RESUMO
The quest for small molecules that strongly bind to G-quadruplex-DNA (G4), so-called G4 ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to discover or rationally design G4 ligands, evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and use them as innovative chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts aimed at developing small molecules that destabilize G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application in neurobiology as many genetic and age-related diseases are caused by an overrepresentation of G4s. Herein, we report on our efforts to develop in vitro assays to reliably identify molecules able to destabilize G4s. This workflow comprises the newly designed G4-unfold assay, adapted from the G4-helicase assay implemented with Pif1, as well as a series of biophysical and biochemical techniques classically used to study G4/ligand interactions (CD, UV-vis, PAGE, and FRET-melting), and a qPCR stop assay, adapted from a Taq-based protocol recently used to identify G4s in the genomic DNA of Schizosaccharomyces pombe. This unique, multipronged approach leads to the characterization of a phenylpyrrolocytosine (PhpC)-based G-clamp analog as a prototype of G4-disrupting small molecule whose properties are validated through many different and complementary in vitro evaluations.
Assuntos
DNA/química , Bibliotecas de Moléculas Pequenas/química , Quadruplex G , Humanos , Ligantes , Estrutura MolecularRESUMO
Social interactions of the nocturnal primates are not well studied. One of the species for which social behavior is scarcely known is the Philippine tarsier (Tarsius [= Carlito] syrichta). We observed a reproducing pair of captive individuals over two mating seasons for two consecutive years. The tarsiers spent approximately 4% of their activity budget on social interactions; ca. 20% of time in 0-1 m proximity to each other; and shared sleeping sites for half of the study time. The majority of the animals' social interactions were peaceful: affiliative and sexual (83%), and the smallest component of the behavior was agonistic (17%). We witnessed two copulation events (one per estrus day), each lasting ca. 5 min, and both occurring just after waking. We revealed temporal - nightly and hourly - fluctuations in the frequency of social interactions, in the distances the individuals spent from each other and in the number of vocalizations. The results present the first assessment of the social behavior of the Philippine tarsier, much needed to improve the captive breeding management for this highly sensitive species threatened with extinction.
Assuntos
Comportamento Sexual Animal , Comportamento Social , Tarsiidae/fisiologia , Agressão , Comunicação Animal , Animais , Feminino , MasculinoRESUMO
Among tarsiers, nocturnal, obligatory faunivorous primates inhabiting islands of South-East Asia, the Philippine tarsier (Tarsius [= Carlito] syrichta) is one of the least studied. To date, activity patterns of this threatened species have not been the subject of any investigation. In the present study, we provide the first quantitative data on how captive male and female T. syrichta apportion their time for various activities in two social contexts: solitary and paired. We found that the sexes do not differ in activity budgets during the non-mating season, both spending most of their time scanning, resting, foraging and travelling. Comparison of activity budgets of the sexes between the mating and non-mating seasons revealed that although both tarsiers noticeably increased travelling time at the expense of time spent resting, the male changed his behaviour to a much greater extent than the female. We also report on fluctuations in the tarsiers' activities throughout a night and compare time budgets of T. syrichta with available data on the western and eastern species of tarsiers. The results extend the current knowledge of tarsier behaviour and may also assist in practical considerations for keeping this highly sensitive, difficult-to-breed species in captivity.
