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1.
J Phys Chem Lett ; 11(11): 4430-4435, 2020 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-32392072

RESUMO

The pandemic outbreak of a new coronavirus (CoV), SARS-CoV-2, has captured the world's attention, demonstrating that CoVs represent a continuous global threat. As this is a highly contagious virus, it is imperative to understand RNA-dependent-RNA-polymerase (RdRp), the key component in virus replication. Although the SARS-CoV-2 genome shares 80% sequence identity with severe acute respiratory syndrome SARS-CoV, their RdRps and nucleotidyl-transferases (NiRAN) share 98.1% and 93.2% identity, respectively. Sequence alignment of six coronaviruses demonstrated higher identity among their RdRps (60.9%-98.1%) and lower identity among their Spike proteins (27%-77%). Thus, a 3D structural model of RdRp, NiRAN, non-structural protein 7 (nsp7), and nsp8 of SARS-CoV-2 was generated by modeling starting from the SARS counterpart structures. Furthermore, we demonstrate the binding poses of three viral RdRp inhibitors (Galidesivir, Favipiravir, and Penciclovir), which were recently reported to have clinical significance for SARS-CoV-2. The network of interactions established by these drug molecules affirms their efficacy to inhibit viral RNA replication and provides an insight into their structure-based rational optimization for SARS-CoV-2 inhibition.


Assuntos
Betacoronavirus/enzimologia , Nucleotidiltransferases/química , RNA Replicase/química , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Amidas/química , Amidas/metabolismo , Antivirais/química , Antivirais/metabolismo , Betacoronavirus/isolamento & purificação , Sítios de Ligação , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Simulação de Acoplamento Molecular , Nucleotidiltransferases/metabolismo , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Pirazinas/química , Pirazinas/metabolismo , Pirrolidinas/química , Pirrolidinas/metabolismo , RNA Replicase/metabolismo
2.
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
3.
Scand J Immunol ; 91(6): e12880, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32219875

RESUMO

Synthetic Toll-like receptor (TLR) 7 agonists have been suggested as immune modulators in a range of conditions. In contrast, self-derived TLR7 activators, such as RNA-containing immune complexes (RNA-IC), can contribute to autoimmune diseases due to endogenous immune activation. The exact difference in immune cell response between synthetic and endogenous TLR7 triggers is only partly known. An understanding of these differences could aid in the development of new therapeutic agents and provide insights into autoimmune disease mechanisms. We therefore compared the stimulatory capacity of two TLR7 agonists, RNA-IC and a synthetic small molecule DSR-6434, on blood leucocytes, plasmacytoid dendritic cells (pDCs) and B cells from healthy individuals. IFN-α, IL-6, IL-8 and TNF levels were measured by immunoassays, and gene expression in pDCs was analysed by an expression array. DSR-6434 triggered 20-fold lower levels of IFN-α by pDCs, but higher production of IL-6, IL-8 and TNF, compared to RNA-IC. Furthermore, IFN-α and TNF production were increased with exogenous IFN-α2b priming, whereas IL-8 synthesis by B cells was reduced for both stimuli. Cocultivation of pDCs and B cells increased the RNA-IC-stimulated IFN-α and TNF levels, while only IL-6 production was enhanced in the DSR-6434-stimulated cocultures. When comparing pDCs stimulated with RNA-IC and DSR-6434, twelve genes were differentially expressed (log2 fold change >2, adjusted P-value <.05). In conclusion, RNA-IC, which mimics an endogenous TLR7 stimulator, and the synthetic TLR7 agonist DSR-6434 trigger distinct inflammatory profiles in immune cells. This demonstrates the importance of using relevant stimuli when targeting the TLR7 pathway for therapeutic purposes.


