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1.
Nucleic Acids Res ; 47(19): 10296-10312, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31495891

RESUMO

Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA/química , Transcrição Genética/efeitos dos fármacos , Uridina/análogos & derivados , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Oxigênio/química , Pseudomonas/química , RNA/genética , Clivagem do RNA/efeitos dos fármacos , Streptomyces/química , Especificidade por Substrato , Timidina/química , Timidina/genética , Transcrição Genética/genética , Fatores de Elongação da Transcrição/genética , Uracila/química , Uridina/síntese química , Uridina/química , Uridina/farmacologia
2.
Nucleosides Nucleotides Nucleic Acids ; 38(12): 980-1005, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31380708

RESUMO

Two series of novel fluorinated nucleosides dimers with an unnatural 1,2,3-triazole linkage were synthesized. The obtained molecules were prepared using "click" chemistry approach based on copper(I) catalyzed Huisgen azide-alkyne cycloaddition. It was performed between 3'- and 5'-azido-nucleosides as the azide components, and the 3'-O- and 5'-O-propargyl-nucleosides as the alkyne components. Based on analysis of the 3 JHH, 3 JH1'C2 and 3 JH1'C6 we estimated conformational preferences of sugar part and orientation around glycosidic bond. All described nucleosides dimers analogs were characterized by spectroscopic methods and evaluated for their in vitro cytotoxicity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7).


Assuntos
Antineoplásicos/síntese química , Floxuridina/química , Nucleosídeos/síntese química , Timidina/química , Triazóis/química , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Química Click/métodos , Reação de Cicloadição/métodos , Dimerização , Células HeLa , Humanos , Células KB , Células MCF-7 , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Nucleosídeos/farmacologia
3.
Chem Pharm Bull (Tokyo) ; 67(7): 707-712, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257326

RESUMO

Hypobromous acid (HOBr) is generated not only by eosinophils but also by neutrophils in the presence of Br- at the plasma concentration. Reactivity of HOBr is greatly modulated by coexistent compounds such as amines and amides. In this study, we investigated effects of urea in the reaction of nucleosides with HOBr. When nucleosides were incubated with HOBr without urea in potassium phosphate buffer at pH 7.4 and 37°C, the reactions almost completed within 10 min, with consumptions in the order of 2'-deoxyguanosine > 2'-deoxycytidine > 2'-deoxythymidine > 2'-deoxyadenosine, generating 8-bromo-2'-deoxyguanosine and 5-bromo-2'-deoxycytidine. In the presence of urea, the reaction of nucleosides with HOBr was relatively slow, continuing over several hours. When HOBr was preincubated without urea in potassium phosphate buffer at pH 7.4 and 37°C for 48 h, the preincubated HOBr solution did not react with nucleosides. However, a similar preincubated solution of HOBr with urea reacted with nucleosides to generate 8-bromo-2'-deoxyguanosine and 5-bromo-2'-deoxycytidine. These results imply that a reactive bromine compound with a long life, probably bromourea, is generated by HOBr in neutral urea solution and reacts with nucleosides, resulting in brominated nucleosides.


Assuntos
Bromatos/química , Nucleosídeos/química , Ureia/química , Cromatografia Líquida de Alta Pressão , Desoxicitidina/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Desoxiguanosina/química , Halogenação , Fosfatos/química , Compostos de Potássio/química , Espectrometria de Massas por Ionização por Electrospray , Timidina/química
4.
Spectrochim Acta A Mol Biomol Spectrosc ; 222: 117165, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31185440

RESUMO

A novel, simple and low-cost nitrogen doped carbon dots (N-CDs) fluorescent sensor for sensitive detection of Cr (VI) was developed via one-step hydrothermal method using thymidine as carbon source. As-prepared N-CDs exhibited the ability of sensitive and selective detection of Cr (VI) through the inner filter effect (IFE). The performances of N-CDs were investigated with the characterization methods of transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). Under the optimized conditions, a good logarithm correlation between the fluorescence intensity of N-CDS and the concentration of Cr (VI) was obtained ranging from 0.1 µM to 430 µM (R2 = 0.992), with a low detection limit (LOD; S/N = 3) of 1.26 nM. The fluorescent sensor showed good repeatability, reproducibility and stability. Furthermore, N-CDs fluorescent sensor had a good applicability for Cr (VI) detection in real water samples with acceptable recoveries, and the detection results were consistent with the inductively coupled plasma mass spectrometry (ICP-MS) results, indicating this fluorescent sensor has a great potential for the environmental monitoring.


