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1.
Nat Commun ; 10(1): 2421, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160600

RESUMO

Translation efficiency can be affected by mRNA stability and secondary structures, including G-quadruplex structures (G4s). The highly conserved DEAH-box helicase DHX36/RHAU resolves G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress.


Assuntos
RNA Helicases DEAD-box/metabolismo , Quadruplex G , RNA Mensageiro/metabolismo , Regiões não Traduzidas , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Fosforilação , Biossíntese de Proteínas , Ribossomos/metabolismo , Estresse Fisiológico , eIF-2 Quinase/metabolismo
2.
Talanta ; 202: 342-348, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171193

RESUMO

A molecular beacons (MBs) loaded on molybdenum disulfide (MoS2) nanosheets as fluorescence probes for sensitive and versatile detection of microRNAs (miRNAs) through hybridization chain reaction (HCR) has been designed. MoS2 was used as a adsorbent to capture the MBs and a selective fluorescence quencher to reduce the background signal. In the absence of miRNAs, HCR could not be triggered due to the stability of MB probes. The probes attached to the MoS2 surface, efficiently quenching fluorescence of the G-quadruplex/Thioflavin T. However, the presence of target miRNAs triggers the HCR process to generate large amount of HCR products. Meanwhile, the HCR products of long nanowires chain with abundant G-quadruplexes could not be adsorbed on the surface of MoS2, and therefore detach from the MoS2. Consequently, Thioflavin T could be embedded in G-quadruplexes and produced strong fluorescence signal. This fluorescence emission signal could achieve detection of miRNA as low as 4.2 pM and a wide linear ranges from 0.1 to 100 nM. In addition, a versatile fluorescence probe has been developed for detection of miRNA-21 by changing the miRNA-recognition domain of MB. Thus, the fluorescent probe would be a potential alternative tool for biomedical research and clinical molecular diagnostics.


Assuntos
Dissulfetos/química , Corantes Fluorescentes/química , Quadruplex G , MicroRNAs/análise , Sondas Moleculares/química , Molibdênio/química , Hibridização de Ácido Nucleico , Humanos
3.
Eur J Med Chem ; 177: 401-413, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158753

RESUMO

Small molecules able to bind non-canonical G-quadruplex DNA structures (G4) have been recently tested as novel potential agents for the treatment of prostate cancer thanks to their repression of aberrant androgen receptor gene. However, metastatic castration-resistant prostate cancer (mCRPC), a letal form of prostate cancer, is still incurable. Here we tested two naphthalenediimide derivatives, previously reported as multitarget agents, on a couple of relevant mCRPC cell models (DU145 and PC-3). We showed that these compounds interfere with the RAS/MEK/ERK and PI3K/AKT pathways. Interestingly, both these two biological processes depend upon Epidermal Growth Factor Receptor (EGFR) activation. By means of biological and analytical tools we showed that our compounds are efficient inducers of the structural transition of the EGFR promoter towards a G-quadruplex conformation, ultimately leading to a reduction of the receptor production. The overall result is an interesting cytotoxic profile for these two derivatives. Thanks to their activity at different steps, these compounds can open the way to novel therapeutic approaches for mCRPC that could contribute to escape resistance to selective treatments.


Assuntos
DNA/metabolismo , Quadruplex G/efeitos dos fármacos , Naftalimidas/farmacologia , Linhagem Celular Tumoral , DNA/genética , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Naftalimidas/química , Naftalimidas/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico
4.
Biochemistry (Mosc) ; 84(5): 562-569, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234770

RESUMO

Amplification of GC-rich regions of genomic DNA is hindered either by high stability of DNA double helix or as a result of alternative structure formation by a guanine-rich DNA strand. Such potential G-quadruplex (G4) sequences are fairly common in promoters of the human genome. The efficiency of PCR amplification of promoter sequences for several human oncogenes (MYC, NRAS, TERT, KRAS, KIT) was studied. We demonstrate that the efficiency of DNA polymerase is reduced in the presence of potassium ions. The primer-extension technique localized DNA polymerase stops at the 3'-ends of potential quadruplex sequences. The structural and thermodynamic properties of short G-rich oligonucleotides corresponding to the stops of DNA polymerase were analyzed. These oligonucleotides formed stable parallel G4 in the presence of potassium ions. Correlation between the stability of G4 structure and efficiency of DNA polymerase stops was revealed. The results provide a method for detecting new G4 structures in extended genomic sequences and also clarify the mechanism of inhibition of DNA polymerase in G-rich regions of DNA.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Quadruplex G , Potássio/metabolismo , Dicroísmo Circular , DNA Polimerase Dirigida por DNA/química , Oligonucleotídeos/metabolismo , Reação em Cadeia da Polimerase , Potássio/química , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Termodinâmica , Proteínas ras/genética
5.
Anal Bioanal Chem ; 411(21): 5555-5561, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31197422