Assuntos
Adenina/farmacologia , Complexo Antígeno-Anticorpo/farmacologia , Linfócitos B/imunologia , Células Dendríticas/imunologia , Complexos Multiproteicos/farmacologia , RNA/farmacologia , Receptor 7 Toll-Like/metabolismo , Adenina/análogos & derivados , Adenina/química , Complexo Antígeno-Anticorpo/química , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Estrutura Molecular , Complexos Multiproteicos/química , RNA/química , Receptor 7 Toll-Like/agonistas
4.
Proc Natl Acad Sci U S A ; 117(9): 4701-4709, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32079721

RESUMO

Proteins' interactions with ancient ligands may reveal how molecular recognition emerged and evolved. We explore how proteins recognize adenine: a planar rigid fragment found in the most common and ancient ligands. We have developed a computational pipeline that extracts protein-adenine complexes from the Protein Data Bank, structurally superimposes their adenine fragments, and detects the hydrogen bonds mediating the interaction. Our analysis extends the known motifs of protein-adenine interactions in the Watson-Crick edge of adenine and shows that all of adenine's edges may contribute to molecular recognition. We further show that, on the proteins' side, binding is often mediated by specific amino acid segments ("themes") that recur across different proteins, such that different proteins use the same themes when binding the same adenine-containing ligands. We identify numerous proteins that feature these themes and are thus likely to bind adenine-containing ligands. Our analysis suggests that adenine binding has emerged multiple times in evolution.


Assuntos
Adenina/metabolismo , Evolução Molecular , Simulação de Acoplamento Molecular/métodos , Conformação Proteica , Adenina/química , Sítios de Ligação , Ligação de Hidrogênio , Ligação Proteica , Análise de Sequência de Proteína/métodos , Software
5.
Phys Chem Chem Phys ; 22(5): 2999-3007, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31957771

RESUMO

Infrared multiple photon dissociation (IRMPD) spectroscopy has been used to probe the structures of the three protonated base-pair mismatches containing 9-ethylguanine (9eG) in the gas phase. Computational chemistry has been used to determine the relative energies and compute the infrared spectra of these complexes. By comparing the IRMPD spectra with the computed spectra, in all cases, it was possible to deduce that what was observed experimentally were the lowest energy computed structures. The protonated complex between 9eG and 1-methylthymine (1mT) is protonated at N7 of 9eG-the most basic site of all three bases in this study-and bound in a Hoogsteen type structure with two hydrogen bonds. The experimental IRMPD spectrum for the protonated complex between 9eG and 9-methyladenine (9mA) is described as arising from a combination of the two lowest energy structures, both which are protonated at N1 of adenine and each containing two hydrogen bonds with 9eG being the acceptor of both. The protonated dimer of 9eG is protonated at N7 with an N7-H+-N7 ionic hydrogen bond. It also contains an interaction between a C-H of protonated guanine and the O6 carbonyl of neutral guanine which is manifested in a slight red shift of that carbonyl stretch. The protonated 9eG/9mA structures have been previously identified by X-ray crystallography in RNA and are contained within the protein database.


Assuntos
Gases/química , Guanina/análogos & derivados , Espectrofotometria Infravermelho , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Pareamento Incorreto de Bases , Cristalografia por Raios X , Guanina/química , Guanina/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Fótons , Timina/análogos & derivados , Timina/química , Timina/metabolismo
6.
J Chem Phys ; 152(3): 035101, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31968979

RESUMO

One-electron oxidation of adenine (A) leads initially to the formation of adenine radical cation (A•+). Subsequent deprotonation of A•+ can provoke deoxyribonucleic acid (DNA) damage, which further causes senescence, cancer formation, and even cell death. However, compared with considerable reports on A•+ reactions in free deoxyadenosine (dA) and duplex DNA, studies in non-B-form DNA that play critical biological roles are rare at present. It is thus of vital importance to explore non-B-form DNA, among which the triplex is an emerging topic. Herein, we investigate the deprotonation behavior of A•+ in the TAT triplex with continuous A bases by time-resolved laser flash photolysis. The rate constants for the one-oxidation of triplex 8.4 × 108 M-1 s-1 and A•+ deprotonation 1.3 × 107 s-1 are obtained. The kinetic isotope effect of A•+ deprotonation in the TAT triplex is 1.8, which is characteristic of a direct release of the proton into the solvent similar to free base dA. It is thus elucidated that the A•+ proton bound with the third strand is most likely to be released into the solvent because of the weaker Hoogsteen H-bonding interaction and the presence of the highly mobile hydration waters within the third strand. Additionally, it is confirmed through Fourier transform infrared spectroscopy that the deprotonation of A•+ results in the dissociation of the third strand and disruption of the secondary structure of the triplex. These results provide valuable kinetic data and in-depth mechanistic insights for understanding the adenine oxidative DNA damage in the triplex.


