RESUMEN
Hydroxyurea (HU; hydroxycarbamide) is a chemotherapy medication used to treat various types of cancer and other diseases such as sickle cell anemia. HU inhibits DNA synthesis by targeting ribonucleotide reductase (RNR). Recent studies have suggested that HU also causes oxidative stress in living systems. In the present study, we investigated if HU could directly affect the activity and/or conformation of DNA. We measured in vitro gene expression in the presence of HU by adapting a cell-free luciferase assay. HU exhibited a bimodal effect on gene expression, where promotion or inhibition were observed at lower or higher concentrations (mM range), respectively. Using atomic force microscopy (AFM), the higher-order structure of DNA was revealed to be partially-thick with kinked-branching structures after HU was added. An elongated coil conformation was observed by AFM in the absence of HU. Single DNA molecules in bulk aqueous solution under fluctuating Brownian motion were imaged by fluorescence microscopy (FM). Both spring and damping constants, mechanical properties of DNA, increased when HU was added. These experimental investigations indicate that HU directly interacts with DNA and provide new insights into how HU acts as a chemotherapeutic agent and targets other diseases.
Asunto(s)
ADN , Hidroxiurea , Microscopía de Fuerza Atómica , Conformación de Ácido Nucleico , Hidroxiurea/farmacología , ADN/metabolismo , ADN/química , Conformación de Ácido Nucleico/efectos de los fármacos , Humanos , Expresión Génica/efectos de los fármacosRESUMEN
The use of insecticides presents a risk to the environment because they can accumulate in the water, soil, air, and organisms, endangering human and animal health. It is therefore essential to investigate the effects of different groups of insecticides on individual biomacromolecules such as DNA. We studied fipronil, which belongs to the group of phenylpyrazole insecticides. The interaction of fipronil with calf thymus DNA was investigated using spectroscopic methods (absorption and fluorescence spectroscopy) complemented with infrared spectroscopy and viscosity measurement. Fluorescence emission spectroscopy showed the formation of a fipronil/DNA complex with a combined static and dynamic type of quenching. The binding constant was 4.15 × 103 L/mol. Viscosity changes were recorded to confirm/disconfirm the intercalation mode of interaction. A slight change in DNA viscosity in the presence of fipronil was observed. The phenylpyrazole insecticide does not cause significant conformational changes in DNA structure or increase of its chain length. We hypothesize that fipronil is incorporated into the minor groove of the DNA macromolecule via hydrogen interactions as indicated by FT-IR and CD measurements.
Asunto(s)
ADN , Insecticidas , Pirazoles , Pirazoles/química , Insecticidas/química , ADN/química , Animales , Viscosidad , Conformación de Ácido Nucleico/efectos de los fármacos , Bovinos , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Mitochondrial RNA editing in trypanosomes represents an attractive target for developing safer and more efficient drugs for treating infections with trypanosomes because this RNA editing pathway is not found in humans. Other workers have targeted several enzymes in this editing system, but not the RNA. Here, we target a universal domain of the RNA editing substrate, which is the U-helix formed between the oligo-U tail of the guide RNA and the target mRNA. We selected a part of the U-helix that is rich in G-U wobble base pairs as the target site for the virtual screening of 262,000 compounds. After chemoinformatic filtering of the top 5000 leads, we subjected 50 representative complexes to 50 nanoseconds of molecular dynamics simulations. We identified 15 compounds that retained stable interactions in the deep groove of the U-helix. The microscale thermophoresis binding experiments on these five compounds show low-micromolar to nanomolar binding affinities. The UV melting studies show an increase in the melting temperatures of the U-helix upon binding by each compound. These five compounds can serve as leads for drug development and as research tools to probe the role of the RNA structure in trypanosomal RNA editing.
Asunto(s)
Edición de ARN , Bibliotecas de Moléculas Pequeñas , Tripanocidas , Trypanosoma , Trypanosoma/efectos de los fármacos , Edición de ARN/efectos de los fármacos , ARN Protozoario/química , ARN Mitocondrial/química , Tripanocidas/química , Tripanocidas/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , Simulación de Dinámica Molecular , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca2+. Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physiological concentration of Ca2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs.
