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1.
Bull Cancer ; 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38824069

RESUMO

A better understanding of the RNA biology and chemistry is necessary to then develop new RNA therapeutic strategies. This review is the synthesis of a series of conferences that took place during the 6th international course on post-transcriptional gene regulation at Institut Curie. This year, the course made a special focus on RNA chemistry.

2.
Nucleic Acids Res ; 52(1): 448-461, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-37986223

RESUMO

Metal ions are essential components for the survival of living organisms. For most species, intracellular and extracellular ionic conditions differ significantly. As G-quadruplexes (G4s) are ion-dependent structures, changes in the [Na+]/[K+] ratio may affect the folding of genomic G4s. More than 11000 putative G4 sequences in the human genome (hg19) contain at least two runs of three continuous cytosines, and these mixed G/C-rich sequences may form a quadruplex or a competing hairpin structure based on G-C base pairing. In this study, we examine how the [Na+]/[K+] ratio influences the structures of G/C-rich sequences. The natural G4 structure with a 9-nt long central loop, CEBwt, was chosen as a model sequence, and the loop bases were gradually replaced by cytosines. The series of CEB mutations revealed that the presence of cytosines in G4 loops does not prevent G4 folding or decrease G4 stability but increases the probability of forming a competing structure, either a hairpin or an intermolecular duplex. Slow conversion to the quadruplex in vitro (in a potassium-rich buffer) and cells was demonstrated by NMR. 'Shape-shifting' sequences may respond to [Na+]/[K+] changes with delayed kinetics.


Assuntos
Quadruplex G , Potássio , Sódio , Humanos , Espectroscopia de Ressonância Magnética , Mutação , Potássio/química , Sódio/química
3.
Nat Genet ; 55(8): 1359-1369, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37400615

RESUMO

Metazoan promoters are enriched in secondary DNA structure-forming motifs, such as G-quadruplexes (G4s). Here we describe 'G4access', an approach to isolate and sequence G4s associated with open chromatin via nuclease digestion. G4access is antibody- and crosslinking-independent and enriches for computationally predicted G4s (pG4s), most of which are confirmed in vitro. Using G4access in human and mouse cells, we identify cell-type-specific G4 enrichment correlated with nucleosome exclusion and promoter transcription. G4access allows measurement of variations in G4 repertoire usage following G4 ligand treatment, HDAC and G4 helicases inhibitors. Applying G4access to cells from reciprocal hybrid mouse crosses suggests a role for G4s in the control of active imprinting regions. Consistently, we also observed that G4access peaks are unmethylated, while methylation at pG4s correlates with nucleosome repositioning on DNA. Overall, our study provides a new tool for studying G4s in cellular dynamics and highlights their association with open chromatin, transcription and their antagonism to DNA methylation.


Assuntos
Cromatina , Quadruplex G , Animais , Humanos , Camundongos , Cromatina/genética , Nucleossomos/genética , DNA/genética , Regiões Promotoras Genéticas
4.
Chembiochem ; 24(8): e202300093, 2023 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-36942862

RESUMO

This symposium is the third PSL (Paris Sciences & Lettres) Chemical Biology meeting (2016, 2019, 2023) held at Institut Curie. This initiative originally started at Institut de Chimie des Substances Naturelles (ICSN) in Gif-sur-Yvette (2013, 2014), under the directorship of Professor Max Malacria, with a strong focus on chemistry. It was then continued at the Institut Curie (2015) covering a larger scope, before becoming the official PSL Chemical Biology meeting. This latest edition was postponed twice for the reasons that we know. This has given us the opportunity to invite additional speakers of great standing. This year, Institut Curie hosted around 300 participants, including 220 on site and over 80 online. The pandemic has had, at least, the virtue of promoting online meetings, which we came to realize is not perfect but has its own merits. In particular, it enables those with restricted time and resources to take part in events and meetings, which can now accommodate unlimited participants. We apologize to all those who could not attend in person this time due to space limitation at Institut Curie.


Assuntos
Biologia , Humanos , Paris
5.
Biochimie ; 214(Pt A): 5-23, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36596406

RESUMO

Besides the well-known DNA double-helix, non-canonical nucleic acid structures regulate crucial biological activities. Among these oddities, guanine-rich DNA sequences can form unusual four-stranded secondary structures called G-quadruplexes (G4s). G4-prone sequences have been found in the genomes of most species, and G4s play important roles in essential processes such as transcription, replication, genome integrity and epigenetic regulation. Here, we present a short overview of G-quadruplexes followed by a detailed description of the biophysical and biochemical methods used to characterize G4s in vitro. The principles, experimental details and possible shortcomings of each method are discussed to provide a comprehensive view of the techniques used to study these structures. We aim to provide a set of guidelines for standardizing research on G-quadruplexes; these guidelines are not meant to be a dogmatic set of rules, but should rather provide useful information on the methods currently used to study these fascinating motifs.


