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1.
Nat Commun ; 11(1): 5115, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037228

RESUMO

Recurrent neural networks have led to breakthroughs in natural language processing and speech recognition. Here we show that recurrent networks, specifically long short-term memory networks can also capture the temporal evolution of chemical/biophysical trajectories. Our character-level language model learns a probabilistic model of 1-dimensional stochastic trajectories generated from higher-dimensional dynamics. The model captures Boltzmann statistics and also reproduces kinetics across a spectrum of timescales. We demonstrate how training the long short-term memory network is equivalent to learning a path entropy, and that its embedding layer, instead of representing contextual meaning of characters, here exhibits a nontrivial connectivity between different metastable states in the underlying physical system. We demonstrate our model's reliability through different benchmark systems and a force spectroscopy trajectory for multi-state riboswitch. We anticipate that our work represents a stepping stone in the understanding and use of recurrent neural networks for understanding the dynamics of complex stochastic molecular systems.


Assuntos
Idioma , Memória , Modelos Estatísticos , Redes Neurais de Computação , Inteligência Artificial , Dipeptídeos/química , Cinética , Cadeias de Markov , Simulação de Dinâmica Molecular , Imagem Individual de Molécula
2.
Nat Commun ; 11(1): 5043, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028863

RESUMO

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.


Assuntos
Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Integrases/ultraestrutura , Proteína Fosfatase 2/ultraestrutura , Vírus Linfotrópico T Tipo 1 de Símios/enzimologia , Proteínas Virais/ultraestrutura , Integração Viral , Motivos de Aminoácidos/genética , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Viral/metabolismo , DNA Viral/ultraestrutura , Vírus Linfotrópico T Tipo 1 Humano/enzimologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Integrases/genética , Integrases/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Paraparesia Espástica Tropical/patologia , Paraparesia Espástica Tropical/virologia , Multimerização Proteica , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Vírus Linfotrópico T Tipo 1 de Símios/genética , Imagem Individual de Molécula , Proteínas Virais/genética , Proteínas Virais/metabolismo
3.
Nat Commun ; 11(1): 5096, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037221

RESUMO

Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.


Assuntos
Fator G para Elongação de Peptídeos/biossíntese , Fator G para Elongação de Peptídeos/química , Dobramento de Proteína , Luminescência , Fator G para Elongação de Peptídeos/genética , Biossíntese de Proteínas , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Imagem Individual de Molécula
4.
Nat Commun ; 11(1): 4476, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900995

RESUMO

Mechanically stable specific heterodimerization between small protein domains have a wide scope of applications, from using as a molecular anchorage in single-molecule force spectroscopy studies of protein mechanics, to serving as force-bearing protein linker for modulation of mechanotransduction of cells, and potentially acting as a molecular crosslinker for functional materials. Here, we explore the possibility to develop heterodimerization system with a range of mechanical stability from a set of recently engineered helix-heterotetramers whose mechanical properties have yet to be characterized. We demonstrate this possibility using two randomly chosen helix-heterotetramers, showing that their mechanical properties can be modulated by changing the stretching geometry and the number of interacting helices. These helix-heterotetramers and their derivatives are sufficiently stable over physiological temperature range. Using it as mechanically stable anchorage, we demonstrate the applications in single-molecule manipulation studies of the temperature dependent unfolding and refolding of a titin immunoglobulin domain and α-actinin spectrin repeats.


Assuntos
Engenharia de Proteínas , Multimerização Proteica , Estabilidade Proteica , Actinina/química , Fenômenos Biomecânicos , Conectina/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Desdobramento de Proteína , Imagem Individual de Molécula , Temperatura
5.
Nat Commun ; 11(1): 4531, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913225

RESUMO

RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5'-to-3' direction, at ~60 nt s-1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.