Assuntos
Carbono/química , Cromo/análise , Corantes Fluorescentes/química , Nitrogênio/química , Timidina/química , Poluentes Químicos da Água/análise , Limite de Detecção , Pontos Quânticos/química , Pontos Quânticos/ultraestrutura , Reprodutibilidade dos Testes , Espectrometria de Fluorescência/métodos , Água/análise
5.
Eur J Med Chem ; 171: 255-264, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30925340

RESUMO

Anticancer anthracyclines are cytotoxic drugs that can induce antitumor immune response as a secondary effect through immunogenic cell death (ICD) mechanism. However, the immunogenic potency is quite limited, possibly due to that these chemotherapeutic agents are not specifically developed as ICD inducers. Thus, new drug entities through studies focusing on enhanced ICD induction would significantly promote antitumor immune response in the vaccination application. We report here a naphthyl quinoxaline thymidine conjugate as a new class of cytotoxic compounds that effectively induced in vivo antitumor activity through the vaccination application. Synthesized naphthyl quinoxaline conjugates were weak fluorescent thymidine analog yet exhibited a pronounced anticancer activity in the low nanomolar range post UVA activation. The potent activity of naphthyl conjugate was able to induce the marked detection of ICD markers including ATP and HMGB1 extracellular and calreticulin intracellularly at 2 h post UVA activation. Most importantly, mice vaccinated with cells treated with naphthyl conjugate plus UVA exhibited complete tumor growth inhibition in the tumor challenge study, and the induced immunogenic inhibition was much more effective than that of mitoxantrone anthracycline drug. All these results demonstrate the high potential of naphthyl quinoxaline conjugate for the cancer cell vaccine against tumor.


Assuntos
Antineoplásicos/farmacologia , Quinoxalinas/farmacologia , Timidina/farmacologia , Raios Ultravioleta , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Quinoxalinas/química , Relação Estrutura-Atividade , Timidina/química , Vacinação
6.
Mikrochim Acta ; 186(4): 216, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30838468

RESUMO

A highly sensitive and selective fluorometric method is described for determination of mercury(II). It is based on (a) the use of graphene oxide (GO) acting as a quencher of the fluoresence of the carboxy-fluorescein (FAM), and (b) of Hg(II)-triggered cleavage of the newly formed nucleic acid sequences harbored blunt 3'-hydroxyl termini by exonuclease III (Exo III) that leads to signal amplification. Two DNA probes are used, viz. a capture probe (CP) and a help probe; HP) that is partially complementary. In the absence of Hg(II), the FAM-labeled hairpin (signal probe, SP) is adsorbed onto the surface of GO via π-stacking interactions. CP blocks the release of the HP for binding to SP. This results in quenching of the green fluorescence of the label. Upon addition of Hg(II), the linear structure of CP is converted to a hairpin structure due to the formation of thymidine-Hg(II)-thymidine duplexes. HP is released from the CP/HP hybrids, and this causes SP to be released from from GO and fluorescence to be recovered. The signal is strongly amplified by using Exo III-assisted targeting and recycling of HP. Hence, Hg(II) can be detected via the strong increase in fluorescence. The method has a linear response in the 0.1 to 30 nM Hg(II) concentration range and a 10 pM detection limit. It was applied to the determination of Hg(II) in three (spiked) Chinese medicines. Graphical abstract Schematic representation of fluorescence sensing strategy for Hg2+ by using graphene oxide as a quencher and exonuclease III-assisted signal amplification.


Assuntos
Exodesoxirribonucleases/química , Grafite/química , Mercúrio/análise , Timidina/química , Técnicas Biossensoriais/métodos , Medicamentos de Ervas Chinesas/análise , Corantes Fluorescentes/química , Fluorometria/métodos , Limite de Detecção , Oxirredução , Sensibilidade e Especificidade
7.
Org Biomol Chem ; 17(9): 2403-2412, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30735210

