RESUMO
A variety of proteins interact with DNA and RNA, including polymerases, histones, ribosomes, transcription factors, and repair enzymes. However, the transient non-covalent nature of these interactions poses challenges for analysis. Introducing a covalent bond between proteins and DNA via photochemical activation of a photosensitive functional group introduced onto nucleic acids offers a means to stabilize these often weak interactions without significantly altering the binding interface. Consequently, photoactivatable oligonucleotides are powerful tools for investigating nucleic acid-protein interactions involved in numerous biological and pathological processes. In this review, we provide a comprehensive overview of the chemical tools developed so far and the different strategies used for incorporating the most commonly used photoreactive reagents into oligonucleotide probes or nucleic acids. Furthermore, we illustrate their application with several examples including protein binding site mapping, identification of protein binding partners, and in cell studies.
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Hydrogen peroxide (H2O2) is responsible for numerous damages when overproduced, and its detection is crucial for a better understanding of H2O2-mediated signaling in physiological and pathological processes. For this purpose, various "off-on" small fluorescent probes relying on a boronate trigger have been prepared, and this design has also been involved in the development of H2O2-activated prodrugs or theranostic tools. However, this design suffers from slow kinetics, preventing activation by H2O2 with a short response time. Therefore, faster H2O2-reactive groups are awaited. To address this issue, we have successfully developed and characterized a prototypic borinic-based fluorescent probe containing a coumarin scaffold. We determined its in vitro kinetic constants toward H2O2-promoted oxidation. We measured 1.9 × 104 m-1â s-1 as a second-order rate constant, which is 10,000-fold faster than its well-established boronic counterpart (1.8 m-1â s-1). This improved reactivity was also effective in a cellular context, rendering borinic acids an advantageous trigger for H2O2-mediated release of effectors such as fluorescent moieties.
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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 , ParisRESUMO
The development of facile methods for conjugating relevant probes, ligands, or delivery agents onto oligonucleotides (ONs) is highly desirable both for fundamental studies in chemical biology and for improving the pharmacology of ONs in medicinal chemistry. Numerous efforts have been focused on the introduction of bioorthogonal groups onto phosphoramidite building blocks, allowing the controlled chemical synthesis of reactive ONs for postsynthetic modifications. Among these building blocks, alkyne, cyclooctynes, trans-cyclooctene, and norbornene have been proved to be compatible with automated solid-phase chemistry. Herein, we present the development of novel 2'-functionalized nucleoside phosphoramidite monomers comprising bioorthogonal methylcyclopropene or sydnone moieties and their introduction for the first time to ON solid-phase synthesis. Traceless ON postsynthetic modifications with reactive complementary probes were successfully achieved through either inverse electron-demand Diels-Alder (iEDDA) reactions or strain-promoted sydnone-alkyne cycloaddition (SPSAC). These results expand the set of bioorthogonal phosphoramidite building blocks to generate ONs for postsynthetic labeling.
Assuntos
Alcinos , Sidnonas , OligonucleotídeosRESUMO
Arylborinic acids represent new, efficient, and underexplored hydrogen peroxide-responsive triggers. In contrast to boronic acids, two concomitant oxidative rearrangements are involved in the complete oxidation of these species, which might represent a major limitation for an efficient effector (drug or fluorophore) release. Herein, a comprehensive study of H2 O2 -mediated unsymmetrical arylborinic acid oxidation to investigate the factors that could selectively guide their oxidative rearrangement is described. The o-CF3 substituent was found to be an excellent directing group allowing a complete regioselectivity on borinic acid models. This result was successfully applied to synthesizing new borinic acid-based fluorogenic probes, which exclusively release the fluorescent moiety upon H2 O2 treatment. These compounds maintained their superior kinetic properties compared to boronic acids, thus further enhancing the potential of arylborinic acids as valuable new H2 O2 -sensitive triggers.
