RESUMO
Fluorescence Resonance Energy Transfer (FRET)-based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super-resolved FRET imaging based on single-molecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.
Assuntos
DNA , Transferência Ressonante de Energia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , DNA/química , Microscopia Confocal , Imagem Individual de MoléculaRESUMO
Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.
Assuntos
Fluorescência , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Imagem Individual de Molécula/métodos , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Microtúbulos/metabolismo , Fotometria/métodosRESUMO
In the past months, the use of the drug hydroxychloroquine has considerably increased in many countries, associated with a proposed treatment for the COVID-19 disease. Although there is no conclusive evidence about the efficacy of the drug for this purpose, surprisingly there are no conclusive studies in the literature concerning its mechanism of action inside cells, which is related to its interaction with nucleic acids. Here, we performed a robust characterization of the interaction between hydroxychloroquine and double-stranded DNA using single-molecule force spectroscopy and gel electrophoresis. Two different binding modes were identified, namely, minor groove binding for low drug concentrations and intercalation for high drug concentrations, and the sets of binding parameters were determined for each of these modes. Such results have unraveled in detail the molecular mechanism of action of the drug as a DNA ligand.
Assuntos
DNA/química , Hidroxicloroquina/química , Substâncias Intercalantes/química , Eletroforese , Pinças Ópticas , Imagem Individual de MoléculaRESUMO
Europium oxide (Eu2O3) was used to evaluate the affinity of this rare earth element for interacting with double-stranded (ds) DNA molecules. To perform the study, we used single molecule force spectroscopy with optical tweezers and gel electrophoresis assays. Force spectroscopy experiments show that Eu2O3 presents a strong interaction with dsDNA, and the binding is independent on the ionic strength used in the surrounding environment. Among the main characteristics of the interaction, Eu2O3 tends to bind in a cooperative way, forming bound clusters of â¼ 3 molecules, and presents a high equilibrium association binding constant on the order of 105 M-1. In addition, gel electrophoresis confirm the weak electrostatic character of the interaction and explicit show that Eu2O3 does not interfere on drug intercalation into the double-helix. Such results demonstrate the potential of europium for interacting with nucleic acids and strongly suggest that this rare earth element may be considered for the design of new metal-based anticancer drugs in the future.
Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Európio/farmacologia , DNA/química , Modelos Moleculares , Imagem Individual de MoléculaRESUMO
By performing single-molecule force spectroscopy with optical tweezers, we have characterized the interaction between the platinum-based compound transplatin and the DNA molecule, establishing a critical comparison with its isomer cisplatin. While transplatin is ineffective against tumor cells, its isomer is one of the most used drugs in current chemotherapies, and a molecular study on this difference performed at the single-molecule level was lacking until the present work. Our experiments show that transplatin binds DNA under low chloride concentrations (a situation usually found inside many cells) with an equilibrium association binding constant about four orders of magnitude lower than cisplatin. In addition, we have found that, at saturation, transplatin binds preferentially forming interstrand cross links and monoadducts, a situation very different from cisplatin, which forms preferentially intrastrand cross links. Such differences explain the ineffectiveness of transplatin in killing tumor cells. From a physical point of view, the present study advances in using the mechanical properties of the DNA molecule as sensors to evaluate the therapeutic efficiency of drugs.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Imagem Individual de Molécula , Antineoplásicos/química , Antineoplásicos/metabolismo , Cisplatino/química , Cisplatino/metabolismo , DNA/metabolismo , Entropia , Pinças ÓpticasRESUMO
Knots are remarkable topological features in nature. The presence of knots in crystallographic structures of proteins have stimulated considerable research to determine the kinetic and thermodynamic consequences of threading a polypeptide chain. By mechanically manipulating MJ0366, a small single domain protein harboring a shallow trefoil knot, we allow the protein to refold from either the knotted or the unknotted denatured state to characterize the free energy profile associated to both folding pathways. By comparing the stability of the native state with reference to the knotted and unknotted denatured state we find that knotting the polypeptide chain of MJ0366 increase the folding energy barrier in a magnitude close to the energy cost of forming a knot randomly in the denatured state. These results support that a protein knot can be formed during a single cooperative step of folding but occurs at the expenses of a large increment on the free energy barrier.