Asunto(s)
Calcio , ADN , Conformación de Ácido Nucleico , ARN Helicasas , Imagen Individual de Molécula , Proteínas no Estructurales Virales , Calcio/metabolismo , Calcio/farmacología , ADN/química , ADN/efectos de los fármacos , ADN/metabolismo , Magnesio/metabolismo , Magnesio/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Replicación Viral , Citosol/metabolismo , Hidrólisis/efectos de los fármacos , ARN Helicasas/efectos de los fármacos , ARN Helicasas/metabolismo , Proteínas no Estructurales Virales/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Electroforesis en Gel de Poliacrilamida , Relación Dosis-Respuesta a Droga , Transcripción GenéticaRESUMEN
Polypod-like structured nucleic acids (polypodnas), which are nanostructured DNAs, are useful for delivering cytosine-phosphate guanine oligodeoxynucleotides (CpG ODNs) to antigen-presenting cells (APCs) expressing Toll-like receptor 9 (TLR9) for immune stimulation. Lipid modification is another approach to deliver ODNs to lymph nodes, where TLR9-positive APCs are abundant, by binding to serum albumin. The combination of these two methods can be useful for delivering CpG ODNs to lymph nodes in vivo. In the present study, CpG1668, a phosphodiester-type CpG ODN, was modified with stearic acid (SA) to obtain SA-CpG1668. Tripodna, a polypodna with three pods, was selected as the nanostructured DNA. Tripodnas loaded with CpG1668 or SA-CpG1668 were obtained in high yields. SA-CpG1668/tripodna bound more efficiently to plasma proteins than CpG1668/tripodna and was more efficiently taken up by macrophage-like RAW264.7 cells than CpG1668/tripodna, whereas the levels of tumor necrosis factor-α released from the cells were comparable between the two. After subcutaneous injection into mice, SA-CpG1668/tripodna induced significantly higher interleukin (IL)-12 p40 production in the draining lymph nodes than SA-CpG1668 or CpG1668/tripodna, with reduced IL-6 levels in plasma. These results indicate that the combination of SA modification and nanostructurization is a useful approach for the targeted delivery of CpG ODNs to lymph nodes.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Nanoestructuras/química , Oligodesoxirribonucleótidos/farmacología , Adyuvantes Inmunológicos/farmacología , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , ADN/inmunología , Sistemas de Liberación de Medicamentos/métodos , Femenino , Inmunización/métodos , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Nanoestructuras/uso terapéutico , Conformación de Ácido Nucleico/efectos de los fármacos , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/metabolismo , Prueba de Estudio Conceptual , Células RAW 264.7 , Ácidos Esteáricos/químicaRESUMEN
The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA-PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings.
Asunto(s)
Antiinfecciosos/farmacología , Biopelículas/efectos de los fármacos , ADN Bacteriano/efectos de los fármacos , Desinfectantes/farmacología , Guanidinas/farmacología , Antiinfecciosos/síntesis química , Antiinfecciosos/química , ADN Bacteriano/química , Desinfectantes/química , Guanidinas/síntesis química , Guanidinas/química , Conformación de Ácido Nucleico/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Relación Estructura-ActividadRESUMEN
CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)-distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Estructuras R-Loop/genética , Sistemas CRISPR-Cas/fisiología , ADN/química , División del ADN/efectos de los fármacos , Desoxirribonucleasa I/metabolismo , Edición Génica , Magnesio/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico/efectos de los fármacos , ARN Guía de Kinetoplastida/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
DNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was applied to study how Anabaena sensory rhodopsin transducer (ASRT) facilitates loop formation of DNA as a possible mechanism for gene regulation. The ASRT-induced DNA looping was maximized at 50 mM of Na+, while Mg2+ also played an essential role in the loop formation.
