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1.
Trends Biochem Sci ; 46(11): 918-930, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34247944

RESUMO

Single-molecule localization microscopy (SMLM) is a potent tool to examine biological systems with unprecedented resolution, enabling the investigation of increasingly smaller structures. At the forefront of these developments is DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), which exploits the stochastic and transient binding of fluorescently labeled DNA probes. In its early stages the implementation of DNA-PAINT was burdened by low-throughput, excessive acquisition time, and difficult integration with live-cell imaging. However, recent advances are addressing these challenges and expanding the range of applications of DNA-PAINT. We review the current state of the art of DNA-PAINT in light of these advances and contemplate what further developments remain indispensable to realize live-cell imaging.


Assuntos
DNA , Imagem Individual de Molécula , DNA/química , Microscopia de Fluorescência/métodos
2.
Nat Methods ; 18(6): 604-617, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34099939

RESUMO

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.


Assuntos
Análise de Sequência de Proteína/métodos , Imagem Individual de Molécula/métodos , Espectrometria de Massas/métodos , Nanotecnologia , Proteínas/química , Proteômica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos
3.
Chemistry ; 28(10): e202103523, 2022 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-34939694

RESUMO

Stimuli-responsive soft materials enable controlled release of loaded drug molecules and biomolecules. Controlled release of potent chemotherapeutic or immunotherapeutic agents is crucial to reduce unwanted side effects. In an effort to develop controlled release strategies that can be triggered by using Cerenkov luminescence, we have developed polymer hydrogels that can release bovine serum albumin and immunoglobulin G by using light (254 nm-375 nm) as a trigger. We describe the synthesis and photochemical characterization of two light sensitive phenacyl bis-azide crosslinkers that are used to prepare transparent self-supporting hydrogel patches. One crosslinker was designed to optimize the overlap with the Cerenkov luminescence emission window, bearing an π-extended phenacyl core, resulting in a high quantum yield (14 %) of photocleavage when irradiated with 375 nm light. We used the extended phenacyl crosslinker for the preparation of protein-loaded dextran hydrogel patches, which showed efficient and selective dosed release of bovine serum albumin or immunoglobulin G after irradiation with 375 nm light. Cerenkov-triggered release is as yet inconclusive due to unexpected side-reactivity. Based on the high quantum yield, efficient release and large overlap with the Cerenkov window, we envision application of these photosensitive soft materials in radiation targeted drug release.


Assuntos
Dextranos , Hidrogéis , Dextranos/química , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Hidrogéis/química , Polímeros/química , Soroalbumina Bovina
4.
Nano Lett ; 21(7): 3295-3301, 2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33739111

RESUMO

Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object, which makes it difficult to apply single-molecule FRET for structural analysis of biomolecules. Here, we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the pair distance with subnanometer precision. The distance between other pairs are determined by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. Our FRET X technology will be a tool for the high-resolution analysis of biomolecules and nanostructures.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Ácidos Nucleicos , DNA/genética , Corantes Fluorescentes , Nanotecnologia
5.
Biophys J ; 120(16): 3253-3260, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34237288

RESUMO

Förster resonance energy transfer (FRET) is a useful phenomenon in biomolecular investigations, as it can be leveraged for nanoscale measurements. The optical signals produced by such experiments can be analyzed by fitting a statistical model. Several software tools exist to fit such models in an unsupervised manner but lack the flexibility to adapt to different experimental setups and require local installations. Here, we propose to fit models to optical signals more intuitively by adopting a semisupervised approach, in which the user interactively guides the model to fit a given data set, and introduce FRETboard, a web tool that allows users to provide such guidance. We show that our approach is able to closely reproduce ground truth FRET statistics in a wide range of simulated single-molecule scenarios and correctly estimate parameters for up to 11 states. On in vitro data, we retrieve parameters identical to those obtained by laborious manual classification in a fraction of the required time. Moreover, we designed FRETboard to be easily extendable to other models, allowing it to adapt to future developments in FRET measurement and analysis.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Software , Nanotecnologia
6.
Proc Natl Acad Sci U S A ; 115(13): 3338-3343, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29531063

RESUMO

Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits unidirectional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Fragmentos de Peptídeos/análise , Mapeamento de Peptídeos/métodos , Proteômica/métodos , Endopeptidase Clp/química , Proteínas de Escherichia coli/química , Fluorescência , Humanos
7.
Nano Lett ; 20(4): 2264-2270, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32168456

RESUMO

Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions.


Assuntos
Proteínas Argonautas/análise , Proteínas de Bactérias/análise , Clostridium butyricum/química , DNA/análise , Imagem Óptica/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Nanoestruturas/química
8.
Angew Chem Int Ed Engl ; 59(24): 9340-9344, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32180306

RESUMO

Supramolecular encapsulation is known to alter chemical properties of guest molecules. We have applied this strategy of molecular encapsulation to temporally control the catalytic activity of a stable copper(I)-carbene catalyst. Encapsulation of the copper(I)-carbene catalyst by the supramolecular host cucurbit[7]uril (CB[7]) resulted in the complete inactivation of a copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. The addition of a chemical signal achieved the near instantaneous activation of the catalyst, by releasing the catalyst from the inhibited CB[7] catalyst complex. To broaden the scope of our on-demand CuAAC reaction, we demonstrated the protein labeling of vinculin with the copper(I)-carbene catalyst, to inhibit its activity by encapsulation with CB[7] and to initiate labeling at any moment by adding a specific signal molecule. Ultimately, this strategy allows for temporal control over copper-catalyzed click chemistry, on small molecules as well as protein targets.

9.
Nat Nanotechnol ; 19(5): 652-659, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38351230

RESUMO

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Intrinsicamente Desordenadas/química , Imagem Individual de Molécula/métodos , Aprendizado de Máquina , Mapeamento de Peptídeos/métodos
10.
iScience ; 24(11): 103239, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34729466

RESUMO

Single-molecule protein identification is an unrealized concept with potentially ground-breaking applications in biological research. We propose a method called FRET X (Förster Resonance Energy Transfer via DNA eXchange) fingerprinting, in which the FRET efficiency is read out between exchangeable dyes on protein-bound DNA docking strands and accumulated FRET efficiencies constitute the fingerprint for a protein. To evaluate the feasibility of this approach, we simulated fingerprints for hundreds of proteins using a coarse-grained lattice model and experimentally demonstrated FRET X fingerprinting on model peptides. Measured fingerprints are in agreement with our simulations, corroborating the validity of our modeling approach. In a simulated complex mixture of >300 human proteins of which only cysteines, lysines, and arginines were labeled, a support vector machine was able to identify constituents with 95% accuracy. We anticipate that our FRET X fingerprinting approach will form the basis of an analysis tool for targeted proteomics.

11.
Nat Commun ; 10(1): 2137, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086181

RESUMO

The in-depth, high-sensitivity characterization of the glycome from complex biological samples, such as biofluids and tissues, is of utmost importance in basic biological research and biomarker discovery. Major challenges often arise from the vast structural diversity of glycans in combination with limited sample amounts. Here, we present a method for the highly sensitive characterization of released N-glycans by combining a capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) approach with linkage-specific derivatization of sialic acids and uniform cationic reducing end labelling of all glycans. This method allows the analysis of glycans at the attomole level, provides information on sialic acid isomers and enables the in-depth characterization of complex samples, even when available in minute amounts.


Assuntos
Glicômica/métodos , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Proteínas Sanguíneas/química , Eletroforese Capilar/métodos , Glicosilação , Humanos , Isomerismo , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Ácidos Siálicos/química
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