Assuntos
Atividade Motora , Comportamento Social , Tarsiidae/fisiologia , Animais , Animais de Zoológico/fisiologia , Espécies em Perigo de Extinção , Feminino , Masculino , Filipinas , Estações do AnoRESUMO
Gd(3+) is increasingly used in EPR spectroscopy due to its increased intracellular stability and signal-to-noise ratios. Here we present the incorporation of Gd(3+)-DOTA into internal positions in DNA. Distance measurements via pulsed Electron Paramagnetic Resonance (EPR) spectroscopy in vitro and in cellula proved enhanced stability and efficiency compared to nitroxide labels.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Gadolínio/química , Compostos Heterocíclicos/química , Oligodesoxirribonucleotídeos/síntese química , Compostos Organometálicos/química , Animais , Química Click , Oligodesoxirribonucleotídeos/efeitos adversos , Oócitos/metabolismo , Marcadores de Spin , Membrana Vitelina/efeitos dos fármacos , Xenopus laevisRESUMO
Herein, we describe the synthesis and spectroscopic properties of five novel pyrrolodeoxycytidine analogs, and the related 5-(1-pyrenylethynyl)-2'-deoxycytidine analog; as well as fluorescence characterization of 5-(p-methoxyphenylethynyl)-2'-deoxyuridine. Within this series of compounds, rigidification of the structure from 6-phenylpyrrolodeoxycytidine to 5,6-benzopyrroldeoxycytidine made remarkable improvement of the fluorescence quantum yield (Φ ~1, EtOH) and substantially increased the Stokes shift. Exchange of the phenyl group of 6-phenylpyrrolodeoxycytidine for other heterocycles (benzofuryl or indolyl) produced an increase in the extinction coefficient at the excitation wavelength while preserving high quantum yields. The steady-state fluorescence response to the environment was determined by sensitivity of Stokes shift to solvent polarity. The effect of solvent polarity on fluorescence emission intensity was concurrently examined and showed that 5,6-benzopyrrolodeoxycytidine is highly sensitive to the presence of water. On the other hand, the previously synthesized 5-(p-methoxyphenylethynyl)-2'-deoxyuridine was found to be sensitive to solvent viscosity indicating molecular rotor behavior.
Assuntos
Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/síntese química , Fluorescência , Estrutura Molecular , Solventes , ViscosidadeRESUMO
EET grown ohm: Excess electron transfer (EET) was observed within a DNA duplex containing π-stacked phenothiazine as an electron donor, phenanthrenes as electron carriers and 5-bromouracil as an electron trap. Increasing the number of phenanthrenyl base pairs increased EET efficiency.
Assuntos
DNA/genética , Transporte de Elétrons/genética , Fenantrenos/química , Fenotiazinas/química , Pareamento de BasesRESUMO
Multiple 2-pyrenyl-C-nucleosides were incorporated into the center of a DNA duplex resulting in stable pyrene self-recognition and excimer formation. This helical pyrene array may find use in DNA-mediated charge transfer and in the creation of DNA-based sensors.
Assuntos
DNA/química , Pirenos/química , Modelos Moleculares , Conformação de Ácido Nucleico , Espectrometria de Fluorescência , TermodinâmicaRESUMO
Pyrene-deoxynucleoside triphosphates (dPTPs), varying by the positioning of the aromatic system, were synthesized. Their ability to function as substrates for the Klenow fragment of Escherichia coli DNA polymerase I and the TdT polymerase was assessed. The dPTPs are all equally well tolerated by the Klenow fragment, and lead to elongation of up to 5 extra nucleotides of a ssDNA primer in a TdT-mediated reaction. The tailing efficiency of the dPTPs compares favorably to other less drastically modified dNTPs.
Assuntos
DNA Polimerase I/metabolismo , Nucleotídeos/metabolismo , Pirenos/química , DNA Nucleotidilexotransferase/metabolismo , Primers do DNA/metabolismo , Nucleotídeos/química , Especificidade por SubstratoRESUMO
Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.
Assuntos
MicroRNAs/antagonistas & inibidores , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismo , Aminoácidos/química , Linhagem Celular , Endocitose , Endossomos , Humanos , Ácidos Nucleicos Peptídicos/análise , Compostos de Sulfidrila/química , Regulação para CimaRESUMO
The complementary Watson-Crick base-pairs, A:T and G:C, have long been recognized as pivotal to both the stability of the DNA double helix and replication/transcription. Recently, the replacement of the Watson-Crick base-pairs with other molecular entities has received considerable attention. In this tutorial review we highlight different approaches used to replace natural base-pairs and equip them with novel function. We also discuss the advantages that non-natural base-pairs convey with respect to practical applications.