Assuntos
Fusobacterium/genética , Regulação Bacteriana da Expressão Gênica , Dobramento de RNA/genética , RNA Bacteriano/genética , Riboswitch/genética , Aminoimidazol Carboxamida/metabolismo , Fusobacterium/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , Ribonucleotídeos/metabolismo , Imagem Individual de Molécula , Transcrição Genética
6.
Nat Commun ; 11(1): 4384, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873796

RESUMO

The ability to detect low concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, we report a biosensor platform using DNA origami featuring a central cavity with a target-specific DNA aptamer coupled with a nanopore read-out to enable individual biomarker detection. We show that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence of the biomarker in the cavity. We exploit this to generate a biosensing platform with a limit of detection of 3 nM and capable of the detection of human C-reactive protein (CRP) in clinically relevant fluids. Future development of this approach may enable multiplexed biomarker detection by using ribbons of DNA origami with integrated barcoding.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , DNA/química , Nanoestruturas/química , Imagem Individual de Molécula/instrumentação , Biomarcadores/análise , Proteína C-Reativa/análise , Desenho de Equipamento , Humanos , Limite de Detecção , Nanotecnologia/métodos
7.
Proc Natl Acad Sci U S A ; 117(37): 22815-22822, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868420

RESUMO

The sensitive and accurate quantification of protein biomarkers plays important roles in clinical diagnostics and biomedical research. Sandwich ELISA and its variants accomplish the capture and detection of a target protein via two antibodies that tightly bind at least two distinct epitopes of the same antigen and have been the gold standard for sensitive protein quantitation for decades. However, existing antibody-based assays cannot distinguish between signal arising from specific binding to the protein of interest and nonspecific binding to assay surfaces or matrix components, resulting in significant background signal even in the absence of the analyte. As a result, they generally do not achieve single-molecule sensitivity, and they require two high-affinity antibodies as well as stringent washing to maximize sensitivity and reproducibility. Here, we show that surface capture with a high-affinity antibody combined with kinetic fingerprinting using a dynamically binding, low-affinity fluorescent antibody fragment differentiates between specific and nonspecific binding at the single-molecule level, permitting the direct, digital counting of single protein molecules with femtomolar-to-attomolar limits of detection (LODs). We apply this approach to four exemplary antigens spiked into serum, demonstrating LODs 55- to 383-fold lower than commercially available ELISA. As a real-world application, we establish that endogenous interleukin-6 (IL-6) can be quantified in 2-µL serum samples from chimeric antigen receptor T cell (CAR-T cell) therapy patients without washing away excess serum or detection probes, as is required in ELISA-based approaches. This kinetic fingerprinting thus exhibits great potential for the ultrasensitive, rapid, and streamlined detection of many clinically relevant proteins.


Assuntos
Ligação Proteica/fisiologia , Imagem Individual de Molécula/métodos , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Especificidade de Anticorpos/fisiologia , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Cinética , Limite de Detecção , Nanotecnologia , Proteínas , Reprodutibilidade dos Testes
8.
PLoS One ; 15(9): e0238470, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32911526

RESUMO

We report a powerful method for capturing the time-resolved concentration profiles, liquid swelling and surface phenomena during the absorption of methane (CH4) in still liquid ethanol (C2D6O) and n-decane (n-C10D22) and at high spatial resolution (pixel size 21.07 µm) using neutron imaging. Absorption of supercritical methane was followed at two temperatures and two pressures of methane, namely 7.0, 37.8 °C and 80, 120 bar. Fick's second law, which was used in the liquid-fixed coordinates, enabled for an adequate parameterization of the observed concentration profiles and liquid levels using simple analytical expressions. For both studied liquids, anomalously slow diffusion was observed in the initial stage of the absorption experiment. This was ascribed to the slow formation of the surface excess on the interface, time constant ranged 130-275 s. The axial symmetry of the cell allowed for the tomographic reconstructions of the profiles of the menisci. Based on these profiles, contact angle and surface tension were evaluated using the Young-Laplace equation. Overall, neutron imaging made it possible to capture time- and space-resolved information from which the methane concentration, liquid level and meniscus shape under high-pressure conditions inside a cylindrical titanium vessel were quantitatively derived. Multiple characteristics of ethanol, a methane hydrate inhibitor, and n-decane, a model constituent of crude oil, were thus measured for the first time under industrially relevant conditions in a one-pot experiment.