Assuntos
Adenina/química , DNA/química , Elétrons , Timina/química , Ligação de Hidrogênio , Oxirredução
7.
J Chromatogr A ; 1612: 460627, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-31668867

RESUMO

This work presents the investigation of utilizing adenine-functionalized polypropylene glycol (APPG) for capillary gas chromatographic separations. The statically coated APPG column (0.25 mm i.d.) showed moderate polarity and high column efficiencies of 4660 plates/m and 4376 plates/m determined by n-octanol and naphthalene, respectively. Remarkably, the APPG column baseline resolved all the components of the Grob test mixture and displayed good peak shapes for some stringent analytes that are prone to peak tailing or severe adsorption. Also, it achieved complete separation of dimethylaniline isomers, which are difficult to be resolved due to their high resemblance in structures and properties. The above results demonstrate the high selectivity and inertness of the APPG column and its distinct advantages over the polypropylene glycol (PPG) column and commercial polyethylene glycol (PEG) column. In addition, its separation performance has good repeatability with the RSD values on retention times below 0.05% for run-to-run, 0.11-0.12% for day-to-day and 1.7-1.9% for column-to-column, respectively. Further, the APPG column was applied to determination of isomer impurities in commercial dimethylaniline products and to determination of the additives of anilines and phenols in a hair-dye product, proving its great potential for practical GC analysis.


Assuntos
Adenina/química , Cromatografia Gasosa/métodos , Polímeros/química , Propilenoglicóis/química , Ácidos , Compostos de Anilina/análise , Compostos de Anilina/química , Isomerismo , Fenóis/análise
8.
Biosens Bioelectron ; 147: 111735, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31634803

RESUMO

Design of suitable nanocomposites with tailored structures was significant in the fabrication of effective and reliable electrochemical sensors. Herein, the copper-nickel@nitrogen, boron-doped reduced graphene oxide (Cu-Ni@N,B-rGO) was successfully synthesized, which exhibited superior electrocatalytic performance towards guanine (G) and adenine (A) oxidation. The Cu-Ni NPs were sequentially decorated on N,B-rGO substrate via an environmentally friendly reduction strategy, which utilized glucose as reducer and stabilizing agent. The nanocomposites with large specific surface area, remarkable conductivity and high catalytic activity showed prominent synergistic effect owning to the uniform dispersion of Cu-Ni NPs on the surface of N,B-rGO. When applied to analysis of G and A using DPV, the wide linear ranges of 1.0-160.0 µM and 1.0-120.0 µM with the determination limits of 0.118 µM and 0.134 µM were obtained, respectively. The sensor was successfully applied to the detection of G and A in calf-thymus DNA with G/A ratio of 0.80. The facile preparation process and attractive sensing properties of the Cu-Ni@N,B-rGO nanocomposites made it a promising candidate for the development of advanced electrochemical sensor.


Assuntos
Adenina/isolamento & purificação , Técnicas Biossensoriais , Técnicas Eletroquímicas , Guanina/isolamento & purificação , Adenina/química , Animais , Catálise , Bovinos , Cobre/química , DNA/química , DNA/genética , Glucose/química , Grafite/química , Guanina/química , Limite de Detecção , Nanosferas/química , Níquel/química , Oxirredução
9.
Phys Chem Chem Phys ; 22(2): 838-853, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31840715

RESUMO

The five fundamental units of the genetic code: uracil (U), thymine (T), cytosine (C), adenine (A) and guanine (G) are known for extremely low vapor pressure and low thermal stability at elevated temperatures. Therefore, application of conventional techniques for the determination of sublimation enthalpies and vapor pressures fails to provide accurate results. Recently, a Fast Scanning Calorimetry method (FSC) for vapor pressure determination was developed for investigation of extremely low volatile, as well as for thermally unstable molecular and ionic molecules. This success has encouraged application of the FSC method for determination of vapor pressures and sublimation enthalpies of the five nucleobases, where available literature data are in disarray. The thermodynamic data of the nucleobases available in the literature were collected, evaluated, and combined with our experimental results to reconcile available experimental data. The set of evaluated thermochemical data on the five nucleobases was recommended as the benchmark properties for these thermally labile compounds.


