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1.
PLoS One ; 15(8): e0237253, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790805

RESUMO

Aptamers are short single-stranded DNA (ssDNA), RNA or synthetic XNA molecules, which are used as a class of affinity binders recognizing target molecules with a very high affinity and specificity. The aim of this study was to generate and characterize ssDNA aptamers for the detection of Newcastle disease virus (NDV). These aptamers were selected using systematic evolution of ligands by exponential enrichment (SELEX) in combination with quantitative high-throughput DNA sequencing. After three rounds of selections, a highly enriched ssDNA pool was sequenced, and the results were analyzed using FASTAptamer Toolkit. Sequencing reads were sorted by copy numbers and clustered into groups, according to their sequence homology. Top aptameric sequences were used to develop a sandwich enzymatic linked aptamer assay (ELAA) for rapid and sensitive detection of NDV in farm samples. The selected aptamers have an affinity within the nanomolar range, and a high specificity with no cross-reactivity towards other avian viruses. Following optimization of the sandwich ELAA method, the results demonstrated that both selected aptamers Apt_NDV01 and Apt_NDV03 with dissociation constant values of 31 nM and 78.1 nM, respectively, showed the highest specificity and affinity for NDV detection. The ELAA results were verified by quantitative real-time PCR, demonstrating strong concordance, and showing outstanding accuracy for detection of NDV in field sample. In summary, combination of SELEX with high-throughput DNA sequencing allowed rapid screening and selection of aptamers. The selected aptamers allowed recognition of NDV with high affinities. This is the first report that uses a validated sandwich ELAA for rapid and specific detection of NDV in poultry samples.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Doença de Newcastle/diagnóstico , Vírus da Doença de Newcastle/isolamento & purificação , Animais , Técnicas Biossensoriais , Sequenciamento de Nucleotídeos em Larga Escala , Doença de Newcastle/virologia , Aves Domésticas/virologia , Técnica de Seleção de Aptâmeros
2.
Nat Commun ; 11(1): 2828, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504003

RESUMO

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.


Assuntos
DNA de Cadeia Simples/metabolismo , RNA Polimerase III/metabolismo , Imagem Individual de Molécula/métodos , Fator de Transcrição TFIIIB/metabolismo , Transcrição Genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Cinética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Sondas Moleculares/ultraestrutura , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Estabilidade Proteica , RNA Polimerase III/química , Proteínas Recombinantes/metabolismo , Proteína de Ligação a TATA-Box/metabolismo
3.
Nucleic Acids Res ; 48(14): 7640-7652, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32558908

RESUMO

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Förster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.


Assuntos
DNA/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , RNA de Cadeia Dupla/química , Pareamento de Bases , DNA de Cadeia Simples/química , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/química
4.
Food Chem ; 330: 127230, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32526651

RESUMO

Chitooligosaccharides are oligosaccharides with many biological activities that can be used in food production for sweeteners, preservatives and humectants, among other products. Chitin, a long-chain polymer of N-acetylglucosamine and a derivative of glucose, can be hydrolyzed by applying chitinase to break down glycosidic bonds to form chitooligosaccharides. Chitinases arising from heterologous gene expression are usually linked to a 6 × His-tag to facilitate easy purification. Heterologously expressed chitinase linked to a 6 × His-tag is a transgenic element, but enzyme activity tests cannot be used to distinguish transgenic elements from natural elements. In this study, we established a rapid and easy method to detect His-tag-containing chitinase using gold nanoparticles (AuNPs) and ssDNA aptamers. Using this method, His-tag-containing chitinase could be detected at concentrations as low as 0.136 nM within 5 min. Color changes of AuNPs showed a positive correlation with His-tag-containing chitinase concentrations.


Assuntos
Aptâmeros de Nucleotídeos/química , Quitinases/metabolismo , DNA de Cadeia Simples/química , Ouro/química , Nanopartículas Metálicas/química , Quitina/análogos & derivados , Quitina/metabolismo , Cor , Hidrólise , Fatores de Tempo
5.
Science ; 368(6495): 1081-1085, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32499435

RESUMO

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome's organization of double-stranded DNA.


Assuntos
Complexos Multiproteicos/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Telômero/química , Microscopia Crioeletrônica , DNA de Cadeia Simples/química , Células HEK293 , Humanos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteína de Replicação A/química
6.
Nucleic Acids Res ; 48(11): 6053-6067, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374866

RESUMO

Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.