RESUMO

G-quadruplexes have been widely researched as new targets for cancer treatment owing to their non-canonical structure and crucial role in biological processes. Although attention has been paid to the development of selective G-quadruplex ligands, few studies have focused on the binding affinity of stereoisomers towards G-quadruplex, which will be conducive to support the optimal design of G-quadruplex ligands in future studies. Here, tetrandrine and isotetrandrine were used to study the binding affinity and difference of stereoisomers towards G-quadruplex structures. The results showed that tetrandrine had a high possibility of binding to the N-myc and Bcl-2 G-quadruplexes through hydrogen bonding, whereas the possibility of binding of isotetrandrine was low and it seemed to have no possibility of forming hydrogen bonds. Our study shows that optical isomerism of ligand molecules has an important effect on G-quadruplex recognition, which is helpful for the design of G-quadruplex ligands in future studies. Graphical abstract.


Assuntos
Alcaloides/metabolismo , Quadruplex G , Benzilisoquinolinas/química , Benzilisoquinolinas/metabolismo , Dicroísmo Circular , Ligações de Hidrogênio , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Estereoisomerismo , Termodinâmica
6.
Chemistry ; 25(47): 11085-11097, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31219221

RESUMO

Naphthalene diimide (NDI) dyads exhibiting a different substitution pattern and linker length have been synthesised and evaluated as G-quadruplex (G4) ligands, by investigating their cytotoxicity in selected cell lines. The dyads with the long C7 linker exhibit extremely low IC50 values, below 10 nm, on different cancer cell lines. Contrary, the dyads with the shorter C4 linker were much less effective, with IC values increasing up to 1 µm. Among the three dyads with the longest linker, small differences in the IC50 values emerge, suggesting that the linker length plays a more important role than the substitution pattern. We have further shown that the dyads are able to induce cellular DNA damage response, which is not limited to the telomeric regions and is likely the origin of their cytotoxicity. Both absorption titration and dynamic light scattering of the most cytotoxic dyads in the presence of hTel22 highlight their ability to induce effective G4 aggregation, acting as non-covalent cross-linking agents.


Assuntos
Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Quadruplex G , Imidas/farmacologia , Naftalenos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidas/síntese química , Imidas/química , Ligantes , Metáfase/efeitos dos fármacos , Microscopia de Fluorescência , Naftalenos/síntese química , Naftalenos/química , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Telômero/efeitos dos fármacos , Telômero/metabolismo
7.
Eur J Med Chem ; 178: 13-29, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31173968

RESUMO

The oncogenic Epstein-Barr virus (EBV) evades the immune system through limiting the expression of its highly antigenic and essential genome maintenance protein, EBNA1, to the minimal level to ensure viral genome replication, thereby also minimizing the production of EBNA1-derived antigenic peptides. This regulation is based on inhibition of translation of the virally-encoded EBNA1 mRNA, and involves the interaction of host protein nucleolin (NCL) with G-quadruplex (G4) structures that form in the glycine-alanine repeat (GAr)-encoding sequence of the EBNA1 mRNA. Ligands that bind to these G4-RNA can prevent their interaction with NCL, leading to disinhibition of EBNA1 expression and antigen presentation, thereby interfering with the immune evasion of EBNA1 and therefore of EBV (M.J. Lista et al., Nature Commun., 2017, 8, 16043). In this work, we synthesized and studied a series of 20 cationic bis(acylhydrazone) derivatives designed as G4 ligands. The in vitro evaluation showed that most derivatives based on central pyridine (Py), naphthyridine (Naph) or phenanthroline (Phen) units were efficient G4 binders, in contrast to their pyrimidine (Pym) counterparts, which were poor G4 binders due to a significantly different molecular geometry. The influence of lateral heterocyclic units (N-substituted pyridinium or quinolinium residues) on G4-binding properties was also investigated. Two novel compounds, namely PyDH2 and PhenDH2, used at a 5 µM concentration, were able to significantly enhance EBNA1 expression in H1299 cells in a GAr-dependent manner, while being significantly less toxic than the prototype drug PhenDC3 (GI50 > 50 µM). Antigen presentation, RNA pull-down and proximity ligation assays confirmed that the effect of both drugs was related to the disruption of NCL-EBNA1 mRNA interaction and the subsequent promotion of GAr-restricted antigen presentation. Our work provides a novel modular scaffold for the development of G-quadruplex-targeting drugs acting through interference with G4-protein interaction.