Assuntos
Ácidos Borínicos , Peróxido de Hidrogênio , Oxirredução , Ácidos Borônicos , Estresse OxidativoRESUMO
Trehalose-based probes are useful tools that allow the detection of the mycomembrane of mycobacteria through the metabolic labeling approach. Trehalose analogues conjugated to fluorescent probes can be used, and other probes are functionalized with a bioorthogonal chemical reporter for a two-step labeling approach. The synthesis of such trehalose-based probes mainly relies on the desymmetrization of natural trehalose using a large number of regioselective protection-deprotection steps to differentiate the eight hydroxyl groups. Herein, in order to avoid these time-consuming steps, we reinvestigated our previously reported tandem protocol mediated by FeCl3·6H2O, with the aim of modifying the ratio of the products to allow the challenging desymmetrization of the C2-symmetrical disaccharide trehalose. We demonstrate the usefulness of this method in providing easy access to trehalose analogues with a bioorthogonal moiety or a fluorophore in C-2, and also present their use in a one-step and two-step labeling approach, either of which can be used to study the mycomembrane in live mycobacteria.
Assuntos
Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Cloretos/farmacologia , Corynebacterium/efeitos dos fármacos , Compostos Férricos/farmacologia , Trealose/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Cloretos/química , Compostos Férricos/química , Testes de Sensibilidade Microbiana , Trealose/síntese química , Trealose/químicaRESUMO
Histone lysine methylation is associated with essential biological functions like transcription activation or repression, depending on the position and the degree of methylation. This post-translational modification is introduced by protein lysine methyltransferases (KMTs) which catalyze the transfer of one to three methyl groups from the methyl donor S-adenosyl-L-methionine (AdoMet) to the amino group on the side chain of lysines. The regulation of protein lysine methylation plays a primary role not only in the basic functioning of normal cells but also in various pathologies and KMT deregulation is associated with diseases including cancer. These enzymes are therefore attractive targets for the development of new antitumor agents, and there is still a need for direct methodology to screen, identify, and characterize KMT inhibitors. We report here a simple and robust in vitro assay to quantify the enzymatic methylation of KMT by MALDI-TOF mass spectrometry. Following this protocol, we can monitor the methylation events over time on a peptide substrate. We detect in the same spectrum the modified and unmodified substrates, and the ratios of both signals are used to quantify the amount of methylated substrate. We first demonstrated the validity of the assay by determining inhibition parameters of two known inhibitors of the KMT SET7/9 ((R)-PFI-2 and sinefungin). Next, based on structural comparison with these inhibitors, we selected 42 compounds from a chemical library. We applied the MALDI-TOF assay to screen their activity as inhibitors of the KMT SET7/9. This study allowed us to determine inhibition constants as well as kinetic parameters of a series of SET7/9 inhibitors and to initiate a structure activity discussion with this family of compounds. This assay is versatile and can be easily adapted to other KMT substrates and enzymes as well as automatized.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adenosina/análogos & derivados , Adenosina/farmacologia , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação/efeitos dos fármacos , Pirrolidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Tetra-Hidroisoquinolinas/farmacologiaRESUMO
Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.
Assuntos
Ensaios Enzimáticos/métodos , Histona-Lisina N-Metiltransferase/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Ensaios Enzimáticos/economia , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Concentração Inibidora 50 , Cinética , Metilação , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia , Fatores de TempoRESUMO
Topoisomerase IB controls DNA topology by cleaving DNA transiently. This property is used by inhibitors, such as camptothecin, that stabilize, by inhibiting the religation step, the cleavage complex, in which the enzyme is covalently attached to the 3'-phosphate of the cleaved DNA strand. These drugs are used in clinics as antitumor agents. Because three-dimensional structural studies have shown that camptothecin derivatives act as base pair mimics and intercalate between two base pairs in the ternary DNA-topoisomerase-inhibitor complex, we hypothesized that base pairs mimics could act like campthotecin and inhibit the religation reaction after the formation of the topoisomerase I-DNA cleavage complex. We show here that three base pair mimics, nucleobases analogues of the aminophenyl-thiazole family, once targeted specifically to a DNA sequence were potent topoisomerase IB inhibitors. The targeting was achieved through covalent linkage to a sequence-specific DNA ligand, a triplex-forming oligonucleotide, and was necessary to position and keep the nucleobase analogue in the cleavage complex. In the absence of triplex formation, only a weak binding to the DNA and topoisomerase I-mediated DNA cleavage was observed. The three compounds were equally active once conjugated, implying that the intercalation of the nucleobase upon triplex formation is the essential feature for the inhibition activity.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA/farmacologia , Marcação de Genes , Mimetismo Molecular , Ácidos Nucleicos Heteroduplexes/farmacologia , Inibidores da Topoisomerase I/farmacologia , Pareamento de Bases/efeitos dos fármacos , Pareamento de Bases/genética , Sequência de Bases , DNA/síntese química , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Pegada de DNA/métodos , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/química , Desoxirribonuclease I/genética , Marcação de Genes/métodos , Mimetismo Molecular/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Ácidos Nucleicos Heteroduplexes/síntese química , Inibidores da Topoisomerase I/síntese químicaRESUMO
DNA methyltransferases (DNMTs) are responsible for DNA methylation, an epigenetic modification involved in gene regulation. Families of conjugates of procainamide, an inhibitor of DNMT1, were conceived and produced by rapid synthetic pathways. Six compounds resulted in potent inhibitors of the murine catalytic Dnmt3A/3L complex and of human DNMT1, at least 50 times greater than that of the parent compounds. The inhibitors showed selectivity for C5 DNA methyltransferases. The cytotoxicity of the inhibitors was validated on two tumour cell lines (DU145 and HCT116) and correlated with the DNMT inhibitory potency. The inhibition potency of procainamide conjugated to phthalimide through alkyl linkers depended on the length of the linker; the dodecane linker was the best.