Assuntos
Pareamento de Bases , DNA/química , DNA/genética , Ligação de Hidrogênio , Interações Hidrofóbicas e HidrofílicasRESUMO
Endogenous 5-methylcytosine ((Me)C) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational 'hotspots' for smoking induced lung cancer. (Me)C enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5'-CCCGGCACCC GC[(15)N(3),(13)C(1)-G]TCCGCG-3', + strand) were prepared containing [(15)N(3), (13)C(1)]-guanine opposite unsubstituted cytosine, (Me)C, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2'-deoxynucleosides, N(2)-BPDE-dG adducts formed at the [(15)N(3), (13)C(1)]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N(2)-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N(2) position of guanine.
Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/análogos & derivados , Citosina/análogos & derivados , Adutos de DNA/química , Desoxiguanosina/análogos & derivados , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/química , Pareamento de Bases , Cromatografia Líquida de Alta Pressão , Desoxiguanosina/química , Genes p53 , Guanina/química , Marcação por Isótopo , Modelos Moleculares , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Overexpression of the aspartyl protease cathepsin D is associated with certain cancers and Alzheimer's disease; thus, it is a potentially useful imaging biomarker for disease. A dual fluorescence/MRI probe for the potential detection of localized cathepsin D activity has been synthesized. The probe design includes both MRI and optical reporter groups connected to a cell penetrating peptide by a cathepsin D cleavable sequence. This design results in the selective intracellular deposition (determined fluorimetrically) of the MRI and optical reporter groups in the presence of overexpressed cathepsin D. The probe also provided clearly detectable in vitro MRI contrast by the mechanism of paramagnetic chemical exchange effects (OPARACHEE).
Assuntos
Catepsina D/química , Corantes Fluorescentes/química , Ácidos Carboxílicos/química , Células Cultivadas , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Imageamento por Ressonância Magnética , Estrutura MolecularRESUMO
Peptide nucleic acids (PNA) containing meta-substituted 6-phenylpyrrolocytosine (PhpC), [mono-m-(aminoethoxy)phenyl]pyrrolocytosine (mmePhpC), [mono-m-(aminopropoxy)phenyl]pyrrolocytosine (mmpPhpC), and [mono-m-(guanidinoethoxy)phenyl]pyrrolocytosine (mmguaPhpC), have been synthesized. Meta-substituted PhpCs have been hybridized with overall higher binding affinity toward DNA and RNA than previously synthesized moePhpC or newly synthesized mopPhpC. The guanidinium-containing nucleobase, mmguaPhpC, exhibited the highest increase in binding affinity toward RNA while fluorometrically responding on the state of hybridization.
Assuntos
Citosina/análogos & derivados , Citosina/síntese química , Corantes Fluorescentes/síntese química , Oligonucleotídeos Antissenso/síntese química , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/síntese química , Citosina/química , DNA/metabolismo , Corantes Fluorescentes/química , Estrutura Molecular , Oligonucleotídeos Antissenso/química , RNA/metabolismo , EstereoisomerismoRESUMO
Peptide nucleic acids (PNAs) efficiently hybridize with DNA and are promoted as versatile gene-targeting analytical tools and pharmaceuticals. However, PNAs have never been exploited as radiopharmaceuticals, and radiation-induced physicochemical modifications of PNA:DNA heteroduplexes have not been studied. Drug- and radiation-induced creation of covalent cross-links in DNA obstruct crucial cell survival processes such as transcription and replication and are thus considered genotoxic events with a high impact in anticancer therapies. Here we report that gamma-irradiation of complementary PNA:DNA heteroduplexes, wherein the PNA contains l-lysine, free amino, or N-methylmorpholinium N- and C-capping groups, results in the formation of irreversible interstrand cross-links (ICL). The number of detected ICL corresponds to the number of available amino functional groups on the PNA. The effect of DNA sequence on the formation of ICL was studied by modifying the terminal nucleotides of the DNA oligonucleotide to create deletions and overhangs. The involvement of abasic sites (ABS) on the DNA strand in the cross-linking reaction was confirmed by independent experiments with synthetic ABS-containing oligonucleotides. Molecular modeling and molecular dynamics (MD) simulations were applied to elucidate the conformation of the N- and C-capping groups of the PNA oligomer and their interactions with the proximal terminus of the DNA. Good agreement between experimental and modeling results was achieved. Modeling indicated that the presence of positively charged capping groups on the PNA increases the conformational flexibility of the PNA:DNA terminal base pairs and often leads to their melting. This disordered orientation of the duplex ends provides conditions for multiple encounters of the short (amino) and bulky (Lys) side chains with nucleobases and the DNA backbone up to the third base pair along the duplex stem. Dangling duplex ends offer favorable conditions for increased accessibility of the radiation-induced free radicals to terminal nucleotides and their damage. It is suggested that the ICL are produced by initial formation of Schiff base adducts between the PNA amino functions and the opposed DNA oxidation-damaged bases or abasic 2'-deoxyribose-derived aldehydic groups. The subsequent reduction by solvated electrons (e(-)(aq)) or other radiation-produced reducing species results in irreversible covalent interstrand cross-links. The simultaneous involvement of oxidizing, (*)OH, and reducing, e(-)(aq), radicals presents a case in which multiple ionization events along a gamma-particle path lead to DNA injuries that also encompass ICL as part of the multiply damaged sites (MDS). The obtained results may find applications in the development of a new generation of gene-targeted radiosensitizers based on PNA vectors.