Asunto(s)
Anabaena/fisiología , ADN/química , ADN/metabolismo , Conformación de Ácido Nucleico , Rodopsinas Sensoriales/metabolismo , Ciclización , ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Modelos Biológicos , Conformación de Ácido Nucleico/efectos de los fármacos , Cloruro de Sodio/farmacología , Análisis EspectralRESUMEN
RNAs have become a well-known target for chemotherapeutic agents in the recent years. The tails of most eukaryotic m-RNA are characterized by the presence of a long polyadenylate sequence which plays an important role in its growth and maturation. This lays emphasis on development of molecular probes that target the polyadenylate sequence. Cryptolepine (hereafter, CRP) is an indoloquinoline alkaloid well known for its anti-malarial activities. A series of spectroscopic experiments namely absorption studies, fluorimetric studies and circular dichroism studies show that cryptolepine binds with single-stranded polyriboadenylic acid (hereafter, ss-poly (rA)) with a binding constant of â¼5 × 103 M-1 at 25 °C. Moreover thermal denaturation experiments show that the bound form of polyriboadenylic acid shows a characteristic transition profile. Such a profile is indicative of the ability of cryptolepine to induce self-assembly in the polyriboadenylic acid sequence on binding to it. Such ability of CRP to modulate the structural conformation of poly (rA), which in turn may cause functional aspects of the RNA to change, may give us a chance to develop effective alkaloid based chemotherapeutic agents.
Asunto(s)
Alcaloides Indólicos/metabolismo , Poli A/metabolismo , Quinolinas/metabolismo , Polarización de Fluorescencia , Alcaloides Indólicos/química , Conformación de Ácido Nucleico/efectos de los fármacos , Transición de Fase , Poli A/química , Quinolinas/química , Espectrometría de Fluorescencia , Termodinámica , Temperatura de TransiciónRESUMEN
RNA is highly negatively charged and often acquires complex structures that require the presence of divalent cations. Subtle changes in conformation resulting from changes in sequence can affect the way ions associate with RNA. Riboswitches are RNA molecules that are involved in the control of gene expression in bacteria and are excellent systems for testing the effects of sequence variations on the conformation of RNA because they contain a highly conserved binding pocket but present sequence variability among different organisms. In this work, we have compared the aptamer domain of a proposed M-box riboswitch from Mycobacterium tuberculosis with the aptamer domain of a validated M-box riboswitch from Bacillus subtilis. We have in vitro transcribed and purified wild-type (WT) M-box riboswitches from M. tuberculosis and B. subtilis as well as a variety of mutated aptamers in which regions from one riboswitch have been replaced with regions from the other riboswitch. We have used ultraviolet unfolding experiments and circular dichroism to characterize the interactions of WT and related M-box riboswitches with divalent cations. Our results show that M-box from M. tuberculosis associates with Mg2+ and Sr2+ in a similar fashion while M-box from B. subtilis discriminates between these two ions and appears to associate better with Mg2+. Our overall results show that M-box from M. tuberculosis interacts differently with cations than M-box from B. subtilis and suggest conformational differences between these two riboswitches.
Asunto(s)
Cationes Bivalentes/metabolismo , Conformación de Ácido Nucleico/efectos de los fármacos , Riboswitch/genética , Aptámeros de Nucleótidos/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sitios de Unión/genética , Cationes Bivalentes/química , Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/genética , Ligandos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , ARN Bacteriano/química , Riboswitch/fisiología , Transcripción Genética/genéticaRESUMEN
The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While â¼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to â¼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.
Asunto(s)
Archaea/enzimología , Magnesio/metabolismo , ARN de Archaea/genética , Ribonucleasa P/genética , Conformación de Ácido Nucleico/efectos de los fármacos , Pyrococcus furiosus/enzimología , Pyrococcus furiosus/genética , Precursores del ARN/genética , ARN de Archaea/ultraestructura , ARN Catalítico , Ribonucleasa P/ultraestructuraRESUMEN
Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson-Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson-Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules.
Asunto(s)
Motivos de Nucleótidos/genética , ARN Ribosómico/ultraestructura , ARN/ultraestructura , Emparejamiento Base/genética , Cristalografía por Rayos X , Enlace de Hidrógeno/efectos de los fármacos , Conformación de Ácido Nucleico/efectos de los fármacos , ARN/efectos de los fármacos , ARN/genética , ARN Ribosómico/efectos de los fármacos , ARN Ribosómico/genética , Ricina/farmacologíaRESUMEN
A new self-cleaving ribozyme was obtained from in vitro selection, displaying site-specific cleavage activity under denaturing conditions, such as high temperatures up to 95 °C, and denaturing solvents (20 M formamide). Adding salt such as Mg2+ also inhibited its activity. The conserved ribozyme sequence was found in the genome of several extremophiles, suggesting its potential biological relevance. This study provides an example of a ribozyme working under exotic conditions, which may expand the application of ribozymes in non-biological environments.