Assuntos
Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Fluorescência , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/química , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Proteínas Intrinsicamente Desordenadas/química , Ligação Proteica , Resposta SOS em Genética
7.
PLoS One ; 15(5): e0226791, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374764

RESUMO

Over the past two decades, measurements of carbon nanotube toxicity and biodistribution have yielded a wide range of results. Properties such as nanotube type (single-walled vs. multi-walled), purity, length, aggregation state, and functionalization, as well as route of administration, greatly affect both the biocompatibility and biodistribution of carbon nanotubes. These differences suggest that generalizable conclusions may be elusive and that studies must be material- and application-specific. Here, we assess the short- and long-term biodistribution and biocompatibility of a single-chirality DNA-encapsulated single-walled carbon nanotube complex upon intravenous administration that was previously shown to function as an in-vivo reporter of endolysosomal lipid accumulation. Regarding biodistribution and fate, we found bulk specificity to the liver and >90% signal attenuation by 14 days in mice. Using near-infrared hyperspectral microscopy to measure single nanotubes, we found low-level, long-term persistence in organs such as the heart, liver, lung, kidney, and spleen. Measurements of histology, animal weight, complete blood count; biomarkers of organ function all suggest short- and long-term biocompatibility. This work suggests that carbon nanotubes can be used as preclinical research tools in-vivo without affecting acute or long-term health.


Assuntos
Materiais Biocompatíveis/farmacologia , Biomarcadores/sangue , Nanotecnologia , Nanotubos de Carbono/efeitos adversos , Animais , Materiais Biocompatíveis/efeitos adversos , Materiais Biocompatíveis/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/farmacologia , Endossomos/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Camundongos , Nanotubos de Carbono/química , Imagem Óptica , Baço/efeitos dos fármacos , Baço/metabolismo , Distribuição Tecidual/efeitos dos fármacos
8.
Nat Methods ; 17(5): 515-523, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32251394

RESUMO

Transcription is a highly dynamic process that generates single-stranded DNA (ssDNA) in the genome as 'transcription bubbles'. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, based on the fast and specific reaction between N3-kethoxal and guanines in ssDNA. KAS-seq allows rapid (within 5 min), sensitive and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ using as few as 1,000 cells. KAS-seq enables definition of a group of enhancers that are single-stranded and enrich unique sequence motifs. These enhancers are associated with specific transcription-factor binding and exhibit more enhancer-promoter interactions than typical enhancers do. Under conditions that inhibit protein condensation, KAS-seq uncovers a rapid release of RNA polymerase II (Pol II) from a group of promoters. KAS-seq thus facilitates fast and accurate analysis of transcription dynamics and enhancer activities simultaneously in both low-input and high-throughput manner.


Assuntos
Aldeídos/química , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , Elementos Facilitadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Animais , DNA de Cadeia Simples/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Transcrição Genética
9.
Nat Commun ; 11(1): 1543, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210235

RESUMO

Field-effect transistor (FET)-based biosensors allow label-free detection of biomolecules by measuring their intrinsic charges. The detection limit of these sensors is determined by the Debye screening of the charges from counter ions in solutions. Here, we use FETs with a deformed monolayer graphene channel for the detection of nucleic acids. These devices with even millimeter scale channels show an ultra-high sensitivity detection in buffer and human serum sample down to 600 zM and 20 aM, respectively, which are ∼18 and ∼600 nucleic acid molecules. Computational simulations reveal that the nanoscale deformations can form 'electrical hot spots' in the sensing channel which reduce the charge screening at the concave regions. Moreover, the deformed graphene could exhibit a band-gap, allowing an exponential change in the source-drain current from small numbers of charges. Collectively, these phenomena allow for ultrasensitive electronic biomolecular detection in millimeter scale structures.


Assuntos
Técnicas Biossensoriais/instrumentação , Sondas de DNA/análise , DNA de Cadeia Simples/análise , Grafite/química , MicroRNAs/análise , Sondas de DNA/química , DNA de Cadeia Simples/química , Estudos de Viabilidade , Humanos , Íons , Limite de Detecção , MicroRNAs/química , Simulação de Dinâmica Molecular , Sensibilidade e Especificidade , Transistores Eletrônicos
10.
Proc Natl Acad Sci U S A ; 117(11): 5801-5809, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32127479

RESUMO

All organisms-bacteria, archaea, and eukaryotes-have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate and define details of stepwise, RNA-extension-driven displacement of the "σ-finger" of the bacterial transcription initiation factor σ. The structures reveal that-for both the primary σ-factor and extracytoplasmic (ECF) σ-factors, and for both 5'-triphosphate RNA and 5'-hydroxy RNA-the "σ-finger" is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5'-end, upon extension of nascent RNA from ∼5 nt to ∼10 nt.


