RESUMEN
Emerging single-molecule protein sensing techniques are ushering in a transformative era in biomedical research. Nevertheless, challenges persist in realizing ultra-fast full-length protein sensing, including loss of molecular integrity due to protein fragmentation, biases introduced by antibodies affinity, identification of proteoforms, and low throughputs. Here, a single-molecule method for parallel protein separation and tracking is introduced, yielding multi-dimensional molecular properties used for their identification. Proteins are tagged by chemo-selective dual amino-acid specific labels and are electrophoretically separated by their mass/charge in custom-designed thin silicon channel with subwavelength height. This approach allows analysis of thousands of individual proteins within a few minutes by tracking their motion during the migration. The power of the method is demonstrated by quantifying a cytokine panel for host-response discrimination between viral and bacterial infections. Moreover, it is shown that two clinically-relevant splice isoforms of Vascular endothelial growth factor (VEGF) can be accurately quantified from human serum samples. Being non-destructive and compatible with full-length intact proteins, this method opens up ways for antibody-free single-protein molecule quantification.
Asunto(s)
Silicio , Factor A de Crecimiento Endotelial Vascular , Silicio/química , Humanos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas/química , Proteínas/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
The ability to routinely identify and quantify the complete proteome from single cells will greatly advance medicine and basic biology research. To meet this challenge of single-cell proteomics, single-molecule technologies are being developed and improved. Most approaches, to date, rely on the analysis of polypeptides, resulting from digested proteins, either in solution or immobilized on a surface. Nanopore biosensing is an emerging single-molecule technique that circumvents surface immobilization and is optimally suited for the analysis of long biopolymers, as has already been shown for DNA sequencing. However, proteins, unlike DNA molecules, are not uniformly charged and harbor complex tertiary structures. Consequently, the ability of nanopores to analyze unfolded full-length proteins has remained elusive. Here, we evaluate the use of heat denaturation and the anionic surfactant sodium dodecyl sulfate (SDS) to facilitate electrokinetic nanopore sensing of unfolded proteins. Specifically, we characterize the voltage dependence translocation dynamics of a wide molecular weight range of proteins (from 14 to 130 kDa) through sub-5 nm solid-state nanopores, using a SDS concentration below the critical micelle concentration. Our results suggest that proteins' translocation dynamics are significantly slower than expected, presumably due to the smaller nanopore diameters used in our study and the role of the electroosmotic force opposing the translocation direction. This allows us to distinguish among the proteins of different molecular weights based on their dwell time and electrical charge deficit. Given the simplicity of the protein denaturation assay and circumvention of the tailor-made necessities for sensing protein of different folded sizes, shapes, and charges, this approach can facilitate the development of a whole proteome identification technique.
Asunto(s)
Nanoporos , Proteoma , ADN/química , Electroósmosis , NanotecnologíaRESUMEN
Accurate identification of both abundant and rare proteins hinges on the development of single-protein sensing methods. Given the immense variation in protein expression levels in a cell, separation of proteins by weight would improve protein classification strategies. Upstream separation facilitates sample binning into smaller groups while also preventing sensor overflow, as may be caused by highly abundant proteins in cell lysates or clinical samples. Here, we scale a bulk analysis method for protein separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), to the single-molecule level using single-photon sensitive widefield imaging. Single-molecule sensing of the electrokinetically moving proteins is achieved by in situ polymerization of the PAGE in a low-profile fluidic channel having a depth of only ~ 0.6 µm. The polyacrylamide gel restricts the Brownian kinetics of the proteins, while the low-profile channel ensures that they remain in focus during imaging, allowing video-rate monitoring of single-protein migration. Calibration of the device involves separating a set of Atto647N-covalently labeled recombinant proteins in the size range of 14-70 kDa, yielding an exponential dependence of the proteins' molecular weights on the measured mobilities, as expected. Subsequently, we demonstrate the ability of our fluidic device to separate and image thousands of proteins directly extracted from a human cancer cell line. Using single-particle image analysis methods, we created detailed profiles of the separation kinetics of lysine and cysteine -labeled proteins. Downstream coupling of the device to single-protein identification sensors may provide superior protein classification and improve our ability to analyze complex biological and medical protein samples.