Assuntos
Antineoplásicos/farmacologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Procainamida/análogos & derivados , Procainamida/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Identification of histone lysine methyltransferase (HKMT) substrates has recently benefited from chemical-biology-based strategies in which artificial S-adenosyl-L-methionine (SAM) cofactors are engineered to allow substrate labeling using either the wild-type target enzyme or designed mutants. Once labeled, substrates can be selectively functionalized with an affinity tag, using a bioorthogonal ligation reaction, to allow their recovery from cell extracts and subsequent identification. In this chapter, we describe steps on how to proceed to set up such an approach to characterize substrates of specific HKMTs of the SET domain superfamily, from the characterization of the HKMT able to accommodate a SAM surrogate containing a bioorthogonal moiety, to the proteomic analysis conducted on a cell extract. We focus in particular on the controls that are necessary to ensure reliable proteomic data analysis. The example of PR-Set7 on which we have implemented this approach is shown.
Assuntos
Metionina , S-Adenosilmetionina , Histona-Lisina N-Metiltransferase/química , Domínios PR-SET , Proteômica , S-Adenosilmetionina/químicaRESUMO
DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Desenvolvimento Embrionário/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Cristalografia por Raios X , DNA (Citosina-5-)-Metiltransferases/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for ß-turn stabilization of different analogs designed as mimics of the ß-turn structure found in tendamistat. The ß-turn conformation of linear ß-amino acid-containing peptides and triazole-cyclized analogs were compared to 'conventional' lactam- and disulfide-bridged hexapeptide analogs. Their 3D structures and their propensity to fold in ß-turns in solution, and for those not structured in solution in the presence of α-amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac-Ser-Trp-Arg-Tyr-NH(2) and both the amide bond-cyclized, c[Pro-Ser-Trp-Arg-Tyr-D-Ala] and the disulfide-bridged, Ac-c[Cys-Ser-Trp-Arg-Tyr-Cys]-NH(2) hexapeptides adopt dominantly in solution a ß-turn conformation closely related to the one observed in tendamistat. On the contrary, the ß-amino acid-containing peptides such as Ac-(R)-ß(3) -hSer-(S)-Trp-(S)-ß(3) -hArg-(S)-ß(3) -hTyr-NH(2) , and the triazole cyclic peptide, c[Lys-Ser-Trp-Arg-Tyr-ßtA]-NH(2) , both specifically designed to mimic this ß-turn, do not adopt stable structures in solution and do not show any characteristics of ß-turn conformation. However, these unstructured peptides specifically interact in the active site of α-amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α-amylase inhibitor. Thus, in contrast to amide-cyclized or disulfide-bridged hexapeptides, ß-amino acid-containing peptides and click-cyclized peptides may not be regarded as ß-turn stabilizers, but can be considered as potential ß-turn inducers.