Assuntos
Proteínas de Escherichia coli/química , Fator sigma/química , Iniciação da Transcrição Genética , Sítios de Ligação , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Simulação de Dinâmica Molecular , Regiões Promotoras Genéticas , Ligação Proteica , RNA/química , RNA/genética , RNA/metabolismo , Fator sigma/metabolismo
11.
Nucleic Acids Res ; 48(7): 3657-3677, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32128579

RESUMO

DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10-30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.


Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteína de Replicação A/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , DNA de Cadeia Simples/química , Ligação Proteica , Vírus 40 dos Símios/imunologia
12.
Nucleic Acids Res ; 48(7): 3692-3707, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32133535

RESUMO

Alkylation is one of the most ubiquitous forms of DNA lesions. However, the motif preferences and substrates for the activity of the major types of alkylating agents defined by their nucleophilic substitution reactions (SN1 and SN2) are still unclear. Utilizing yeast strains engineered for large-scale production of single-stranded DNA (ssDNA), we probed the substrate specificity, mutation spectra and signatures associated with DNA alkylating agents. We determined that SN1-type agents preferably mutagenize double-stranded DNA (dsDNA), and the mutation signature characteristic of the activity of SN1-type agents was conserved across yeast, mice and human cancers. Conversely, SN2-type agents preferably mutagenize ssDNA in yeast. Moreover, the spectra and signatures derived from yeast were detectable in lung cancers, head and neck cancers and tumors from patients exposed to SN2-type alkylating chemicals. The estimates of mutation loads associated with the SN2-type alkylation signature were higher in lung tumors from smokers than never-smokers, pointing toward the mutagenic activity of the SN2-type alkylating carcinogens in cigarettes. In summary, our analysis of mutations in yeast strains treated with alkylating agents, as well as in whole-exome and whole-genome-sequenced tumors identified signatures highly specific to alkylation mutagenesis and indicate the pervasive nature of alkylation-induced mutagenesis in cancers.


Assuntos
Alquilantes/toxicidade , Mutagênese , Mutação , Neoplasias/genética , Adenina/química , Animais , DNA Glicosilases/metabolismo , DNA Fúngico/química , DNA de Cadeia Simples/química , Humanos , Camundongos , Leveduras/efeitos dos fármacos , Leveduras/genética , Leveduras/metabolismo
13.
Nanotechnology ; 31(25): 255102, 2020 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176872

RESUMO

DNA-displaying nanoparticles comprised of conjugates of single-stranded DNA (ssDNA) and elastin-like polypeptide (ELP) were developed. ssDNA was enzymatically conjugated to ELPs via a catalytic domain of Porcine Circovirus type 2 replication initiation protein (pRep) fused to ELPs. Nanoparticles were formed upon heating to temperatures above the phase transition temperature due to the hydrophobicity of ELPs and the hydrophilicity of conjugated ssDNA. We demonstrated the applicability of the resultant nanoparticles as drug carriers with tumor-targeting properties by conjugating a DNA aptamer, which is known to bind to Mucin 1 (MUC1), to ELPs. DNA aptamer-displaying nanoparticles encapsulating the anti-cancer drug paclitaxel were able to bind to cells overexpressing MUC1 and induce cell death.


Assuntos
DNA de Cadeia Simples/química , Elastina/química , Paclitaxel/farmacologia , Peptídeos/química , Proteínas Virais/química , Aptâmeros de Nucleotídeos/química , Sobrevivência Celular/efeitos dos fármacos , Circovirus/genética , Circovirus/metabolismo , Replicação do DNA , Portadores de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células MCF-7 , Nanopartículas , Paclitaxel/química
14.
Nucleic Acids Res ; 48(3): 1353-1371, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31943071

RESUMO

The human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons, and other viruses such as hepatitis B virus, but can cause a mutator phenotype in many cancers. While structural information exists for several A3 proteins, the precise details regarding deamination target selection are not fully understood. Here, we report the first parallel, comparative analysis of site selection of A3 deamination using six of the seven purified A3 member enzymes, oligonucleotides having 5'TC3' or 5'CT3' dinucleotide target sites, and different flanking bases within diverse DNA secondary structures. A3A, A3F and A3H were observed to have strong preferences toward the TC target flanked by A or T, while all examined A3 proteins did not show a preference for a TC target flanked by a G. We observed that the TC target was strongly preferred in ssDNA regions rather than dsDNA, loop or bulge regions, with flanking bases influencing the degree of preference. CT was also shown to be a potential deamination target. Taken together, our observations provide new insights into A3 enzyme target site selection and how A3 mutagenesis impacts mutation rates.