RESUMO

TBA is a 15-mer DNA aptamer for human α-thrombin, and its three T-rich loops are involved in the binding interactions with thrombin differently. In order to clarify their specific spatial locations in the binding interactions and search for more favourable positions, here a systematic investigation of all the loop residues was conducted with 3'-inverted thymidine (iT), by which both unnatural 3'-3'- and 5'-5'-linkages for each incorporation were introduced in the tertiary structure. The changes in Tm values and CD spectra revealed that motifs T3T12 and T4T13 are structurally distinct. Longer anti-clotting time was obtained for the T3 and T12 modifications, respectively, while T4 and T13 were completely intolerant with such changes, in terms of stability and binding to thrombin. In particular, the increased affinity bindings and longer anti-clotting time were obtained with the replacement on the central loop T7G8T9, which were closely related to the existence of a monovalent ion, K+ or Na+, consistently with the supposed binding site of these ions in TBA. It is worthwhile to note that both the subtle variations of the loop residues induced by iT and the monovalent ions determined the interacting residues of TBA and the binding strength rather than the thermal stability of the TBA structure.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Trombina/metabolismo , Timidina/metabolismo , Anticoagulantes/química , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Sequência de Bases , Sítios de Ligação , Dicroísmo Circular , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Temperatura Ambiente , Timidina/química , Timidina/farmacologia
8.
Chem Pharm Bull (Tokyo) ; 67(2): 130-134, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30713273

RESUMO

When a neutral solution of a nucleoside mixture was irradiated with UV light having wavelength longer than 300 nm, addition of salicylic acid to the solution greatly accelerated the reaction of thymidine. The UV light irradiation of thymidine solution in the presence of salicylic acid resulted in four major product peaks in HPLC. All the products were identified as isomers of cyclobutane thymidine dimers by MS and NMR. The cyclobutane thymidine dimers were generated from thymidine almost exclusively. UV irradiation with the longer wavelength of 350 nm induced almost no reaction. The results indicate that salicylic acid is a photosensitizer for thymidine dimerization excited by UV light of wavelength 300 to 350 nm.


Assuntos
Fármacos Fotossensibilizantes/química , Ácido Salicílico/química , Timidina/química , Cromatografia Líquida de Alta Pressão , Processos Fotoquímicos , Multimerização Proteica/efeitos da radiação , Espectrometria de Massas em Tandem , Raios Ultravioleta
9.
Phys Chem Chem Phys ; 21(4): 2006-2016, 2019 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-30633272

RESUMO

Thio-substituted nucleobases have received long-standing interest from experimental and theoretical scientists due to their potential applications in photodynamic therapy and crosslinking studies. Though the thymidine:4-thiothymidine dimer is an important structure in the DNA duplex, the molecular-level photoreaction mechanisms are still obscure. Herein, high-level QM/MM methods were adopted to investigate the photoinduced cycloaddition and (6-4) reactions of the thymidine:4-thiothymidine dimer in the DNA duplex, namely, d(ACCT(4ST)CGC:TGGAAGCG). Based on the calculated results, we identified five efficient nonadiabatic decay pathways to populate the T1 state from the initially occupied S2 state of Tp4ST via two crucial intersection structures, i.e., S2/S1 and S2/T2/S1/T1. Such photophysical processes are mainly localized on the 4-thiothymidine chromophore. After hopping to the T1 state, the light-induced [2+2] cycloaddition reaction could take place via a stepwise and nonadiabatic reaction pathway, which starts from Tp4ST via T1cc or T1cs intermediates in the T1 state and ends up with S5-thietane in the S0 state. By contrast, the concerted and thermal cycloaddition pathway in the ground state has a remarkable energy barrier, which is mechanistically less important. The subsequent generation of S5-(6-4) from S5-thietane is a concerted process in the S0 state with the simultaneous fission of the C4-S8 bond and the formation of the S8-H9 bond. In the end, we believe our present work will provide important mechanistic insights into photo-isomerization of thio-substituted nucleobases in DNA duplexes.


Assuntos
DNA/química , Timidina/análogos & derivados , Reação de Cicloadição , Dimerização , Modelos Químicos , Conformação de Ácido Nucleico , Timidina/química
10.
Anal Chim Acta ; 1049: 115-122, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30612642

RESUMO

Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.


Assuntos
Enzimas Imobilizadas/química , Núcleosídeo-Difosfato Quinase/química , Núcleosídeo-Fosfato Quinase/química , Timidina Quinase/química , Timidina/química , Análise de Injeção de Fluxo/métodos , Humanos , Cinética , Espectrometria de Massas/métodos , Nanopartículas/química , Fosforilação
11.
Nat Biotechnol ; 37(2): 169-178, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30607034

RESUMO

Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA-protein interactions. We also characterize dynamic changes in RNA-protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism.