Assuntos
Metano/química , Imagem Individual de Molécula/métodos , Alcanos/química , Difusão , Etanol/química , Modelos Estatísticos , Nêutrons , Petróleo , Fenômenos Físicos , Imagem Individual de Molécula/instrumentação , Temperatura , Água
9.
Nat Commun ; 11(1): 4768, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958747

RESUMO

Detection and identification of proteins are typically achieved by analyzing protein size, charge, mobility and binding to antibodies, which are critical for biomedical research and disease diagnosis and treatment. Despite the importance, measuring these quantities with one technology and at the single-molecule level has not been possible. Here we tether a protein to a surface with a flexible polymer, drive it into oscillation with an electric field, and image the oscillation with a near field optical imaging method, from which we determine the size, charge, and mobility of the protein. We also measure antibody binding and conformation changes in the protein. The work demonstrates a capability for comprehensive protein analysis and precision protein biomarker detection at the single molecule level.


Assuntos
Proteínas/química , Proteínas/metabolismo , Imagem Individual de Molécula/métodos , Análise de Fourier , Ligantes , Polietilenoglicóis/química , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Compostos de Estanho/química
10.
Mol Cell ; 80(1): 114-126.e8, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32916094

RESUMO

DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis. Here, we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, redefining the architecture of the active eukaryotic replisome.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Células Eucarióticas/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleo Celular/metabolismo , Cinética , Modelos Biológicos , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Fatores de Tempo
11.
Nucleic Acids Res ; 48(19): 10820-10831, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32997109

RESUMO

DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.


Assuntos
DNA/química , Proteínas HMGN/química , Proteínas de Saccharomyces cerevisiae/química , DNA/metabolismo , Proteínas HMGN/metabolismo , Simulação de Dinâmica Molecular , Movimento (Física) , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula
12.
ACS Nano ; 14(10): 13964-13974, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-32930583

RESUMO

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.


Assuntos
Técnicas Biossensoriais/métodos , Nanoporos , RNA Mensageiro/análise , Imagem Individual de Molécula/métodos , Betacoronavirus/genética , Técnicas Biossensoriais/normas , Células HCT116 , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/genética , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Imagem Individual de Molécula/normas , Transativadores/genética , Transativadores/metabolismo
13.
Nat Commun ; 11(1): 4321, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859904

RESUMO

Bacterial colonization of the human intestine requires firm adhesion of bacteria to insoluble substrates under hydrodynamic flow. Here we report the molecular mechanism behind an ultrastable protein complex responsible for resisting shear forces and adhering bacteria to cellulose fibers in the human gut. Using single-molecule force spectroscopy (SMFS), single-molecule FRET (smFRET), and molecular dynamics (MD) simulations, we resolve two binding modes and three unbinding reaction pathways of a mechanically ultrastable R. champanellensis (Rc) Dockerin:Cohesin (Doc:Coh) complex. The complex assembles in two discrete binding modes with significantly different mechanical properties, with one breaking at ~500 pN and the other at ~200 pN at loading rates from 1-100 nN s-1. A neighboring X-module domain allosterically regulates the binding interaction and inhibits one of the low-force pathways at high loading rates, giving rise to a catch bonding mechanism that manifests under force ramp protocols. Multi-state Monte Carlo simulations show strong agreement with experimental results, validating the proposed kinetic scheme. These results explain mechanistically how gut microbes regulate cell adhesion strength at high shear stress through intricate molecular mechanisms including dual-binding modes, mechanical allostery and catch bonds.


Assuntos
Aderência Bacteriana/fisiologia , Microbioma Gastrointestinal/fisiologia , Fenômenos Mecânicos , Fenômenos Físicos , Bactérias , Aderência Bacteriana/genética , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Trato Gastrointestinal/microbiologia , Técnicas de Inativação de Genes , Humanos , Cinética , Simulação de Dinâmica Molecular , Método de Monte Carlo , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula , Estresse Mecânico
14.
Nat Commun ; 11(1): 4259, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848156

RESUMO

The plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available to manipulate membrane protein location at the single-molecule level. Here, we use fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to control their movement. FMNPs allow single-particle tracking (SPT) at 10 nm and 5 ms spatiotemporal resolution, and using a magnetic needle, we pull membrane components laterally with femtonewton-range forces. In this way, we drag membrane proteins over the surface of living cells. Doing so, we detect barriers which we could localize to the submembrane actin cytoskeleton by super-resolution microscopy. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion.