Assuntos
Citidina Desaminase/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Desaminação/genética , Sítios de Ligação/genética , Linhagem Celular , Citidina Desaminase/química , Citosina Desaminase/química , Citosina Desaminase/genética , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , HIV-1/genética , HIV-1/patogenicidade , Vírus da Hepatite B/genética , Humanos , Mutagênese/genética , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , Retroelementos/genética
15.
Nucleic Acids Res ; 48(4): 1701-1714, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31919510

RESUMO

Replication protein A (RPA) plays a critical role in all eukaryotic DNA processing involving single-stranded DNA (ssDNA). Contrary to the notion that RPA provides solely inert protection to transiently formed ssDNA, the RPA-ssDNA complex acts as a dynamic DNA processing unit. Here, we studied the diffusion of RPA along 60 nt ssDNA using a coarse-grained model in which the ssDNA-RPA interface was modeled by both aromatic and electrostatic interactions. Our study provides direct evidence of bulge formation during the diffusion of ssDNA along RPA. Bulges can form at a few sites along the interface and store 1-7 nt of ssDNA whose release, upon bulge dissolution, leads to propagation of ssDNA diffusion. These findings thus support the reptation mechanism, which involves bulge formation linked to the aromatic interactions, whose short range nature reduces cooperativity in ssDNA diffusion. Greater cooperativity and a larger diffusion coefficient for ssDNA diffusion along RPA are observed for RPA variants with weaker aromatic interactions and for interfaces homogenously stabilized by electrostatic interactions. ssDNA propagation in the latter instance is characterized by lower probabilities of bulge formation; thus, it may fit the sliding-without-bulge model better than the reptation model. Thus, the reptation mechanism allows ssDNA mobility despite the extensive and high affinity interface of RPA with ssDNA. The short-range aromatic interactions support bulge formation while the long-range electrostatic interactions support the release of the stored excess ssDNA in the bulge and thus the overall diffusion.


Assuntos
Replicação do DNA/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteína de Replicação A/genética , Estruturas Cromossômicas/genética , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Humanos , Ligação Proteica/genética , Proteína de Replicação A/química
16.
Talanta ; 209: 120550, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31891998

RESUMO

Developing the high selectivity and sensitivity strategy for nucleic acid detection is crucial for early diagnosis and therapy of diseases. In this work, a novel low back-ground fluorescent sensor platform for the detection of nucleic acid has been developed based on δ-FeOOH nanosheets integrating with exonuclease III-assisted target-recycling signal amplification. Because of the strong binding ability between the single-strand DNA (ssDNA) and the δ-FeOOH nanosheets, the dye-labeled ssDNA probe would be quenched by δ-FeOOH nanosheets through fluorescence resonance energy transfer (FRET). By using magnetic separate properties of δ-FeOOH, the background signal was separated from the sensor system, and the low background sensor system was obtained. After adding the target DNA, a double-strand DNA complex (dsDNA) would be formed between the target DNA and dye-labeled ssDNA probe. Then, the dye-labeled ssDNA probe in the dsDNA complex would be stepwise hydrolyzed into short fragments from 3'-terminus by Exonuclease III, and the fluorescence signal was recovered due to the weak bind affinity between the short fragments and δ-FeOOH nanosheets. By using the fluorescence quenching ability of δ-FeOOH nanosheets and enzyme-assisted target-recycling signal amplification, this strategy could show an excellent selectivity toward hepatitis C virus DNA with a low detection limit of 10 pM. By simply changing the dye-labeled ssDNA probe sequence, this sensing platform can be developed as a universal approach for the simple, sensitive, and selective detection of different target DNA.


Assuntos
DNA Viral/sangue , Exodesoxirribonucleases/química , Compostos Férricos/química , Hepacivirus/isolamento & purificação , Hepatite C/sangue , Técnicas Biossensoriais/métodos , Sondas de DNA/química , DNA de Cadeia Simples/química , DNA Viral/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Hepatite C/virologia , Humanos , Limite de Detecção , Nanoestruturas/química
17.
Nucleic Acids Res ; 48(4): 2173-2188, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31925419

RESUMO

The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.