Assuntos
Quadruplex G , Epigênese Genética , DNA/química , Genoma
6.
Nucleic Acids Res ; 50(16): e93, 2022 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-35670668

RESUMO

Algorithms have been widely used to predict G-quadruplexes (G4s)-prone sequences. However, an experimental validation of these predictions is generally required. We previously reported a high-throughput technique to evidence G4 formation in vitro called FRET-MC. This method, while convenient and reproducible, has one known weakness: its inability to pin point G4 motifs of low thermal stability. As such quadruplexes may still be biologically relevant if formed at physiological temperature, we wanted to develop an independent assay to overcome this limitation. To this aim, we introduced an isothermal version of the competition assay, called iso-FRET, based on a duplex-quadruplex competition and a well-characterized bis-quinolinium G4 ligand, PhenDC3. G4-forming competitors act as decoys for PhenDC3, lowering its ability to stabilize the G4-forming motif reporter oligonucleotide conjugated to a fluorescence quencher (37Q). The decrease in available G4 ligand concentration restores the ability of 37Q to hybridize to its FAM-labeled short complementary C-rich strand (F22), leading to a decrease in fluorescence signal. In contrast, when no G4-forming competitor is present, PhenDC3 remains available to stabilize the 37Q quadruplex, preventing the formation of the F22 + 37Q complex. Iso-FRET was first applied to a reference panel of 70 sequences, and then used to investigate 23 different viral sequences.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Quadruplex G , Ligantes
7.
Chemistry ; 28(35): e202200734, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35441438

RESUMO

Combining the selectivity of G-quadruplex (G4) ligands with the spatial and temporal control of photochemistry is an emerging strategy to elucidate the biological relevance of these structures. In this work, we developed six novel V-shaped G4 ligands that can, upon irradiation, form stable covalent adducts with G4 structures via the reactive intermediate, quinone methide (QM). We thoroughly investigated the photochemical properties of the ligands and their ability to generate QMs. Subsequently, we analyzed their specificity for various topologies of G4 and discovered a preferential binding towards the human telomeric sequence. Finally, we tested the ligand ability to act as photochemical alkylating agents, identifying the covalent adducts with G4 structures. This work introduces a novel molecular tool in the chemical biology toolkit for G4s.


Assuntos
Quadruplex G , Indolquinonas , Alquilantes/química , Humanos , Ligantes
8.
Nucleic Acids Res ; 50(5): 2719-2735, 2022 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-35234933

RESUMO

Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite.


Assuntos
Quadruplex G , Nematoides , Parasitos/genética , Platelmintos , Animais , Bovinos , Genoma , Helmintos/genética , Humanos , Ligantes , Nematoides/genética , Platelmintos/genética
9.
Nucleic Acids Res ; 49(22): 12644-12660, 2021 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-34875077

RESUMO

G-quadruplexes (G4s) are secondary structures forming in G-rich nucleic acids. G4s are assumed to play critical roles in biology, nonetheless their detection in cells is still challenging. For tracking G4s, synthetic molecules (G4 ligands) can be used as reporters and have found wide application for this purpose through chemical functionalization with a fluorescent tag. However, this approach is limited by a low-labeling degree impeding precise visualization in specific subcellular regions. Herein, we present a new visualization strategy based on the immuno-recognition of 5-bromo-2'-deoxyuridine (5-BrdU) modified G4 ligands, functionalized prior- or post-G4-target binding by CuAAC. Remarkably, recognition of the tag by antibodies leads to the detection of the modified ligands exclusively when bound to a G4 target both in vitro, as shown by ELISA, and in cells, thereby providing a highly efficient G4-ligand Guided Immunofluorescence Staining (G4-GIS) approach. The obtained signal amplification revealed well-defined fluorescent foci located in the perinuclear space and RNase treatment revealed the preferential binding to G4-RNA. Furthermore, ligand treatment affected significantly BG4 foci formation in cells. Our work headed to the development of a new imaging approach combining the advantages of immunostaining and G4-recognition by G4 ligands leading to visualization of G4/ligands species in cells with unrivaled precision and sensitivity.