Assuntos
Adenina/química , Calorimetria , Citosina/química , Guanina/química , Termodinâmica , Timina/química , Uracila/química , Pressão , Volatilização
10.
Acta Biochim Pol ; 66(4): 585-588, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804790

RESUMO

Fluorescent tri-cyclic purine analogs, derivatives of isoguanine and adenine, were examined as potential substrates of purine-nucleoside phosphorylase. It was found previously that etheno- derivatives of both compounds are ribosylated in phosphate-free media, but ribosylation places in some instances differ from purine N9. New ribosides are examined as potential substrates of human blood PNP and indicators of this enzyme. Of these, N6-riboside of 1,N6-etheno-adenine was found the most promising.


Assuntos
Adenina/química , Glicosídeos/química , Guanina/química , Purina-Núcleosídeo Fosforilase/sangue , Adenina/farmacologia , Fluorescência , Guanina/farmacologia , Humanos , Cinética , Purina-Núcleosídeo Fosforilase/isolamento & purificação , Purinas/química
11.
Org Biomol Chem ; 17(46): 9913-9923, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31720670

RESUMO

Nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) inhibitors have been suggested as a potential treatment for calcium pyrophosphate dihydrate (CPPD) deposition disease. Here, we targeted the development of improved NPP1 inhibitors based on acyclic mimics of Pα,α-phosphorodithioate-substituted adenine nucleotides, 7-10. The latter were obtained in a facile two-step synthesis from adenine-(methoxy)ethanol. Among analogs 7-10, adenine-(methoxy)ethoxy-Pα,α-dithio-triphosphate, 8, was the most potent NPP1 inhibitor both with purified enzyme (IC50 0.645 µM) and in osteoarthritic human chondrocytes (IC50 0.033 µM). Furthermore, it efficaciously (10-fold vs. control) inhibited ATP-induced CPPD in human articular chondrocytes. Importantly, 8 was a highly selective NPP1 inhibitor which showed only minor inhibition of NPP3, CD39 and CD73, and did not inhibit TNAP (tissue nonspecific alkaline phosphatase) activity in human chondrocytes. Furthermore, 8 did not activate P2Y1,2,6 receptors. Analog 8 was not toxic to cultured chondrocytes at 100 µM. Therefore, 8 may be suitable for further development as a drug candidate for the treatment of CPPD arthritis and other NPP1-related diseases.


Assuntos
Adenina/farmacologia , Pirofosfato de Cálcio/antagonistas & inibidores , Condrócitos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Osteoartrite do Joelho/tratamento farmacológico , Polifosfatos/farmacologia , Pirofosfatases/antagonistas & inibidores , Compostos de Sulfidrila/farmacologia , Adenina/síntese química , Adenina/química , Pirofosfato de Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Diester Fosfórico Hidrolases/metabolismo , Polifosfatos/química , Pirofosfatases/metabolismo , Relação Estrutura-Atividade , Compostos de Sulfidrila/química
12.
Drug Deliv ; 26(1): 1125-1139, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31736389