Assuntos
Reparo do DNA/genética , Modelos Moleculares , Proteína de Replicação A/química , Proteína de Xeroderma Pigmentoso Grupo A/química , DNA/química , DNA/genética , Dano ao DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica/genética , Proteína de Replicação A/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética
18.
Biochem Biophys Res Commun ; 524(1): 103-108, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980174

RESUMO

SP_0782 from Streptococcus pneumoniae is a dimeric PC4-like protein binding single-stranded DNA (ssDNA), and is potentially involved in maintenance of genome stability and natural transformation. SP_0782 binds with different lengths of ssDNA in various patterns through accommodating nucleotides differently in its two DNA-binding regions (DBRs). Here, we report the characterization of a novel site, leucine 20 (L20), which is not located in the DBRs but impairs the DNA binding when mutated to alanine (L20A). The L20A mutation markedly reduced the DNA-binding affinity of SP_0782 for ssDNA dT19G1, and affected the formation of high-order SP_0782:dT19G1 complexes. The side chain of L20 shows interactions with several residues at the backside of the DBRs in apo SP_0782 structure, and the L20A mutation led to a change of circular dichroism (CD) spectrum and broad chemical shift perturbations (CSPs) in NMR spectrum compared with the wild type. The most affected residues in NMR spectrum included F39 and R49 located in DBR2, as well as K60 in DBR1, which was suggested to be important for cooperative binding of ssDNA by the two subunits in SP_0782 dimer. Thus, the L20A mutation caused a local conformational change of SP_0782, which exerted an indirect effect on the DNA-binding interface and therefore impaired the affinity for ssDNA dT19G1. Interestingly, this L20 site is conserved in bacterial but not eukaryotic PC4-like proteins, suggesting an evolutionary divergence. This study provides an insight into the structure-function relationship of SP_0782, and an amino-acid site probably targeted for inhibiting bacteria selectively.


Assuntos
Proteínas de Bactérias/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Leucina/química , Proteínas Mutantes/química , Streptococcus pneumoniae/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Modelos Moleculares , Conformação Molecular , Proteínas Mutantes/genética , Mutação , Ligação Proteica
19.
Nucleic Acids Res ; 48(1): 445-459, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31724707

RESUMO

Bacterial viruses encode a vast number of ORFan genes that lack similarity to any other known proteins. Here, we present a 2.20 Å crystal structure of N4-related Pseudomonas virus LUZ7 ORFan gp14, and elucidate its function. We demonstrate that gp14, termed here as Drc (ssDNA-binding RNA Polymerase Cofactor), preferentially binds single-stranded DNA, yet contains a structural fold distinct from other ssDNA-binding proteins (SSBs). By comparison with other SSB folds and creation of truncation and amino acid substitution mutants, we provide the first evidence for the binding mechanism of this unique fold. From a biological perspective, Drc interacts with the phage-encoded RNA Polymerase complex (RNAPII), implying a functional role as an SSB required for the transition from early to middle gene transcription during phage infection. Similar to the coliphage N4 gp2 protein, Drc likely binds locally unwound middle promoters and recruits the phage RNA polymerase. However, unlike gp2, Drc does not seem to need an additional cofactor for promoter melting. A comparison among N4-related phage genera highlights the evolutionary diversity of SSB proteins in an otherwise conserved transcription regulation mechanism.


Assuntos
DNA de Cadeia Simples/química , DNA Viral/química , Proteínas de Ligação a DNA/química , Fagos de Pseudomonas/genética , Pseudomonas/virologia , Proteínas Virais/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Clonagem Molecular , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Fagos de Pseudomonas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcrição Genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
J Agric Food Chem ; 68(1): 402-408, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31809034

RESUMO

The functional ingredients of microalgal biomass are receiving substantial recognition as the global demands for health supplements produced from natural sources are on the rise. Paramylon, a conglomerate of ß-1,3-glucans, is one of the major valuable sources derived from Euglena gracilis having multiple applications, thus necessitating the development of an efficient quantification method. Here, we employed a DNA aptamer to quantify the amount of paramylon produced by E. gracilis. Paramylon-specific aptamers were isolated by the systematic evolution of ligands by exponential enrichment (SELEX) process. To evaluate the potential aptamers, the binding affinity between aptamer candidates and paramylon granules was confirmed by a confocal laser scanning microscope and the dissociation constants of the selected aptamers were determined by nonlinear regression analysis. The selected DNA aptamer was successfully used for the quantification of paramylon, and the results were compared to those obtained by the standard methods. The new approach was also used for quantification of paramylon from E. gracilis cells cultured to different cell stages and physiologies. It can be concluded that the aptamer-based protocol for the measurement of paramylon proposed in this study is highly accurate and comparatively less time-consuming.


Assuntos
Aptâmeros de Nucleotídeos/genética , DNA de Cadeia Simples/genética , Euglena gracilis/química , Glucanos/análise , Extratos Vegetais/análise , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Euglena gracilis/genética , Euglena gracilis/metabolismo , Glucanos/metabolismo , Microalgas/química , Microalgas/genética , Microalgas/metabolismo , Extratos Vegetais/metabolismo
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