Assuntos
Hidrazonas/farmacologia , Evasão da Resposta Imune/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Quadruplex G , Herpesvirus Humano 4/genética , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Fatores Imunológicos/síntese química , Fatores Imunológicos/química , Ligantes , Camundongos , RNA Mensageiro/genética
8.
Talanta ; 200: 57-66, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036225

RESUMO

In our work, aptamers and hemin/G-quadruplex DNAzyme modified sandwich-rod graphene quantum dots @ graphene oxide @ carbon fiber composite (DNAzyme/L-Apt/GQDs@GO@CF) was successfully prepared for sensitive and selective chemiluminescence (CL) detection of lysozyme (LZM). Initially, GQDs@GO@CF was successfully prepared and characterized. Lysozyme aptamers (L-Apt) as a recognition element and hemin/G-quadruplex DNAzyme (DNAzyme) as a catalyst of luminal - H2O2 were modified on the surface of GQDs@GO@CF, sequentially. The immobilization properties of GQDs@GO@CF to L-Apt and the adsorption properties of L-Apt/GQDs@GO@CF to DNAzyme were also researched, respectively. Then, the modified sandwich-rod carbon fiber composite was applied to the construction of CL biosensor for LZM detection. When LZM existed, DNAzyme would be released from the surface of L-Apt/GQDs@GO@CF and catalyzed the reaction of luminal - H2O2. Under optimized conditions, the CL biosensor for LZM detection showed wide linear range of 2.64 × 10-10 to 6.6 × 10-8 g/L and low detection limit of 1.25 × 10-11 g/L (3δ). Finally, the CL biosensor was successfully used for LZM detection in human urine samples and illustrated the potential application in pratical samples.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Carbono/química , DNA Catalítico/química , Hemina/química , Muramidase/análise , Adsorção , DNA Catalítico/metabolismo , Quadruplex G , Luminescência , Muramidase/metabolismo , Propriedades de Superfície
9.
Chem Commun (Camb) ; 55(45): 6453-6456, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31099349

RESUMO

In this work, a novel fluorescent assay was proposed for the ultrasensitive detection of microRNA-21 (miRNA-21) based on the efficient immobilization of protoporphyrin IX (PPIX) as signal indicators in massive G-quadruplex structures obtained by target recycling, three-dimensional DNA walker and a rolling circle amplification (RCA) coupled cascade nucleic acid amplification strategy.


Assuntos
Quadruplex G , MicroRNAs/análise , Fármacos Fotossensibilizantes/química , Protoporfirinas/química , Espectrometria de Fluorescência/métodos , Sequência de Bases , Técnicas Biossensoriais , Humanos , Técnicas de Amplificação de Ácido Nucleico
10.
Talanta ; 200: 424-431, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036205

RESUMO

A 15-mer thrombin-binding aptamer (TBA) was discovered with specificity for thrombin. It forms a unique G-quadruplex (G4), which is postulated to be the molecular basis for its binding specificity. Many analytical methods make use of affinity binding between the thrombin and TBA as they form a very stable complex. We develop a strategy to stabilize TBA/G4's structure by introducing G4-interactive molecules, which may enhance its ability to recognize the target. Herein, a fast screening ESI-MS assay was employed to determine potential binding of natural products molecules with the TBA/G4 complex. The experimental results showed that four investigated natural alkaloids had apparent binding affinities. One of them, jatrorrhizine (L1), has been shown to bind strongly to the TBA/G4 mainly in 1:2 M ratio. Once the working conditions were established, the interaction of the jatrorrhizine with the TBA/G4 was explored using a combination of ESI-MS and spectroscopic techniques. Ligand-induced effects on TBA/G4 structure and its stability were examined by means of circular dichroism (CD). Jatrorrhizine inducing the G4 formation seems also to be the more effective in terms of thermal stabilization under the experimental conditions used. Both results of UV and fluorescence experiments undoubtedly showed a good binding affinity with the binding constant around 105 L mol-1. The stacking interactions of jatrorrhizine with the G-tetrads in TBA/G4 were further confirmed by competition experiment. ESI-MS was carried out to determine the coexistence of 1:1 and 1:2 complexes in TBA/G4-L1 system, and showed a dynamical shift from 1:1 to 1:2 complex in minutes.