Asunto(s)
Análisis por Matrices de Proteínas/métodos , Proteínas/química , Resinas Acrílicas/química , Calibración , Línea Celular Tumoral , Cisteína/química , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Lisina/química , Peso Molecular , Neoplasias/patología , Proteómica , Dodecil Sulfato de Sodio/químicaRESUMEN
RNA quantification methods are broadly used in life science research and in clinical diagnostics. Currently, real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most common analytical tool for RNA quantification. However, in cases of rare transcripts or inhibiting contaminants in the sample, an extensive amplification could bias the copy number estimation, leading to quantification errors and false diagnosis. Single-molecule techniques may bypass amplification but commonly rely on fluorescence detection and probe hybridization, which introduces noise and limits multiplexing. Here, we introduce reverse transcription quantitative nanopore sensing (RT-qNP), an RNA quantification method that involves synthesis and single-molecule detection of gene-specific cDNAs without the need for purification or amplification. RT-qNP allows us to accurately quantify the relative expression of metastasis-associated genes MACC1 and S100A4 in nonmetastasizing and metastasizing human cell lines, even at levels for which RT-qPCR quantification produces uncertain results. We further demonstrate the versatility of the method by adapting it to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA against a human reference gene. This internal reference circumvents the need for producing a calibration curve for each measurement, an imminent requirement in RT-qPCR experiments. In summary, we describe a general method to process complicated biological samples with minimal losses, adequate for direct nanopore sensing. Thus, harnessing the sensitivity of label-free single-molecule counting, RT-qNP can potentially detect minute expression levels of RNA biomarkers or viral infection in the early stages of disease and provide accurate amplification-free quantification.
Asunto(s)
Técnicas Biosensibles/métodos , Nanoporos , ARN Mensajero/análisis , Imagen Individual de Molécula/métodos , Betacoronavirus/genética , Técnicas Biosensibles/normas , Células HCT116 , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteína de Unión al Calcio S100A4/genética , Proteína de Unión al Calcio S100A4/metabolismo , SARS-CoV-2 , Imagen Individual de Molécula/normas , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Solid-state nanopore sensing of ultralong genomic DNA molecules has remained challenging, as the DNA must be controllably delivered by its leading end for efficient entry into the nanopore. Herein, we introduce a nanopore sensor device designed for electro-optical detection and sorting of ultralong (300+ kilobase pair) genomic DNA. The fluidic device, fabricated in-silicon and anodically bonded to glass, uses pressure-induced flow and an embedded pillar array for controllable DNA stretching and delivery. Extremely low concentrations (50 fM) and sample volumes (â¼1 µL) of DNA can be processed. The low height profile of the device permits high numerical aperture, high magnification imaging of DNA molecules, which remain in focus over extended distances. We demonstrate selective DNA sorting based on sequence-specific nick translation labeling and imaging at high camera frame rates. Nanopores are fabricated directly in the assembled device by laser etching. We show that uncoiling and stretching of the ultralong DNA molecules permits efficient nanopore capture and threading, which is simultaneously and synchronously imaged and electrically measured. Furthermore, our technique provides key insights into the translocation behavior of ultralong DNA and promotes the development of all-in-one micro/nanofluidic platforms for nanopore sensing of biomolecules.
Asunto(s)
ADN/genética , Electricidad , Genoma Humano , Nanoporos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenómenos Ópticos , Electrodos , Fluorescencia , Células HCT116 , Humanos , Rayos LáserRESUMEN
Multicolor fluorescence substantially expands the sensing capabilities of nanopores by complementing or substituting the resistive pulsing signals. However, to date single-fluorophore detection in multiple color channels has proven to be challenging primarily due to high photoluminescence (PL) emanating from the silicon nitride (SiN x) membrane. We hypothesize that the high bandgap of titanium oxide (TiO2) would eliminate the PL background when used as a substrate for a nanopore, and hence enable individual fluorophore sensing during the fast passage of biomolecules through the pore. Herein, we introduce a method for fabricating locally supported, free-standing, TiO2 membranes, in which solid-state nanopores can be readily drilled. These devices produce essentially no PL in the blue-to-red visible spectral range, even when excited by multiple lasers simultaneously. At the same time, the TiO2 nanopores exhibit low electrical noise comparable with standard SiN x devices. Importantly, the optical signal-to-background ratio (SBR) in single-molecule sensing is improved by an order of magnitude, enabling the differentiation among labeled DNA molecules of similar length based solely on their labeling scheme. Finally, the increased SBR of the TiO2 devices allows detection of single fluorophores conjugated to the lysine or cysteine residues of short polypeptides, thus introducing the possibility for optical based peptide/protein discrimination in nanopores.