Asunto(s)
ARN Catalítico/química , ARN Catalítico/metabolismo , Temperatura , Formamidas/farmacología , Cinética , Magnesio/farmacología , Modelos Moleculares , Conformación de Ácido Nucleico/efectos de los fármacos , Desnaturalización de Ácido Nucleico/efectos de los fármacosRESUMEN
The MYCN gene encodes the transcription factor N-Myc, a driver of neuroblastoma (NB). Targeting G-quadruplexes (G4s) with small molecules is attractive strategy to control the expression of undruggable proteins such as N-Myc. However, selective binders to G4s are challenging to identify due to the structural similarity of many G4s. Here, we report the discovery of a small molecule ligand (4) that targets the noncanonical, hairpin containing G4 structure found in the MYCN gene using small molecule microarrays (SMMs). Unlike many G4 binders, the compound was found to bind to a pocket at the base of the hairpin region of the MYCN G4. This compound stabilizes the G4 and has affinity of 3.5 ± 1.6 µM. Moreover, an improved analog, MY-8, suppressed levels of both MYCN and MYCNOS (a lncRNA embedded within the MYCN gene) in NBEB neuroblastoma cells. This work indicates that the approach of targeting complex, hybrid G4 structures that exist throughout the human genome may be an applicable strategy to achieve selectivity for targeting disease-relevant genes including protein coding (MYCN) as well as non-coding (MYCNOS) gene products.
Asunto(s)
ADN/química , G-Cuádruplex , Proteína Proto-Oncogénica N-Myc/genética , Conformación de Ácido Nucleico/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Bases , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Dicroismo Circular , ADN/genética , ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Estructura Molecular , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-ActividadRESUMEN
Recently, the 1H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing the characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess the application potential of 1H and 19F-detected in-cell NMR spectroscopy to profile drugs/ligands targeting DNA G-quadruplexes, arguably the most studied class of anti-cancer drugs targeting nucleic acids. We show that the extension of the original in-cell NMR approach is not straightforward. The severe signal broadening and overlap of 1H in-cell NMR spectra of polymorphic G-quadruplexes and their complexes complicate their quantitative interpretation. Nevertheless, the 1H in-cell NMR can be used to identify drugs that, despite strong interaction in vitro, lose their ability to bind G-quadruplexes in the native environment. The in-cell NMR approach is adjusted to a recently developed 3,5-bis(trifluoromethyl)phenyl probe to monitor the intracellular interaction with ligands using 19F-detected in-cell NMR. The probe allows dissecting polymorphic mixture in terms of number and relative populations of individual G-quadruplex species, including ligand-bound and unbound forms in vitro and in cellulo. Despite the probe's discussed limitations, the 19F-detected in-cell NMR appears to be a promising strategy to profile G-quadruplex-ligand interactions in the complex environment of living cells.
Asunto(s)
ADN/efectos de los fármacos , G-Cuádruplex/efectos de los fármacos , Conformación de Ácido Nucleico/efectos de los fármacos , Preparaciones Farmacéuticas/química , Sitios de Unión/efectos de los fármacos , ADN/química , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , ProtonesRESUMEN
The involvement of G-quadruplex (G4) structures in nucleic acids in various molecular processes in cells such as replication, gene-pausing, the expression of crucial cancer-related genes and DNA damage repair is well known. The compounds targeting G4 usually bind directly to the G4 structure, but some ligands can also facilitate the G4 folding of unfolded G-rich sequences and stabilize them even without the presence of monovalent ions such as sodium or potassium. Interestingly, some G4-ligand complexes can show a clear induced CD signal, a feature which is indirect proof of the ligand interaction. Based on the dichroic spectral profile it is not only possible to confirm the presence of a G4 structure but also to determine its topology. In this study we examine the potential of the commercially available Rhodamine 6G (RhG) as a G4 ligand. RhG tends to convert antiparallel G4 structures to parallel forms in a manner similar to that of Thiazole Orange. Our results confirm the very high selectivity of this ligand to the G4 structure. Moreover, the parallel topology of G4 can be verified unambiguously based on the specific induced CD profile of the G4-RhG complex. This feature has been verified on more than 50 different DNA sequences forming various non-canonical structural motifs.