Assuntos
RNA Mensageiro/química , Proteínas de Ligação a RNA/isolamento & purificação , RNA/isolamento & purificação , Linhagem Celular Tumoral , Análise por Conglomerados , Reagentes para Ligações Cruzadas/química , Escherichia coli , Glicoproteínas/química , Células HEK293 , Humanos , Nocodazol/química , Ligação Proteica , Proteoma , Proteômica , RNA/química , RNA Bacteriano/química , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/química , Análise de Sequência de RNA , Timidina/química , Transcriptoma
12.
Artigo em Inglês | MEDLINE | ID: mdl-30588866

RESUMO

We describe a simple method for the synthesis of modified dinucleosides containing pyrimidine nucleoside analogues (2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine). Six different dimers with a 1,2,3-triazole linkage were obtained by azide-alkyne 1,3-dipolar cycloaddition (click reaction), starting from propargylated 2'-deoxyuridine and 5'-azido-nucleoside derivatives. Their cytotoxic activity was tested in five human cancer cell lines: cervical (HeLa), high grade gliomas (U-118 MG, U-87 MG, T98G), liver (HepG2), and normal human fibroblast cell line (MRC-5) using the sulforhodamine B (SRB) assay. The experiment showed that the obtained dimers with a 1,2,3-triazole moiety were very stable compounds, also in the physiological-like media, and had no anticancer activity.


Assuntos
Antineoplásicos/síntese química , Desoxiuridina/síntese química , Nucleosídeos/síntese química , Triazóis/química , Alquinos/química , Antineoplásicos/farmacologia , Azidas/química , Bioensaio/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Química Click/métodos , Reação de Cicloadição/métodos , Desoxiuridina/farmacologia , Dimerização , Descoberta de Drogas , Humanos , Nucleosídeos/farmacologia , Timidina/química
13.
Anal Chem ; 90(24): 14111-14115, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30500177

RESUMO

A wide spectrum of DNA lesions can be generated from byproducts of endogenous metabolism and/or from environmental exposure. A DNA adductomic approach for the robust quantification of DNA adducts in cellular and tissue DNA may facilitate the use of DNA adducts for biomonitoring studies and enable comprehensive assessment about DNA repair. Normalized retention time (iRT) has been widely used in scheduled selected-reaction monitoring (SRM) methods for highly sensitive and high-throughput analyses of protein samples in complicated matrices. By using a similar method, we established the iRT scores for 36 modified nucleosides from the retention times of the four canonical 2'-deoxynucleosides on a nanoflow liquid chromatography-nanospray ionization-tandem mass spectrometry (nLC-NSI-MS/MS) system. The iRT scores facilitated reliable prediction of retention time and were employed for establishing a scheduled SRM method for quantitative assessment of a subset of the DNA adductome. The quantification results of the scheduled SRM method were more accurate and precise than those from an unscheduled method.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Adutos de DNA/análise , Espectrometria de Massas em Tandem , Desoxiadenosinas/química , Desoxicitidina/química , Desoxiguanosina/química , Interações Hidrofóbicas e Hidrofílicas , Nanotecnologia , Estereoisomerismo , Timidina/química
14.
Org Biomol Chem ; 16(40): 7488-7497, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30272759

RESUMO

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was reported to participate in the development of a variety of tumors. BC15 is a DNA aptamer targeting hnRNP A1. Firstly, through sequence truncation, we identified 31-mer sequence BC15-31 as the core sequence of BC15 with a strong binding affinity and high selectivity to the hnRNP A1 protein. Isothymidine (isoT) modification was then applied for the structural optimization of BC15-31, systematic modification and biological evaluation were carried out. Incorporation of isoT in the 1,3 sites at the 5'-end of BC15-31 can significantly enhance the protein affinity. Chemical modifications close to the 3'-end can greatly improve the stability of the aptamer. Furthermore, BC15-31 modified with isoT at both the 5'-end and 3'-end displayed an additive effect with enhanced bioactivity and stability at the same time. Our study strategy on BC15 provides a useful guideline for chemical modification and optimization of the aptamer for further clinical application.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Timidina/química , Células A549 , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Células Hep G2 , Humanos , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
15.
Chemistry ; 24(71): 18890-18896, 2018 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-30338582