Asunto(s)
ADN/química , Nanoporos , Péptidos/química , Titanio/química , Mediciones Luminiscentes , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Monitoring individual proteins in solution while simultaneously obtaining tertiary and quaternary structural information is challenging. In this study, translocation of the vascular endothelial growth factor (VEGF) protein through a solid-state nanopore (ssNP) produces distinct ion-current blockade amplitude levels and durations likely corresponding to monomer, dimer, and higher oligomeric states. Upon changing from a non-reducing to a reducing condition, ion-current blockage events from the monomeric state dominate, consistent with the expected reduction of the two inter-chain VEGF disulfide bonds. Cleavage by plasmin and application of either a positive or a negative NP bias results in nanopore signals corresponding either to the VEGF receptor recognition domain or to the heparin binding domain, accordingly. Interestingly, multi-level analysis of VEGF events reveals how individual domains affect their translocation pattern. Our study shows that careful characterization of ssNP results elucidates real-time structural information about the protein, thereby complementing classical techniques for structural analysis of proteins in solution with the added advantage of quantitative single-molecule resolution of native proteins.
Asunto(s)
ADN/química , Fibrinolisina/química , Factor A de Crecimiento Endotelial Vascular/química , Sitios de Unión , Técnicas Electroquímicas , Humanos , Modelos Moleculares , Nanoporos/ultraestructura , Oxidación-Reducción , Fosfinas/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/químicaRESUMEN
PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH(-/-) cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH(-/-) cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Proteínas Aviares/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Segregación Cromosómica/genética , ADN Helicasas/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitosis/genética , Animales , Proteínas Aviares/metabolismo , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Pollos , Inestabilidad Cromosómica/genética , ADN Helicasas/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Técnicas de Inactivación de Genes , Humanos , Linfocitos/metabolismoRESUMEN
Nanopore sensing involves an electrophoretic transport of analytes through a nanoscale pore, permitting label-free sensing at the single-molecule level. However, to date, the detection of individual small proteins has been challenging, primarily due to the poor signal/noise ratio that these molecules produce during passage through the pore. Here, we show that fine adjustment of the buffer pH, close to the isoelectric point, can be used to slow down the translocation speed of the analytes, hence permitting sensing and characterization of small globular proteins. Ubiquitin (Ub) is a small protein of 8.5 kDa, which is well conserved in all eukaryotes. Ub conjugates to proteins as a posttranslational modification called ubiquitination. The immense diversity of Ub substrates, as well as the complexity of Ub modification types and the numerous physiological consequences of these modifications, make Ub and Ub chains an interesting and challenging subject of study. The ability to detect Ub and to identify Ub linkage type at the single-molecule level may provide a novel tool for investigation in the Ub field. This is especially adequate because, for most ubiquitinated substrates, Ub modifies only a few molecules in the cell at a given time. Applying our method to the detection of mono- and poly-Ub molecules, we show that we can analyze their characteristics using nanopores. Of particular importance is that two Ub dimers that are equal in molecular weight but differ in 3D structure due to their different linkage types can be readily discriminated. Thus, to our knowledge, our method offers a novel approach for analyzing proteins in unprecedented detail using solid-state nanopores. Specifically, it provides the basis for development of single-molecule sensing of differently ubiquitinated substrates with different biological significance. Finally, our study serves as a proof of concept for approaching nanopore detection of sub-10-kDa proteins and demonstrates the ability of this method to differentiate among native and untethered proteins of the same mass.
Asunto(s)
Técnicas Biosensibles/métodos , Nanoporos , Ubiquitina/química , Técnicas Biosensibles/instrumentación , Multimerización de ProteínaRESUMEN
Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA strands and play an important role in the maintenance of genomic integrity. Here we used single-molecule FRET to define the mechanism of interaction of BLM helicase with intra-stranded G4 structures. We show that the activity of BLM is substrate dependent, and highly regulated by a short-strand DNA (ssDNA) segment that separates the G4 motif from double-stranded DNA. We demonstrate cooperativity between the RQC and HRDC domains of BLM during binding and unfolding of the G4 structure, where the RQC domain interaction with G4 is stabilized by HRDC binding to ssDNA. We present a model that proposes a unique role for G4 structures in modulating the activity of DNA processing enzymes.
Asunto(s)
ADN de Cadena Simple/genética , G-Cuádruplex , RecQ Helicasas/genética , Síndrome de Bloom/genética , Línea Celular , Reparación del ADN/genética , Exodesoxirribonucleasas/genética , Transferencia Resonante de Energía de Fluorescencia , Humanos , Modelos Genéticos , Estructura Terciaria de Proteína , Helicasa del Síndrome de WernerRESUMEN
The eukaryotic cell cycle is conventionally viewed as comprising several discrete steps, each of which must be completed before the next one is initiated. However, emerging evidence suggests that incompletely replicated, or unresolved, chromosomes from S-phase can persist into mitosis, where they present a potential threat to the faithful segregation of sister chromatids. In this review, we provide an overview of the different classes of loci where this 'unfinished S-phase business' can lead to a variety of cytogenetically distinct DNA structures throughout the various steps of mitosis. Furthermore, we discuss the potential ways in which cells might not only tolerate this inevitable aspect of chromosome biology, but also exploit it to assist in the maintenance of genome stability.
