We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H2O2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.
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A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.