Assuntos
Bromodesoxiuridina , Imunofluorescência/métodos , Quadruplex G , Células A549 , Linhagem Celular , Química Click , Ensaio de Imunoadsorção Enzimática , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes
10.
Biopolymers ; 112(4): e23415, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33368198

RESUMO

G-quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G-quartets, the length and positions of loops, flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4-prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G-quadruplex or not. In this article, we adapted the well-known FRET-melting assay to characterize G4 in batch, where the sequence to be tested is added, as an unlabeled competitor, to a system composed of a dual-labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G-quadruplexes. In this so-called FRET-MC (melting competition) assay, G4-forming competitors lead to a marked decrease of the ligand-induced stabilization effect (∆Tm ), while non-specific competitors (e.g., single- or double-stranded sequences) have little effect. Sixty-five known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized.


Assuntos
Compostos de Anéis Fundidos/química , Quadruplex G , Oligonucleotídeos/química , Bioensaio/métodos , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Humanos , Técnicas In Vitro , Desnaturação de Ácido Nucleico
11.
Biomolecules ; 10(9)2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32967357

RESUMO

The importance of unusual DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes (G4s) have gained in popularity during the last decade, and their presence and functional relevance at the DNA and RNA level has been demonstrated in a number of viral, bacterial, and eukaryotic genomes, including humans. Here, we performed the first systematic search of G4-forming sequences in all archaeal genomes available in the NCBI database. In this article, we investigate the presence and locations of G-quadruplex forming sequences using the G4Hunter algorithm. G-quadruplex-prone sequences were identified in all archaeal species, with highly significant differences in frequency, from 0.037 to 15.31 potential quadruplex sequences per kb. While G4 forming sequences were extremely abundant in Hadesarchaea archeon (strikingly, more than 50% of the Hadesarchaea archaeon isolate WYZ-LMO6 genome is a potential part of a G4-motif), they were very rare in the Parvarchaeota phylum. The presence of G-quadruplex forming sequences does not follow a random distribution with an over-representation in non-coding RNA, suggesting possible roles for ncRNA regulation. These data illustrate the unique and non-random localization of G-quadruplexes in Archaea.


Assuntos
Archaea/genética , DNA/química , Quadruplex G , Genoma Arqueal/genética , RNA/química , Archaea/classificação , Archaea/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Dicroísmo Circular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genômica/métodos , Conformação de Ácido Nucleico , Filogenia , RNA/genética , RNA/metabolismo , Especificidade da Espécie
12.
Cell Chem Biol ; 26(12): 1681-1691.e5, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31668518

RESUMO

Heme is an essential cofactor for many enzymes, but free heme is toxic and its levels are tightly regulated. G-quadruplexes bind heme avidly in vitro, raising the possibility that they may sequester heme in vivo. If so, then treatment that displaces heme from quadruplexes is predicted to induce expression of genes involved in iron and heme homeostasis. Here we show that PhenDC3, a G-quadruplex ligand structurally unrelated to heme, displaces quadruplex-bound heme in vitro and alters transcription in cultured human cells, upregulating genes that support heme degradation and iron homeostasis, and most strikingly causing a 30-fold induction of heme oxidase 1, the key enzyme in heme degradation. We propose that G-quadruplexes sequester heme to protect cells from the pathophysiological consequences of free heme.


Assuntos
Compostos de Anéis Fundidos , Quadruplex G , Heme/metabolismo , Sítios de Ligação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA Catalítico/metabolismo , Heme/química , Humanos , Ferro/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Transcrição Gênica/efeitos dos fármacos
13.
Nucleic Acids Res ; 47(12): 6098-6113, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31114920

RESUMO

G-quadruplexes play various roles in multiple biological processes, which can be positive when a G4 is involved in the regulation of gene expression or detrimental when the folding of a stable G4 impairs DNA replication promoting genome instability. This duality interrogates the significance of their presence within genomes. To address the potential biased evolution of G4 motifs, we analyzed their occurrence, features and polymorphisms in a large spectrum of species. We found extreme bias of the short-looped G4 motifs, which are the most thermodynamically stable in vitro and thus carry the highest folding potential in vivo. In the human genome, there is an over-representation of single-nucleotide-loop G4 motifs (G4-L1), which are highly conserved among humans and show a striking excess of the thermodynamically least stable G4-L1A (G3AG3AG3AG3) sequences. Functional assays in yeast showed that G4-L1A caused the lowest levels of both spontaneous and G4-ligand-induced instability. Analyses across 600 species revealed the depletion of the most stable G4-L1C/T quadruplexes in most genomes in favor of G4-L1A in vertebrates or G4-L1G in other eukaryotes. We discuss how these trends might be the result of species-specific mutagenic processes associated to a negative selection against the most stable motifs, thus neutralizing their detrimental effects on genome stability while preserving positive G4-associated biological roles.