Assuntos
Aptâmeros de Nucleotídeos/química , Produtos Biológicos/química , Quadruplex G , Sítios de Ligação , Estrutura Molecular , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray
11.
Chemistry ; 25(41): 9691-9700, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31087710

RESUMO

Guanine-rich sequences of DNA are known to readily fold into tetra-stranded helical structures known as G-quadruplexes (G4). Due to their biological relevance, G4s are potential anticancer drug targets and therefore there is significant interest in molecules with high affinity for these structures. Most G4 binders are polyaromatic planar compounds which π-π stack on the G4's guanine tetrad. However, many of these compounds are not very selective since they can also intercalate into duplex DNA. Herein we report a new class of binder based on an octahedral cobalt(III) complex that binds to G4 via a different mode involving hydrogen bonding, electrostatic interactions and π-π stacking. We show that this new compound binds selectivity to G4 over duplex DNA (particularly to the G-rich sequence of the c-myc promoter). This new octahedral complex also has the ability to template the formation of G4 DNA from the unfolded sequence. Finally, we show that upon binding to G4, the complex prevents helicase Pif1-p from unfolding the c-myc G4 structure.


Assuntos
Cobalto/química , Cobalto/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , DNA/química , Quadruplex G/efeitos dos fármacos , Animais , Bovinos , DNA/genética , DNA/metabolismo , DNA Helicases/metabolismo , Genes myc/efeitos dos fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Fenilenodiaminas/química , Fenilenodiaminas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos
12.
Anal Bioanal Chem ; 411(20): 5197-5207, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31119345

RESUMO

Human telomeric G-quadruplexes are emerging targets in anticancer drug discovery since they are able to efficiently inhibit telomerase, an enzyme which is greatly involved in telomere instability and immortalization process in malignant cells. G-quadruplex (G4) DNA is highly polymorphic and can adopt different topologies upon addition of electrolytes, additives, and ligands. The study of G-quadruplex forms under various conditions, however, might be quite challenging. In this work, surface-enhanced Raman scattering (SERS) spectroscopy has been applied to study G-quadruplexes formed by human telomeric sequences, d[A3G3(TTAGGG)3A2] (Tel26) and d[(TTAGGG)4T2] (wtTel26), under dilute and crowding conditions. The SERS spectra distinctive of hybrid-1 and hybrid-2 G-quadruplexes of Tel26 and wtTel26, respectively, were observed for the sequences folded in the presence of K+ ions (110 mM) in a buffered solution, representing the diluted medium. Polyethylene glycol (5, 10, 15, 20, and 40% v/v PEG) was used to create a molecular-crowded environment, resulting in the formation of the parallel G-quadruplexes of both studied human telomeric sequences. Despite extensive overlap by the crowding agent bands, the SERS spectral features indicative of parallel G4 form of Tel26 were recognized. The obtained results implied that SERS of G-quadruplexes reflected not only the primary structure of the studied human telomeric sequence, including its nucleobase composition and sequence, but also its secondary structure in the sense of Hoogsteen hydrogen bonds responsible for the guanine tetrad formation, and finally its tertiary structure, defining a three-dimensional DNA shape, positioned close to the enhancing metallic surface. Graphical abstract.