Assuntos
DNA/química , Raios gama , Ácidos Nucleicos Heteroduplexes , Ácidos Nucleicos Peptídicos/química , Sequestradores de Radicais Livres/química , Concentração de Íons de Hidrogênio , Modelos MolecularesRESUMO
A new pyrrolocytosine derivative has been designed to selectively interact with guanine and has been evaluated in peptide nucleic acid where it imparts increased selective binding affinity for complementary oligonucleotides. The modified nucleobase also possesses an exceptionally high fluorescence quantum yield that is responsive to hybridization.
Assuntos
Citosina/análogos & derivados , Corantes Fluorescentes/química , Guanina/química , Hibridização de Ácido Nucleico/métodos , Oligonucleotídeos/química , Ácidos Nucleicos Peptídicos/química , Pirróis/química , Citosina/química , DNA Complementar/química , Ligação de Hidrogênio , Modelos Moleculares , Teoria Quântica , RNA/químicaRESUMO
A phenylpyrrolocytosine derivative possessing bis-(ortho-aminoethoxy) substitution has been designed, synthesized and incorporated into PNA resulting in oligomers that demonstrate increased binding affinity for DNA targets. The phenylpyrrolocytosine is an exceptionally fluorescent modified nucleobase possessing a high quantum yield while the fluorescence intensity displays useful environmental sensitivity.
Assuntos
Citosina/análogos & derivados , Corantes Fluorescentes/química , Ácidos Nucleicos Peptídicos/química , Pirróis/química , Citosina/síntese química , Citosina/química , DNA/química , Corantes Fluorescentes/síntese química , Ácidos Nucleicos Peptídicos/síntese química , Pirróis/síntese químicaRESUMO
A simple and practical synthesis of the benzyl, allyl, and 4-nitrobenzyl esters of N-[2-(Fmoc)aminoethyl]glycine is described starting from the known N-(2-aminoethyl)glycine. These esters are stored as stable hydrochloride salts and were used in the synthesis of peptide nucleic acid monomers possessing bis-N-Boc-protected nucleobase moieties on the exocyclic amino groups of ethyl cytosin-1-ylacetate, ethyl adenin-9-ylacetate and ethyl (O(6)-benzylguanin-9-yl)acetate. Upon ester hydrolysis, the corresponding nucleobase acetic acids were coupled to N-[2-(Fmoc)aminoethyl]glycine benzyl ester or to N-[2-(Fmoc)aminoethyl]glycine allyl ester in order to retain the O(6) benzyl ether protecting group of guanine. The Fmoc/bis-N-Boc-protected monomers were successfully used in the Fmoc-mediated solid-phase peptide synthesis of mixed sequence 10-mer PNA oligomers and are shown to be a viable alternative to the currently most widely used Fmoc/Bhoc-protected peptide nucleic acid monomers.