RESUMO

Luminescent materials are of great interest in many fields, such as fluorescent sensing and medical imaging. Here, the construction of lanthanide-based luminescent ultralong microfibers through supramolecular self-assembly (SSA) is reported. Nucleosides (thymidine in particular), the building blocks of nucleic acids, were used as new ligands to mediate the formation of luminescent microfibers in water. The length of microfibers from thymidine-lanthanide ion (Eu and Tb) SSA was on the centimeter scale. Notably, the microfibers exhibited strong luminescence because the lanthanide ions had been chelated, sensitized and effectively protected by thymidine molecules in water. Only when the stoichiometric ratio of lanthanide ion to thymidine was 1:3 and the pH of the solution was 7, are luminescent microfibers formed. Other nucleosides, such as adenosine, cytidine, and guanosine, could not form microfibers with the lanthanide ions. This work opens a new avenue for constructing nucleoside-lanthanide SSA architectures, which hold great potential in biological and optical related applications.


Assuntos
Európio/química , Substâncias Luminescentes/química , Térbio/química , Timidina/química , Íons/química , Luminescência , Modelos Moleculares , Água/química
16.
Biochimie ; 154: 164-175, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30171884

RESUMO

Photoaffinity labeling (PAL) in combination with recent developments in mass spectrometry is a powerful tool for studying nucleic acid-protein interactions, enabling crosslinking of both partners through covalent bond formation. Such a strategy requires a preliminary study of the most judicious photoreactive group to crosslink efficiently with the target protein. In this study, we report a survey of three different photoreactive nucleobases (including a guanine functionalized with a benzophenone or a diazirine and the zero-length agent 4-thiothymine) incorporated in 30-mer oligonucleotides (ODN) containing a biotin moiety for selective trapping and enrichment of single-stranded DNA binding proteins (SSB). First, the conditions and efficiency of the photochemical reaction with a purified protein using human replication protein A as the relevant model was studied. Secondly, the ability of the probe as bait to photocrosslink and enrich SSB in cell lysate was addressed. Among the different ODN probes studied, we showed that 4-thiothymine was the most relevant: i) it allows efficient and specific trapping of SSB in whole cell extracts in a similar extent as the widely used diazirine, ii) it features the advantages of a zero-length agent thus retaining the physicochemical properties of the ODN bait; iii) ODN including this photochemical agent are easily accessible. In combination with mass spectrometry, the probes incorporating this nucleobase are powerful tools for PAL strategies and can be added in the toolbox of the traditional photocrosslinkers for studying DNA-protein interactions.


Assuntos
Sondas Moleculares/química , Oligonucleotídeos/química , Proteína de Replicação A/química , Timidina/análogos & derivados , Humanos , Timidina/química
17.
Nucleic Acids Res ; 46(18): 9764-9775, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30102387

RESUMO

Sensing of nucleic acids for molecular discrimination between self and non-self is a challenging task for the innate immune system. RNA acts as a potent stimulus for pattern recognition receptors including in particular human Toll-like receptor 7 (TLR7). Certain RNA modifications limit potentially harmful self-recognition of endogenous RNA. Previous studies had identified the 2'-O-methylation of guanosine 18 (Gm18) within tRNAs as an antagonist of TLR7 leading to an impaired immune response. However, human tRNALys3 was non-stimulatory despite lacking Gm18. To identify the underlying molecular principle, interferon responses of human peripheral blood mononuclear cells to differentially modified tRNALys3 were determined. The investigation of synthetic modivariants allowed attributing a significant part of the immunosilencing effect to the 2'-O-methylthymidine (m5Um) modification at position 54. The effect was contingent upon the synergistic presence of both methyl groups at positions C5 and 2'O, as shown by the fact that neither Um54 nor m5U54 produced any effect alone. Testing permutations of the nucleobase at ribose-methylated position 54 suggested that the extent of silencing and antagonism of the TLR7 response was governed by hydrogen patterns and lipophilic interactions of the nucleobase. The results identify a new immune-modulatory endogenous RNA modification that limits TLR7 activation by RNA.