Assuntos
Aminoácidos/química , Dissulfetos/química , Lactamas/química , Peptídeos/química , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Ciclização , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/genéticaRESUMO
Using an automated coupled colorimetric assay for the Escherichia coli cyclopropane fatty acid synthase (CFAS), we have screened an academic chemical library of 3040 compounds, to identify new inhibitors of this enzyme. We identified 8 compounds as potent inhibitors of this enzyme, with IC(50) ranging from 1 to 10 microM, in the presence of 750 microM S-adenosyl-l-methionine and 1 mg/mL phospholipids. We conducted kinetic analyses of the inhibition of the CFAS using dioctylamine and three inhibitors identified in this report: sinefungin, 1, a synthetic S-adenosyl-l-homocysteine analog, 2, and an indoloquinolizine derivative, 3. The inhibition patterns observed were interpreted assuming that the E. coli CFAS operated via an ordered Bi Bi mechanism with binding of S-adenosyl-l-methionine first. Dioctylamine was the most potent inhibitor with a competitive inhibition constant of 130 nM with respect to the phospholipids. Compound 2 bound to the two substrate-binding sites of the enzyme suggesting that it acted as a bisubstrate analog (apparent inhibition constant, K(I)=6 microM). Compound 2 was also found to completely inhibit cyclopropanation of the phospholipids in growing E. coli cells, at 150 microM. This molecule is thus the first inhibitor of a cyclopropane synthase that is active in vivo, contrary to sinefungin and other analogs that are only active on the isolated enzyme.
Assuntos
Inibidores Enzimáticos/isolamento & purificação , Escherichia coli/enzimologia , Metiltransferases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/análise , Adenosina/análogos & derivados , Adenosina/farmacologia , Antibacterianos/farmacologia , Catálise/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Ácidos Graxos/metabolismo , Metiltransferases/metabolismo , Modelos BiológicosRESUMO
Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/efeitos dos fármacos , DNA/metabolismo , Oligonucleotídeos/farmacologia , Antineoplásicos Fitogênicos/química , Sequência de Bases , Camptotecina/química , Células Cultivadas , DNA Topoisomerases Tipo I/metabolismo , Vaga-Lumes/enzimologia , Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Humanos , Luciferases/análise , Luciferases/genética , Dados de Sequência Molecular , Oligonucleotídeos/química , Células Tumorais CultivadasRESUMO
In this study, we report the first synthesis of an alkyne-based trehalose monomycolate probe containing a ß-hydroxylated fatty acid and an α-branched chain similar to those of the natural mycolic acid. We demonstrate its utility for the labeling of the mycomembrane of Corynebacteria as well as for the study of mycoloyltransferases.
Assuntos
Aciltransferases/análise , Membrana Celular/química , Corynebacterium/enzimologia , Corantes Fluorescentes/química , Ácidos Micólicos/química , Aciltransferases/metabolismo , Membrana Celular/metabolismo , Corynebacterium/citologia , Corantes Fluorescentes/síntese química , Estrutura Molecular , Ácidos Micólicos/síntese químicaRESUMO
Human topoisomerase II (topo II) is the cellular target for a number of widely used antitumor agents, such as etoposide (VP16). These agents 'poison' the enzyme and induce it to generate DNA breaks that are lethal to the cell. Topo II-targeted drugs show a limited sequence preference, triggering double-stranded breaks throughout the genome. Circumstantial evidence strongly suggests that some of these breaks induce chromosomal translocations that lead to specific types of leukaemia (called treatment-related or secondary leukaemia). Therefore, efforts are ongoing to decrease these secondary effects. An interesting option is to increase the sequence-specificity of topo II-targeted drugs by attaching them to triplex-forming oligonucleotides (TFO) that bind to DNA in a highly sequence-specific manner. Here five derivatives of VP16 were attached to TFOs. The active topo II poisons, once linked, induced cleavage 13-14 bp from the triplex end where the drug was attached. The use of triple-helical DNA structures offers an efficient strategy for targeting topo II-mediated cleavage to DNA specific sequences. Finally, drug-TFO conjugates are useful tools to investigate the mechanistic details of topo II poisoning.
Assuntos
Antineoplásicos Fitogênicos/química , DNA/química , Etoposídeo/análogos & derivados , Inibidores da Topoisomerase II , Antineoplásicos Fitogênicos/toxicidade , Dano ao DNA , Pegada de DNA , DNA Topoisomerases Tipo II/metabolismo , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Etoposídeo/toxicidade , Humanos , Oligodesoxirribonucleotídeos/químicaRESUMO
Surface-confined host-guest chemistry at the air/solid interface is used for trapping a functionalized 3D Zn-phthalocyanine complex into a 2D porous supramolecular template allowing the large area functionalization of an sp2-hybridized carbon-based substrate as evidenced by STM, resonance Raman spectroscopy, and water contact angle measurements.
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/químicaRESUMO
The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.