Assuntos
Dobramento de Proteína , Desdobramento de Proteína , Dicroísmo Circular , Cinética , Methanocaldococcus/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Pinças Ópticas , Conformação Proteica , Desnaturação Proteica , Proteínas Recombinantes/química , Imagem Individual de Molécula , TermodinâmicaRESUMO
Gram-positive bacteria use their adhesive pili to attach to host cells during early stages of a bacterial infection. These extracellular hair-like appendages experience mechanical stresses of hundreds of picoNewtons; however, the presence of an internal isopeptide bond prevents the pilus protein from unfolding. Here, we describe a method to interfere with nascent pili proteins through a peptide that mimics one of the ß-strands of the molecule. By using AFM-based force spectroscopy, we study the isopeptide bond formation and the effect of the peptide in the elasticity of the pilus protein. This method could be used to afford a new strategy for mechanically targeted antibiotics by simply blocking the folding of the bacterial pilus protein.
Assuntos
Proteínas de Fímbrias/metabolismo , Desdobramento de Proteína/efeitos dos fármacos , Imagem Individual de Molécula/métodos , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/metabolismo , Bactérias Gram-Positivas/metabolismo , Microscopia de Força Atômica/métodos , Peptídeos/farmacologia , Dobramento de Proteína , Streptococcus pyogenes/química , Streptococcus pyogenes/metabolismo , Estresse MecânicoRESUMO
Eukaryotic genomes are folded in a hierarchical organization that reflects and possibly regulates their function. Genomewide studies revealed a new level of organization at the kilobase-to-megabase scale termed "topological associating domains" (TADs). TADs are characterized as stable units of chromosome organization that restrict the action of regulatory sequences within one "functional unit." Consequently, TADs are expected to appear as physical entities in most cells. Very recent single-cell studies have shown a notable variability in genome architecture at this scale, raising concerns about this model. Furthermore, the direct and simultaneous observation of genome architecture and transcriptional output showed the lack of stable interactions between regulatory sequences in transcribing cells. These findings are consistent with a large body of evidence suggesting that genome organization is highly heterogeneous at different scales. In this review, we discuss the main strategies employed to image chromatin organization, present the latest state-of-the-art developments, and propose an interpretation reconciling population-based findings with direct single-cell chromatin organization observations. All in all, we propose that TADs are made of multiple, low-frequency, low-affinity interactions that increase the probability, but are not deterministic, of regulatory interactions.
Assuntos
Cromatina/química , Cromatina/metabolismo , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Conformação Molecular , Imagem Individual de Molécula , Eucariotos , MicroscopiaRESUMO
In this work we use single molecule force spectroscopy performed with optical tweezers in order to characterize the complexes formed between the anticancer drug Pixantrone (PIX) and the DNA molecule, at two very different ionic strengths. Firstly, the changes of the mechanical properties of the DNA-PIX complexes were studied as a function of the drug concentration in the sample. Then, a quenched-disorder statistical model of ligand binding was used in order to determine the physicochemical (binding) parameters of the DNA-PIX interaction. In particular, we have found that the PIX molecular mechanism of action involves intercalation into the double helix, followed by a significant compaction of the DNA molecule due to partial neutralization of the phosphate backbone. Finally, this scenario of interaction was quantitatively compared to that found for the related drug Mitoxantrone (MTX), which binds to DNA with a considerably higher equilibrium binding constant and promotes a much stronger DNA compaction. The comparison performed between the two drugs can bring clues to the development of new (and more efficient) related compounds.
Assuntos
Antineoplásicos/química , DNA/química , Substâncias Intercalantes/química , Isoquinolinas/química , Ligantes , Pinças Ópticas , Imagem Individual de Molécula/métodosRESUMO
Here, we investigated the features of replication in Trypanosoma cruzi epimastigotes based on fork speed progression, which is influenced by distinct features such as DNA polymerase rate, susceptibility to DNA damage and repair, secondary structures, transcription and chromatin state. Although T. cruzi exhibits a mean fork speed (2.05 ± 0.10 kb/min) very similar to other trypanosomatids, we found that the majority of DNA molecules replicated more slowly, with a frequency distribution approximately 1 kb/min. This frequency distribution analysis provides more information about the replication profile of this organism.