Assuntos
Quadruplex G , Análise Espectral Raman/métodos , Telômero , Dicroísmo Circular , Humanos , Conformação de Ácido Nucleico
13.
Molecules ; 24(9)2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31052562

RESUMO

The role of local DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, the significance of G-quadruplexes was demonstrated in the last decade, and their presence and functional relevance has been demonstrated in many genomes, including humans. In this study, we analyzed the presence and locations of G-quadruplex-forming sequences by G4Hunter in all complete bacterial genomes available in the NCBI database. G-quadruplex-forming sequences were identified in all species, however the frequency differed significantly across evolutionary groups. The highest frequency of G-quadruplex forming sequences was detected in the subgroup Deinococcus-Thermus, and the lowest frequency in Thermotogae. G-quadruplex forming sequences are non-randomly distributed and are favored in various evolutionary groups. G-quadruplex-forming sequences are enriched in ncRNA segments followed by mRNAs. Analyses of surrounding sequences showed G-quadruplex-forming sequences around tRNA and regulatory sequences. These data point to the unique and non-random localization of G-quadruplex-forming sequences in bacterial genomes.


Assuntos
Bactérias/genética , DNA Bacteriano/química , Quadruplex G , Genoma Bacteriano , Humanos , Conformação de Ácido Nucleico , Filogenia
14.
Chem Commun (Camb) ; 55(42): 5882-5885, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-31037281

RESUMO

Spinach aptamer fluorescence requires formation of a tripartite complex composed of folded RNA, a GFP-like fluorophore, and selective cation coordination. 2'F pyrimidine modified Spinach has retained fluorescence, increased chemical stability, and accelerated cation association via increased G-quadruplex dynamics, thereby reducing readout time and enhancing Spinach utility for aqueous Pb2+ detection.


Assuntos
Aptâmeros de Nucleotídeos/química , Chumbo/química , Ribose/química , Cátions Bivalentes , Dicroísmo Circular , Fluorescência , Corantes Fluorescentes/química , Quadruplex G , Técnicas In Vitro , Conformação de Ácido Nucleico , Espectrometria de Fluorescência
15.
Eur J Med Chem ; 175: 20-33, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31071547

RESUMO

In this report, we synthesized a series of TO conjugates containing different amino side chains and investigated their binding to telomeric G-quadruplex DNA (G4) using several biophysical methods including fluorometric titration and thermal denaturation monitored by fluorescence and circular dichroism. The composition of side chains strongly affects the binding of these molecules to G-quadruplex DNA. Incorporation of amino side chains increases the binding affinity of TO toward G4 but has a minimal effect on its selectivity for G4 over duplex DNA. The plausible binding modes are a synergistic effect of end-stacking and groove interactions as indicated by docking studies. Inhibition of human telomerase activity by TO derivatives was determined in vitro by the TRAP assay. Several derivatives can selectively inhibit the activity of telomerase over DNA polymerase at low concentrations. More significantly, TO-spermine conjugate (16) exhibits a remarkable effect on telomerase inhibition in the submicromolar range, which is comparable to the inhibition effect of a well-known G4 ligand, BRACO-19. Our results here provide guidance of utilizing TO derivatives as a viable scaffold to design novel G4 ligands, G4 probes, and potent telomerase inhibitors.


Assuntos
Acridinas/farmacologia , Benzotiazóis/química , Benzotiazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Espermina/química , Espermina/farmacologia , Telomerase/antagonistas & inibidores , Fenômenos Biofísicos , Dicroísmo Circular , Quadruplex G , Humanos , Espectrometria de Fluorescência , Telômero
16.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067825

RESUMO

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.


Assuntos
Proteínas de Ligação a DNA/química , Quadruplex G , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Conformação de Ácido Nucleico , Ligação Proteica , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Ribossômicas/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica
17.
Soft Matter ; 15(22): 4454-4459, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31073583

RESUMO

Designing ligands that selectively target G-quadruplex DNAs has gained attention due to their possible roles in regulation of gene expression and as anti-cancer agents. In this article, we report irradiation-induced ligand binding to G-quadruplex DNAs which offers a novel approach to targeting specific G-quadruplexes. Photoinduced binding to G-quadruplex DNAs was observed for copolymers of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG). This molecule has an aromatic ring with cationic charge, which after irradiation becomes a binding site for G-quadruplex DNA. PVAMGs acted as neutral polymers with no binding affinity under dark conditions. The photoinduced binding was revealed by fluorescence spectroscopy, NMR spectroscopy, UV melting curve, and DNA polymerase stop assay. PVAMGs showed preference to parallel G-quadruplex structures over mixed parallel/antiparallel structures. PVAMGs were found to be noncytotoxic under both dark and irradiated conditions up to a concentration of 20 µM.