RESUMO

Stability in systemic circulation, effective tumor accumulation, and the subsequent crucial subcellular targeting are significant elements that maximize the therapeutic efficacy of a drug. Accordingly, novel nanoparticles based on polysaccharides that simultaneously presented prolonged systemic circulation and mitochondrial-targeted drug release were synthesized. First, the mitochondrial-targeted polymer, 3,4-dihydroxyphenyl propionic acid-chitosan oligosaccharide-dithiodipropionic acid-berberine (DHPA-CDB), was synthesized, which was used to form self-assembled curcumin (Cur)-encapsulated cationic micelles (DHPA-CDB/Cur). Negatively charged oligomeric hyaluronic acid-3-carboxyphenylboronic acid (oHA-PBA), a ligand to sialic acid and CD44, was further added to the surface of the preformed DHPA-CDB/Cur core to shield the positive charges and to prolong blood persistence. oHA-PBA@DHPA-CDB/Cur formed a covalent polyplex of oHA-PBA and DHPA-CDB/Cur via the pH-responsive borate ester bond between PBA and DHPA. The mildly acidic tumor environment led to the degradation of borate ester bonds, thereby realizing the exposure of the cationic micelles and causing a charge reversal from -19.47 to +12.01 mV, to promote cell internalization and mitochondrial localization. Compared with micelles without the oHA-PBA modification, the prepared oHA-PBA@DHPA-CDB/Cur showed enhanced cytotoxicity to PANC-1 cells and greater cellular uptake via receptor-mediated endocytosis. oHA-PBA@DHPA-CDB/Cur was effectively targeted to the mitochondria, which triggered mitochondrial membrane depolarization. In mice xenografted with PANC-1 cells, compared with control mice, oHA-PBA@DHPA-CDB/Cur resulted in more effective tumor suppression and greater biosafety with preferential accumulation in the tumor tissue. Thus, the long-circulating oHA-PBA@DHPA-CDB/Cur, with mitochondrial targeting and tumor environment charge-reversal capabilities, was shown to be an excellent candidate for subcellular-specific drug delivery.


Assuntos
Antineoplásicos/química , Preparações de Ação Retardada/química , Mitocôndrias/efeitos dos fármacos , Nanopartículas/química , Polissacarídeos/química , Adenina/análogos & derivados , Adenina/química , Animais , Antineoplásicos/farmacologia , Berberina/química , Linhagem Celular , Quitosana/química , Curcumina/química , Preparações de Ação Retardada/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Camundongos , Camundongos Nus , Micelas , Polímeros/química , Polissacarídeos/farmacologia
13.
J Biomol NMR ; 73(8-9): 509-518, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31606878

RESUMO

Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s-1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s-1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s-1. The µs-ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.


Assuntos
Adenina/química , Aptâmeros de Nucleotídeos/química , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Riboswitch , Cristalografia por Raios X , Modelos Moleculares
14.
Nat Commun ; 10(1): 4413, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562325

RESUMO

The synthesis of nucleobases in natural environments, especially in interstellar molecular clouds, is the focus of a long-standing debate regarding prebiotic chemical evolution. Here we report the simultaneous detection of all three pyrimidine (cytosine, uracil and thymine) and three purine nucleobases (adenine, xanthine and hypoxanthine) in interstellar ice analogues composed of simple molecules including H2O, CO, NH3 and CH3OH after exposure to ultraviolet photons followed by thermal processes, that is, in conditions that simulate the chemical processes accompanying star formation from molecular clouds. Photolysis of primitive gas molecules at 10 K might be one of the key steps in the production of nucleobases. The present results strongly suggest that the evolution from molecular clouds to stars and planets provides a suitable environment for nucleobase synthesis in space.


Assuntos
Adenina/química , Citosina/química , Hipoxantina/química , Timina/química , Uracila/química , Xantina/química , Adenina/síntese química , Amônia/química , Monóxido de Carbono/química , Citosina/síntese química , Evolução Química , Meio Ambiente Extraterreno , Hipoxantina/síntese química , Gelo , Metanol/química , Estrutura Molecular , Processos Fotoquímicos/efeitos da radiação , Timina/síntese química , Raios Ultravioleta , Uracila/síntese química , Água/química , Xantina/síntese química
15.
Arch Biochem Biophys ; 675: 108120, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31563510