Assuntos
Ésteres/síntese química , Glicina/análogos & derivados , Ácidos Nucleicos Peptídicos/química , Ésteres/química , Glicina/química , Estrutura Molecular , EstereoisomerismoRESUMO
Tissue temperature is a fundamental physiological parameter that can provide insight into pathological processes. The purpose of this study was to develop and characterize a novel paramagnetic chemical exchange saturation transfer (CEST) agent suitable for in vivo temperature mapping at 9.4T. The CEST properties of the europium (Eu(3+)) complex of the DOTAM-Glycine (Gly)-Phenylalanine (Phe) ligand were studied in vitro at 9.4T as a function of temperature, pH, and agent concentration. The transfer of magnetization (CEST effect) from the bound water to bulk water pools was approximately 75% greater for Eu(3+)-DOTAM-Gly-Phe compared to Eu(3+)-DOTAM-Gly at physiologic temperature (38 degrees C) and pH (7.0 pH units) when using power level sufficiently low for in vivo imaging. Unlike Eu(3+)-DOTAM-Gly, whose CEST effect decreased with increasing temperature in the physiologic range, the CEST effect of Eu(3+)-DOTAM-Gly-Phe was optimal at body temperature. A strong linear dependence of the chemical shift of the bound water pool on temperature was observed (0.3 ppm/ degrees C), which was insensitive to pH and agent concentration. Temperature maps with SDs < 1 degrees C were acquired at 9.4T in phantoms containing: 1) phantom A, an aqueous solution of 10 mM Eu(3+)-DOTAM-Gly-Phe; 2) phantom B, 5% bovine serum albumin (BSA) with 15 mM Eu(3+)-DOTAM-Gly-Phe; and 3) phantom C, mouse brain tissue with 4 mM Eu(3+)-DOTAM-Gly-Phe. The temperature sensitivity combined with the high CEST effect observed at low concentration using low saturation power (B(1)) suggests this compound may be a good choice for in vivo temperature mapping at 9.4T.
Assuntos
Meios de Contraste/química , Imageamento por Ressonância Magnética , Acetamidas/química , Meios de Contraste/síntese química , Európio/química , Glicina/química , Compostos Heterocíclicos com 1 Anel/química , Concentração de Íons de Hidrogênio , Estrutura Molecular , Imagens de Fantasmas , Fenilalanina/química , TemperaturaRESUMO
A novel approach to the introduction of chirality into peptide nucleic acid (PNA) by replacement of the methylene carbonyl linker by an alpha-amino acid derived moiety is described. A monomer compatible with Fmoc-based oligomerization chemistry possessing an L-serine derived linker has been synthesized and incorporated into PNA oligomers. A single, central substitution in a hexathymine PNA strongly destabilized triple helix formation whereas a central substitution in a mixed sequence is much better tolerated. We have investigated the influence of this substitution on the selectivity for strand composition (DNA versus RNA complement) and strand orientation (antiparallel versus parallel) in the context of duplex formation. A PNA 11-mer with a single substitution demonstrates a preference for an antiparallel RNA complement, as judged by thermal denaturation analysis of the complexes.
Assuntos
Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/síntese química , Sequência de Bases , Estabilidade de Medicamentos , Conformação Molecular , Estrutura Molecular , Estereoisomerismo , TermodinâmicaRESUMO
Pyrimidine nucleobases bearing 5-phenylethynyl substitution represent compact and intrinsically fluorescent nucleobases. Such nucleobases are capable of selective recognition of a complementary base and may fluorimetrically report on hybridization events. Our past work has demonstrated that the fluorescence of 5-phenylethynyluracils is sensitive to substitution on the phenyl ring, however these are relatively weak fluorophores. We currently are pursuing the functionalization of the phenyl group of these modified nucleobases in order to further improve their fluorescence response, increase their aqueous solubility and stabilize hybrids formed with complementary nucleic acids. As an example of this work, we have synthesized the 5-((9H-fluoren-2-yl)ethynyl)uracil PNA monomer that will be incorporated into oligomers using Fmoc-based chemistry. Initial evaluation of the fluorescence of the 5-((9H-fluoren-2-yl)ethynyl)uracil derivative shows that the fluorescence intensity is approximately 50 times greater than a similar 5-phenylethynyluracil derivative when under identical conditions.