Assuntos
Quadruplex G , Genoma , Animais , Eucariotos/genética , Genoma Humano , Humanos , Camundongos , Motivos de Nucleotídeos , Polimorfismo Genético , Termodinâmica
14.
Analyst ; 144(11): 3518-3524, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31020955

RESUMO

Mass spectrometry provides exquisite details on ligand and cation binding stoichiometries with a DNA target. The next important step is to develop reliable methods to determine the cation and ligand binding sites in each complex separated by using a mass spectrometer. To circumvent the caveat of ligand derivatization for cross-linking, which may alter the ligand binding mode, we explored a tandem mass spectrometry (MS/MS) method that does not require ligand derivatization, and is therefore also applicable to localize metal cations. By putting more negative charge states on the complexes using supercharging agents, and by creating radical ions by electron photodetachment, oligonucleotide bonds become weaker than the DNA-cation or DNA-ligand noncovalent bonds upon collision-induced dissociation of the radicals. This electron photodetachment (EPD) method allows one to locate the binding regions of cations and ligands by top-down sequencing of the oligonucleotide target. The very potent G-quadruplex ligands 360A and PhenDC3 were found to replace a potassium cation and bind close to the central loop of 4-repeat human telomeric sequences.


Assuntos
DNA/química , Quadruplex G , Potássio/química , Sequência de Bases , Sítios de Ligação , DNA/genética , DNA/metabolismo , Humanos , Ligantes , Potássio/metabolismo , Piridinas/química , Quinolinas/química , Análise de Sequência de DNA/métodos , Espectrometria de Massas em Tandem/métodos
15.
Molecules ; 24(3)2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30678027

RESUMO

Guanine-rich DNA can form four-stranded structures called G-quadruplexes (G4s) that can regulate many biological processes. Metal complexes have shown high affinity and selectivity toward the quadruplex structure. Here, we report the comparison of a panel of platinum (II) complexes for quadruplex DNA selective recognition by exploring the aromatic core around terpyridine derivatives. Their affinity and selectivity towards G4 structures of various topologies have been evaluated by FRET-melting (Fluorescence Resonance Energy Transfert-melting) and Fluorescent Intercalator Displacement (FID) assays, the latter performed by using three different fluorescent probes (Thiazole Orange (TO), TO-PRO-3, and PhenDV). Their ability to bind covalently to the c-myc G4 structure in vitro and their cytotoxicity potential in two ovarian cancerous cell lines were established. Our results show that the aromatic surface of the metallic ligands governs, in vitro, their affinity, their selectivity for the G4 over the duplex structures, and platination efficiency. However, the structural modifications do not allow significant discrimination among the different G4 topologies. Moreover, all compounds were tested on ovarian cancer cell lines and normal cell lines and were all able to overcome cisplatin resistance highlighting their interest as new anticancer drugs.


Assuntos
Quadruplex G/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Platina/química , Proteínas Proto-Oncogênicas c-myc/química , Anticarcinógenos/química , Anticarcinógenos/uso terapêutico , Cisplatino/efeitos adversos , Cisplatino/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Ligantes , Conformação de Ácido Nucleico/efeitos dos fármacos , Piridinas/química
16.
J Med Chem ; 61(23): 10502-10518, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30457335

RESUMO

Heptaheteroaryl compounds comprised of oxazole and pyridine units (TOxaPy) are quadruplex DNA (G4)-interactive compounds. Herein, we report on the synthesis of parent compounds bearing either amino side chains (TOxaPy-1-5) or featuring an isomeric oxazole-pyridine central connectivity (iso-TOxapy, iso-TOxapy 1-3) or a bipyridine core (iso-TOxabiPy). The new isomeric series showed significant G4-binding activity in vitro, and remarkably, three compounds (iso-TOxaPy, iso-TOxaPy-1, and iso-TOxabiPy) exhibited high antiproliferative activity toward a tumor panel of cancer cell lines. However, these compounds do not behave as typical G-quadruplex (G4) binders, and the kinase profiling assay revealed that the best antiproliferative molecule iso-TOxaPy selectively inhibited Rock-2. The targeting of Rock kinase was confirmed in cells by the dephosphorylation of Rock-2 substrates, the decrease of stress fibers, and peripheral focal adhesions, as well as the induction of long neurite-like extensions. Remarkably, two of these molecules were able to inhibit the growth of cells organized as spheroids.