RESUMO

Dpo4 is a representative model of Y-family DNA polymerase and is therefore one of the most intensively studied DNA polymerase. 6 mA, an epigenetic marker, plays important roles in regulation of various biological processes. However, its effects on DNA replication by Dpo4 is completely unknown. Here, we found that 6 mA and its intermediate Hyp inhibits primer extension by Dpo4, showing an obvious blockage just one nucleotide before 6 mA or Hyp. 6 mA reduces dTTP incorporation efficiency, next-base extension efficiency, binding affinity of DNA to Dpo4, binding affinity of dTTP to Dpo4-DNA complex, the fraction of productive Dpo4 or productive ternary complex, and the burst incorporation rate, explaining the inhibition effects of 6 mA on DNA replication by Dpo4. Hyp is similar to G and dCTP is preferentially incorporated opposite Hyp by Dpo4, resulting in A:T to G:C mutation. Relative to dTTP incorporation opposite unmodified A, Hyp reduces dCTP incorporation efficiency, next-base extension efficiency, the priority in extension beyond correct pair, binding affinity of Dpo4 to DNA, binding of dCTP to Dpo4-DNA complex, and the burst incorporation efficiency, explaining the inhibition effects of Hyp on DNA replication by Dpo4. This work provides insight in the effects of epigenetically modified 6 mA and Hyp on DNA replication by a representative Y-family DNA polymerase Dpo4.


Assuntos
Adenina/análogos & derivados , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , Epigênese Genética , Sulfolobus solfataricus/enzimologia , Adenina/química
16.
Chemistry ; 25(67): 15288-15294, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31483908

RESUMO

Nanoscale assemblies of DNA strands are readily designed and can be generated in a wide range of shapes and sizes. Turning them into solids that bind biomolecules reversibly, so that they can act as active material in flow cells, is a challenge. Among the biomolecular ligands, cofactors are of particular interest because they are often the most expensive reagents of biochemical transformations, for which controlled release and recycling are desirable. We have recently described DNA triplex motifs that bind adenine-containing cofactors, such as NAD, FAD and ATP, reversibly with low micromolar affinity. We sought ways to convert the soluble DNA motifs into a macroporous solid for cofactor binding. While assemblies of linear and branched DNA motifs produced hydrogels with undesirable properties, long DNA triplexes treated with protamine gave materials suitable for flow cells. Using exchangeable cells in a flow system, thermally controlled loading and discharge were demonstrated. Employing a flow cell loaded with ATP, bioluminescence was induced through thermal release of the cofactor. The results show that materials generated from functional DNA structures can be successfully employed in macroscopic devices.


Assuntos
Adenina/química , DNA/química , Nanopartículas/química , Trifosfato de Adenosina/química , Sítios de Ligação , Flavina-Adenina Dinucleotídeo/química , Ligantes , NAD/química , Motivos de Nucleotídeos , Espectrometria de Fluorescência/métodos , Termodinâmica
17.
Nucleic Acids Res ; 47(16): 8362-8374, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31392985

RESUMO

Many nucleic acid aptamers that bind to target molecules have been reported as antibody alternatives. However, while the affinities of aptamers vary widely, little is known about the relationship between the affinities and their applicabilities for practical use. Here, we developed molecular affinity rulers: a series of DNA aptamers with different affinities that bind to the same area of target molecules, to measure the aptamer and its device applicabilities. For the ruler preparation, we used high-affinity DNA aptamers containing a hydrophobic unnatural base (Ds) as the fifth base. By replacing Ds bases with A bases in Ds-DNA aptamers targeting VEGF165 and interferon-γ, we prepared two sets of DNA aptamers with dissociation constants (KD) ranging from 10-12 to 10-8 M. Using these molecular affinity rulers, we evaluated the sensitivity of DNA aptamers in ELISA (enzyme-linked immunosorbent assay), which showed the clear relationship between aptamer affinities and their detection sensitivities. In sandwich-type ELISA using combinations of aptamers and antibodies, aptamers with KD values lower than ∼10-9 M were required for sufficient sensitivities (limit of detection (LOD) < 10 pM) and signal intensities, but optimizations improved the lower-affinity aptamers' applicabilities. These aptamer affinity rulers could be useful for evaluating and improving aptamer applicabilities.