Asunto(s)
Mitosis/fisiología , Fase S/fisiología , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Cromátides/genética , Cromátides/metabolismo , Cromátides/fisiología , Cromosomas/metabolismo , Cromosomas/fisiología , Replicación del ADN/genética , Replicación del ADN/fisiología , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Humanos , Mitosis/genética , Modelos Biológicos , Fase S/genéticaRESUMEN
The Plk1-interacting checkpoint helicase (PICH) protein localizes to ultrafine anaphase bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including the Bloom's syndrome protein (BLM). However, very little is known about the function of PICH or how it is recruited to UFBs. Using a combination of microfluidics, fluorescence microscopy, and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to duplex DNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding DNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a model in which PICH recognizes and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids.
Asunto(s)
Anafase/genética , ADN Helicasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Cromátides/metabolismo , ADN Helicasas/química , ADN Helicasas/genética , Humanos , Microscopía Fluorescente/métodos , Ácidos Nucleicos Heterodúplex/metabolismo , Nucleosomas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Replicative DNA damage bypass promotes cell viability in the presence of genotoxic agents but at the same time may lead to mutations, thereby contributing to genomic instability. In eukaryotes, DNA damage bypass is mediated by damage-induced ubiquitylation of the sliding clamp protein, proliferating cell nuclear antigen (PCNA). We have recently shown that replication protein A (RPA), a single-stranded (ss)DNA-binding protein essential for DNA replication, repair and recombination, is required for PCNA ubiquitylation in budding yeast. Both in yeast and in mammalian cells, RPA physically interacts with Rad18, the ubiquitin ligase responsible for PCNA mono-ubiquitylation. The association of Rad18 with chromatin correlates with that of RPA, and purified RPA can recruit the ligase to ssDNA in vitro. Here we have examined in more detail the interactions between Rad18, RPA and DNA and discuss their contribution to the activation of DNA damage bypass.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/metabolismo , Proteína de Replicación A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Replicación del ADN , ADN de Cadena Simple/metabolismo , Modelos Biológicos , Concentración Osmolar , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , UbiquitinaciónRESUMEN
Replicative DNA damage bypass, mediated by the ubiquitylation of the sliding clamp protein PCNA, facilitates the survival of a cell in the presence of genotoxic agents, but it can also promote genomic instability by damage-induced mutagenesis. We show here that PCNA ubiquitylation in budding yeast is activated independently of the replication-dependent S phase checkpoint but by similar conditions involving the accumulation of single-stranded DNA at stalled replication intermediates. The ssDNA-binding replication protein A (RPA), an essential complex involved in most DNA transactions, is required for damage-induced PCNA ubiquitylation. We found that RPA directly interacts with the ubiquitin ligase responsible for the modification of PCNA, Rad18, both in yeast and in mammalian cells. Association of the ligase with chromatin is detected where RPA is most abundant, and purified RPA can recruit Rad18 to ssDNA in vitro. Our results therefore implicate the RPA complex in the activation of DNA damage tolerance.
Asunto(s)
Daño del ADN , ADN de Cadena Simple/metabolismo , Proteína de Replicación A/metabolismo , Ubiquitina/metabolismo , Animales , Ciclo Celular/fisiología , Línea Celular , Replicación del ADN , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína de Replicación A/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina/genéticaRESUMEN
Posttranslational modification of proliferating cell nuclear antigen (PCNA), an essential processivity clamp for DNA polymerases, by ubiquitin and SUMO contributes to the coordination of DNA replication, damage tolerance, and mutagenesis. Whereas ubiquitination in response to DNA damage promotes the bypass of replication-blocking lesions, sumoylation during S phase is damage independent. As both modifiers target the same site on PCNA, an antagonistic action of SUMO on ubiquitin-dependent DNA damage tolerance has been proposed. We now present evidence that the apparent negative effect of SUMO on lesion bypass is not due to competition with ubiquitination but is rather mediated by the helicase Srs2p, which affects genome stability by suppressing unscheduled homologous recombination. We show that Srs2p physically interacts with sumoylated PCNA, which contributes to the recruitment of the helicase to replication forks. Our findings suggest a mechanism by which SUMO and ubiquitin cooperatively control the choice of pathway for the processing of DNA lesions during replication.