Assuntos
Desenho de Fármacos , Quadruplex G/efeitos dos fármacos , Oxazóis/química , Oxazóis/farmacologia , Piridinas/química , Quinases Associadas a rho/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Ligantes , Oxazóis/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade
17.
Molecules ; 23(9)2018 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-30154319

RESUMO

Non-macrocyclic heteroaryls represent a valuable class of ligands for nucleic acid recognition. In this regard, non-macrocyclic pyridyl polyoxazoles and polyoxadiazoles were recently identified as selective G-quadruplex stabilizing compounds with high cytotoxicity and promising anticancer activity. Herein, we describe the synthesis of a new family of heteroaryls containing oxadiazole and pyridine moieties targeting DNA G-quadruplexes. To perform a structure⁻activity analysis identifying determinants of activity and selectivity, we followed a convergent synthetic pathway to modulate the nature and number of the heterocycles (1,3-oxazole vs. 1,2,4-oxadiazole and pyridine vs. benzene). Each ligand was evaluated towards secondary nucleic acid structures, which have been chosen as a prototype to mimic cancer-associated G-quadruplex structures (e.g., the human telomeric sequence, c-myc and c-kit promoters). Interestingly, heptapyridyl-oxadiazole compounds showed preferential binding towards the telomeric sequence (22AG) in competitive conditions vs. duplex DNA. In addition, G4-FID assays suggest a different binding mode from the classical stacking on the external G-quartet. Additionally, CD titrations in the presence of the two most promising compounds for affinity, TOxAzaPy and TOxAzaPhen, display a structural transition of 22AG in K-rich buffer. This investigation suggests that the pyridyl-oxadiazole motif is a promising recognition element for G-quadruplexes, combining seven heteroaryls in a single binding unit.


Assuntos
Oxidiazóis/química , Piridinas/química , Dicroísmo Circular , Desenho de Fármacos , Estabilidade de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Quadruplex G , Ligantes , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/farmacologia , Piridinas/síntese química , Piridinas/farmacologia
18.
Angew Chem Int Ed Engl ; 55(34): 10131-5, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27392211

RESUMO

DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High-fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high-fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template-dependent DNA synthesis, despite this "cargo" being more than 100-fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked-eye detection of DNA and RNA at single nucleotide resolution.


Assuntos
DNA Polimerase Dirigida por DNA , Nucleotídeos , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Moleculares , Estrutura Molecular , Nucleotídeos/química , Nucleotídeos/metabolismo , Especificidade por Substrato
19.
Bioorg Med Chem Lett ; 26(3): 841-844, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26774580

RESUMO

DNA polymerases can efficiently and sequence selectively incorporate oligonucleotide (ODN)-modified nucleotides and the incorporated oligonucleotide strand can be employed as primer in rolling circle amplification (RCA). The effective amplification of the DNA primer by Φ29 DNA polymerase allows the sequence-selective hybridisation of the amplified strand with a G-quadruplex DNA sequence that has horse radish peroxidase-like activity. Based on these findings we develop a system that allows DNA detection with single-base resolution by naked eye.


Assuntos
DNA/análise , Técnicas de Amplificação de Ácido Nucleico , Oligonucleotídeos/metabolismo , Biotina/química , DNA Catalítico/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Oligonucleotídeos/química
20.
Angew Chem Int Ed Engl ; 53(4): 994-8, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24338872

RESUMO

We have developed a straightforward synthetic pathway to a set of six photoactivatable G-quadruplex ligands with a validated G4-binding motif (the bisquinolinium pyridodicarboxamide PDC-360A) tethered through various spacers to two different photo-cross-linking groups: benzophenone and an aryl azide. The high quadruplex-versus-duplex selectivity of the PDC core was retained in the new derivatives and resulted in selective alkylation of two well-known G-quadruplexes (human telomeric G4 and oncogene promoter c-myc G4) under conditions of harsh competition. The presence of two structurally different photoactivatable functions allowed the selective alkylation of G-quadruplex structures at specific nucleobases and irreversible G4 binding. The topology and sequence of the quadruplex matrix appear to influence strongly the alkylation profile, which differs for the telomeric and c-myc quadruplexes. The new compounds are photoactive in cells and thus provide new tools for studying G4 biology.


Assuntos
Azidas/química , Benzofenonas/química , Reagentes de Ligações Cruzadas/química , Quadruplex G , Humanos , Ligantes , Estrutura Molecular , Processos Fotoquímicos
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