Assuntos
Adenina/química , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Ensaio de Imunoadsorção Enzimática/métodos , Interferon gama/análise , Fator A de Crescimento do Endotélio Vascular/análise , Animais , Anticorpos Monoclonais/química , Aptâmeros de Nucleotídeos/síntese química , Pareamento de Bases , Sequência de Bases , Biotina/química , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Interferon gama/química , Cinética , Limite de Detecção , Conformação de Ácido Nucleico , Ligação Proteica , Padrões de Referência , Técnica de Seleção de Aptâmeros , Estreptavidina/química , Fator A de Crescimento do Endotélio Vascular/química
18.
Biosens Bioelectron ; 143: 111596, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31442754

RESUMO

Plasmonic-dielectic hybrid substrates of Ag-islands on three-dimensional photonic crystals are fabricated through magnetron sputtering of silver onto hydrophobized silica photonic crystals, free from etching process. Without typical "hot-spots" such as nanogaps, significant Raman enhancements can be achieved, attributed to the enhanced electromagnetic field and scattering of the plasmonic nanoparticles as well as the enhanced light-matter interaction by the slow photon effects. The detection limit for adenine by the hybrid substrates reaches nM level, with a calculated enhancement factor of 1.13 × 107, which is three orders of magnitude higher than the conventional noble metal film over nanosphere (FON) control group. Furthermore, microchips based on the hybrid substrates are facilely achieved, enabling micro-detection through super hydrophobic concentration. The facile fabrication and effective Raman enhancements make the Ag-islands on 3D photonic crystals promising candidates in the field of chemical sensors, Raman mapping and bioassays.


Assuntos
Adenina/isolamento & purificação , Técnicas Biossensoriais , Nanopartículas Metálicas/química , Óptica e Fotônica , Adenina/química , Campos Eletromagnéticos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Nanosferas/química , Nanoestruturas/química , Dióxido de Silício/química , Análise Espectral Raman , Propriedades de Superfície
19.
Adv Mater ; 31(37): e1903443, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31379091

RESUMO

The targeted and sustained drug release from stimuli-responsive nanodelivery systems is limited by the irreversible and uncontrolled disruption of the currently used nanostructures. Bionic nanocapsules are designed by cross-linking polythymine and photoisomerized polyazobenzene (PETAzo) with adenine-modified ZnS (ZnS-A) nanoparticles (NPs) via nucleobase pairing. The ZnS-A NPs convert X-rays into UV radiation that isomerizes the azobenzene groups, which allows controlled diffusion of the active payloads across the bilayer membranes. In addition, the nucleobase pairing interactions between PETAzo and ZnS-A prevent drug leakage during their in vivo circulation, which not only enhances tumor accumulation but also maintains stability. These nanocapsules with tunable permeability show prolonged retention, remotely controlled drug release, enhanced targeted accumulation, and effective antitumor effects, indicating their potential as an anticancer drug delivery system.


Assuntos
Pareamento de Bases , Materiais Biomiméticos/química , Portadores de Fármacos/química , Nanocápsulas/química , Adenina/química , Adsorção , Animais , Compostos Azo/química , Linhagem Celular Tumoral , Humanos , Isomerismo , Camundongos , Permeabilidade , Processos Fotoquímicos , Soroalbumina Bovina/química , Sulfetos/química , Timina/química , Raios X , Compostos de Zinco/química
20.
Nucleic Acids Res ; 47(14): 7223-7234, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31276590

RESUMO

The catalytic strategies of small self-cleaving ribozymes often involve interactions between nucleobases and the ribonucleic acid (RNA) backbone. Here we show that multiply protonated, gaseous RNA has an intrinsic preference for the formation of ionic hydrogen bonds between adenine protonated at N3 and the phosphodiester backbone moiety on its 5'-side that facilitates preferential phosphodiester backbone bond cleavage upon vibrational excitation by low-energy collisionally activated dissociation. Removal of the basic N3 site by deaza-modification of adenine was found to abrogate preferential phosphodiester backbone bond cleavage. No such effects were observed for N1 or N7 of adenine. Importantly, we found that the pH of the solution used for generation of the multiply protonated, gaseous RNA ions by electrospray ionization affects phosphodiester backbone bond cleavage next to adenine, which implies that the protonation patterns in solution are at least in part preserved during and after transfer into the gas phase. Our study suggests that interactions between protonated adenine and phosphodiester moieties of RNA may play a more important mechanistic role in biological processes than considered until now.


Assuntos
Adenina/química , Prótons , Clivagem do RNA , RNA/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Modelos Químicos , Estrutura Molecular , Conformação de Ácido Nucleico
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