Assuntos
Quadruplex G , Polímeros/química , Corantes de Rosanilina/química , Polímeros/efeitos da radiação , Álcool de Polivinil/química , Corantes de Rosanilina/efeitos da radiação , Raios Ultravioleta
18.
BMC Genomics ; 20(1): 382, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31096907

RESUMO

BACKGROUND: Several lines of evidence suggest that recombination plays a central role in replication and evolution of herpes simplex virus-1 (HSV-1). G-quadruplex (G4)-motifs have been linked to recombination events in human and microbial genomes, but their role in recombination has not been studied in DNA viruses. RESULTS: The availability of near full-length sequences from 40 HSV-1 recombinant strains with exact position of the recombination breakpoints provided us with a unique opportunity to investigate the role of G4-motifs in recombination among herpes viruses. We mapped the G4-motifs in the parental and all the 40 recombinant strains. Interestingly, the genome-wide distribution of breakpoints closely mirrors the G4 densities in the HSV-1 genome; regions of the genome with higher G4 densities had higher number of recombination breakpoints. Biophysical characterization of oligonucleotides from a subset of predicted G4-motifs confirmed the formation of G-quadruplex structures. Our analysis also reveals that G4-motifs are enriched in regions flanking the recombination breakpoints. Interestingly, about 11% of breakpoints lie within a G4-motif, making these DNA secondary structures hotspots for recombination in the HSV-1 genome. Breakpoints within G4-motifs predominantly lie within G4-clusters rather than individual G4-motifs. Of note, we identified the terminal guanosine of G4-clusters at the boundaries of the UL (unique long) region on either side of the OriL (origin of replication within UL) represented the commonest breakpoint among the HSV-1 recombinants. CONCLUSION: Our findings suggest a correlation between the HSV-1 recombination landscape and the distribution of G4-motifs and G4-clusters, with possible implications for the evolution of DNA viruses.


Assuntos
Pontos de Quebra do Cromossomo , DNA Viral/genética , Quadruplex G , Genoma Viral , Herpesvirus Humano 1/genética , Recombinação Genética , Replicação do DNA , Humanos
19.
Biosci Biotechnol Biochem ; 83(9): 1697-1702, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31062645

RESUMO

An enhancer located upstream of the transcriptional start site of Ccnb1ip1 containing two GGA-rich regions and a 14-GGA repeat (GGA)14 region has been previously identified. Three copies of four GGA repeats in the c-myb promoter that form a tetrad:heptad:heptad:tetrad (T:H:H:T) dimerized G-quadruplex (G4) structure reportedly functions as both a transcriptional repressor and activator. Here, the secondary structures of the two GGA-rich and (GGA)14 regions were analyzed using circular dichroism spectral analysis, which indicated that the two GGA-rich DNAs formed parallel-type G4 structures, whereas (GGA)14 DNA formed the T:H:H:T dimerized G4 structure. Reporter assays demonstrated that individual regions did not show enhancer activity; however, the deletion of the (GGA)14 region resulted in 1.5-fold higher enhancer activity than that of the whole enhancer. These results indicate that the (GGA)14 region that forms the T:H:H:T dimerized G4 structure functions as a negative regulator of the Ccnb1ip1 enhancer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Elementos Facilitadores Genéticos , Quadruplex G , Sequências Repetitivas de Ácido Nucleico , Animais , Dicroísmo Circular , Humanos , Camundongos , Células NIH 3T3
20.
Chem Commun (Camb) ; 55(35): 5131-5134, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30973555

RESUMO

A novel approach to program target-responsive devices by incorporating the split G4 motifs in a DNA nanocage has been developed. The rigid prism outcompetes the flexible one in reaction kinetics and signal/background ratios, which can be easily internalized by cells and successfully applied in microRNA imaging in live cells.


Assuntos
DNA/química , Quadruplex G , MicroRNAs/análise , Nanopartículas/química , Benzotiazóis/química , Linhagem Celular Tumoral , DNA/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Humanos , Substâncias Intercalantes/química , Cinética , MicroRNAs/genética , Microscopia Confocal/métodos , Hibridização de Ácido Nucleico
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