Assuntos
Nanopartículas de Magnetita , Proteínas de Membrana/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Proteínas de Fluorescência Verde/metabolismo , Campos Magnéticos , Lipídeos de Membrana/metabolismo , Microscopia de Fluorescência , Nanotecnologia , Imagem Individual de Molécula/métodos
15.
Nat Commun ; 11(1): 4281, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855416

RESUMO

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Conformational transitions of the transcription initiation complex must be central for such control, but how the conformational dynamics progress throughout transcription initiation remains unknown. Here, we use single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the transcriptional system of yeast mitochondria with single-base resolution. We show that the yeast mitochondrial transcriptional complex dynamically transitions among closed, open, and scrunched states throughout the initiation stage. Then abruptly at position +8, the dynamic states of initiation make a sharp irreversible transition to an unbent conformation with associated promoter release. Remarkably, stalled initiation complexes remain in dynamic scrunching and unscrunching states without dissociating the RNA transcript, implying the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation suggests a kinetically driven regulation of mitochondrial transcription.


Assuntos
Mitocôndrias/genética , Saccharomyces cerevisiae/genética , Iniciação da Transcrição Genética , Trifosfato de Adenosina , DNA Fúngico/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Nucleic Acids Res ; 48(17): e97, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32756898

RESUMO

Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques.


Assuntos
DNA/química , Espectrometria de Massas/métodos , Fotometria/métodos , Imagem Individual de Molécula/métodos , DNA/análise
17.
Nucleic Acids Res ; 48(16): 9195-9203, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32810236

RESUMO

G-quadruplex (GQ) is formed at various regions of DNA, including telomeres of chromosomes and regulatory regions of oncogenes. Since GQ is important in both gene regulation and genome instability, the biological and medical implications of this abnormal DNA structure have been intensively studied. Its formation mechanisms, however, are not clearly understood yet. We report single-molecule fluorescence experiments to monitor the cotranscriptional GQ formation coupled with R-loop formation using T7 RNA polymerase. The GQ is formed very rarely per single-round transcription. R-loop formation precedes and facilitates GQ formation. Once formed, some GQs are extremely stable, resistant even to RNase H treatment, and accumulate in multiple-round transcription conditions. On the other hand, GQ existing in the non-template strand promotes the R-loop formation in the next rounds of transcription. Our study clearly shows the existence of a positive feedback mechanism of GQ and R-loop formations, which may possibly contribute to gene regulation and genome instability.


Assuntos
DNA/ultraestrutura , Quadruplex G , Estruturas R-Loop/genética , Imagem Individual de Molécula/métodos , RNA Polimerases Dirigidas por DNA/ultraestrutura , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Humanos , Telômero/ultraestrutura , Proteínas Virais/ultraestrutura
18.
Proc Natl Acad Sci U S A ; 117(35): 21346-21353, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817549

RESUMO

Cells continually sample their mechanical environment using exquisite force sensors such as talin, whose folding status triggers mechanotransduction pathways by recruiting binding partners. Mechanical signals in biology change quickly over time and are often embedded in noise; however, the mechanics of force-sensing proteins have only been tested using simple force protocols, such as constant or ramped forces. Here, using our magnetic tape head tweezers design, we measure the folding dynamics of single talin proteins in response to external mechanical noise and cyclic force perturbations. Our experiments demonstrate that talin filters out external mechanical noise but detects periodic force signals over a finely tuned frequency range. Hence, talin operates as a mechanical band-pass filter, able to read and interpret frequency-dependent mechanical information through its folding dynamics. We describe our observations in the context of stochastic resonance, which we propose as a mechanism by which mechanosensing proteins could respond accurately to force signals in the naturally noisy biological environment.


Assuntos
Mecanotransdução Celular , Talina/fisiologia , Domínios Proteicos , Dobramento de Proteína , Imagem Individual de Molécula
19.
Nucleic Acids Res ; 48(17): 9550-9570, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32810208

RESUMO

Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer-gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer-gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Neurônios/fisiologia , RNA/fisiologia , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Cromatina/metabolismo , Células HEK293 , Humanos , Neurônios/citologia , Proteínas Proto-Oncogênicas c-fos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Ratos , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Imagem Individual de Molécula
20.
Proc Natl Acad Sci U S A ; 117(31): 18540-18549, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675239

RESUMO

Once described as mere "bags of enzymes," bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imagem Individual de Molécula , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
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