Assuntos
Imunidade Inata/genética , Ácidos Nucleicos/imunologia , RNA de Transferência/imunologia , Receptor 7 Toll-Like/genética , Guanosina/química , Guanosina/imunologia , Humanos , Hidrogênio/química , Interferons/genética , Leucócitos Mononucleares/química , Leucócitos Mononucleares/imunologia , Metilação , Ácidos Nucleicos/química , Ácidos Nucleicos/genética , RNA de Transferência/genética , Timidina/análogos & derivados , Timidina/química , Timidina/genética , Receptor 7 Toll-Like/imunologia
18.
Nucleic Acids Res ; 46(16): 8069-8078, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30085103

RESUMO

There are five canonical bases in DNA and RNA. Each base has its particular molecular recognition properties and base pairing strength. Thymine and uracil form only two hydrogen bonds when pairing with adenine, and duplexes rich in A:T base pairs are more labile than duplexes rich in C and G, making some sequences difficult to detect via hybridization in a genomic context. Here we report the synthesis of an ethynylmethylpyridone C-nucleoside, abbreviated 'W', that presents a similar recognition surface as thymidine in the major groove but pairs with A about as strongly as C pairs with G. A phosphoramidite building block was synthesized that allows for incorporation of W residues via automated synthesis in high yield. Melting point increases over duplexes containing T:A pairs of up to 17.5°C, or up to 5.8°C per residue were measured for oligonucleotides containing W. Further, the new base shows excellent fidelity, with a single mismatched G opposite W causing a melting point depression of up to 20.5°C. The strongly pairing replacement for thymidine is only slightly larger than its natural counterpart and performs well in different sequence contexts. It can be used to target weakly pairing A-rich sequences in biological studies.


Assuntos
Pareamento de Bases , Timidina/análogos & derivados , Cromatografia Líquida de Alta Pressão , DNA/química , Ligações de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Oligonucleotídeos/síntese química , Compostos Organofosforados , Relação Estrutura-Atividade , Timidina/química , Temperatura de Transição
19.
Phys Chem Chem Phys ; 20(24): 16428-16436, 2018 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-29873362

RESUMO

The decay of the triplet state of photosensitizers is essential to their performance in singlet-oxygen generation. Experiments have shown that in thionucleosides, this decay is enhanced compared to that in the corresponding thionucleobases. In this work, we applied quantum-chemical methods and chemical-kinetic modeling to investigate the effects of the sugar substituent on the triplet decay of thionucleosides. The computed rates for the energetically favored conformers of thiothymidine, thiouridine, and thioguanosine (and the respective thionucleobases) show a remarkable quantitative agreement with the experimental results. We additionally show that the triplet decay enhancement is caused by the repulsion interaction between the sugar group and the sulfur atom, which reduces the activation energy for intersystem crossing by destabilizing the T1 minimum. In some instances, an intramolecular hydrogen bond stabilizes the energy of the T1/S0 crossing point, also reducing the activation energy. This molecular understanding of the mechanism of enhanced triplet decay provides a guideline to control the triplet decay rate, which was tested in new thiothymidine derivatives.


Assuntos
Fármacos Fotossensibilizantes/química , Tionucleosídeos/química , Desoxirribose/química , Glicosilação , Cinética , Modelos Químicos , Conformação Molecular , Teoria Quântica , Tioguanina/química , Timidina/análogos & derivados , Timidina/química
20.
Nucleic Acids Res ; 46(13): 6470-6479, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29901748

RESUMO

Binding reactions of HgII and AgI to pyrimidine-pyrimidine mismatches in duplex DNA were characterized using fluorescent nucleobase analogs, thermal denaturation and 1H NMR. Unlike AgI, HgII exhibited stoichiometric, site-specific binding of C-T mismatches. The on- and off-rates of HgII binding were approximately 10-fold faster to C-T mismatches (kon ≈ 105 M-1 s-1, koff ≈ 10-3 s-1) as compared to T-T mismatches (kon ≈ 104 M-1 s-1, koff ≈ 10-4 s-1), resulting in very similar equilibrium binding affinities for both types of 'all natural' metallo base pairs (Kd ≈ 10-150 nM). These results are in contrast to thermal denaturation analyses, where duplexes containing T-T mismatches exhibited much larger increases in thermal stability upon addition of HgII (ΔTm = 6-19°C), as compared to those containing C-T mismatches (ΔTm = 1-4°C). In addition to revealing the high thermodynamic and kinetic stabilities of C-HgII-T base pairs, our results demonstrate that fluorescent nucleobase analogs enable highly sensitive detection and characterization of metal-mediated base pairs - even in situations where metal binding has little or no impact on the thermal stability of the duplex.


Assuntos
Pareamento Incorreto de Bases , Citosina/química , Mercúrio/química , Timidina/química , Ressonância Magnética Nuclear Biomolecular , Desnaturação de Ácido Nucleico , Prata/química
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