Asunto(s)
G-Cuádruplex/efectos de los fármacos , Conformación de Ácido Nucleico/efectos de los fármacos , Rodaminas/farmacología , Benzotiazoles/química , ADN/química , Reparación del ADN/efectos de los fármacos , Ligandos , Ácidos Nucleicos/química , Quinolinas/química , Rodaminas/química , Rodaminas/metabolismoRESUMEN
Pseudoisocytosine (J), a neutral analogue of protonated cytosine, is currently the gold standard modified nucleobase in peptide nucleic acids (PNAs) for the formation of J·G-C triplets that are stable at physiological pH. This study shows that triple-helical recognition of RNA and DNA is significantly improved by using 2-aminopyridine (M) instead of J. The positively charged M forms 3-fold stronger M+·G-C triplets than J with uncompromised sequence selectivity. Replacement of six Js with Ms in a PNA 9-mer increased its binding affinity by â¼2 orders of magnitude. M-modified PNAs prefer binding double-stranded RNA over DNA and disfavor off-target binding to single-stranded nucleic acids. Taken together, the results show that M is a promising modified nucleobase that significantly improves triplex-forming PNAs and may provide breakthrough developments for therapeutic and biotechnology applications.
Asunto(s)
Aminopiridinas/química , Conformación de Ácido Nucleico/efectos de los fármacos , Ácidos Nucleicos de Péptidos/metabolismo , Aminopiridinas/metabolismo , Citosina/análogos & derivados , Citosina/química , ADN/química , ADN/metabolismo , ARN BicatenarioRESUMEN
Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC-nevirapine, and RTIC-efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA-tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.
Asunto(s)
Transcriptasa Inversa del VIH/química , VIH-1/efectos de los fármacos , ARN de Transferencia de Lisina/química , ARN Viral/química , Inhibidores de la Transcriptasa Inversa/química , Alquinos/química , Alquinos/farmacología , Benzoxazinas/química , Benzoxazinas/farmacología , Dominio Catalítico , Microscopía por Crioelectrón , Ciclopropanos/química , Ciclopropanos/farmacología , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , VIH-1/genética , VIH-1/metabolismo , Modelos Moleculares , Nevirapina/química , Nevirapina/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , ARN de Transferencia de Lisina/genética , ARN Viral/genética , Inhibidores de la Transcriptasa Inversa/farmacologíaRESUMEN
The use of a small molecule compound to reduce toxic repeat RNA transcripts or their translated aberrant proteins to target repeat-expanded RNA/DNA with a G4C2 motif is a promising strategy to treat C9orf72-linked disorders. In this study, the crystal structures of DNA and RNA-DNA hybrid duplexes with the -GGGCCG- region as a G4C2 repeat motif were solved. Unusual groove widening and sharper bending of the G4C2 DNA duplex A-DNA conformation with B-form characteristics inside was observed. The G4C2 RNA-DNA hybrid duplex adopts a more typical rigid A form structure. Detailed structural analysis revealed that the G4C2 repeat motif of the DNA duplex exhibits a hydration shell and greater flexibility and serves as a 'hot-spot' for binding of the anthracene-based nickel complex, NiII(Chro)2 (Chro = Chromomycin A3). In addition to the original GGCC recognition site, NiII(Chro)2 has extended specificity and binds the flanked G:C base pairs of the GGCC core, resulting in minor groove contraction and straightening of the DNA backbone. We have also shown that Chro-metal complexes inhibit neuronal toxicity and suppresses locomotor deficits in a Drosophila model of C9orf72-associated ALS. The approach represents a new direction for drug discovery against ALS and FTD diseases by targeting G4C2 repeat motif DNA.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Proteína C9orf72/genética , ADN de Forma A/ultraestructura , Demencia Frontotemporal/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Antracenos/química , Antracenos/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , ADN/efectos de los fármacos , ADN/ultraestructura , ADN de Forma A/efectos de los fármacos , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Humanos , Conformación de Ácido Nucleico/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
We demonstrated that a synthetic ligand NA, which selectively binds to a 5'-CAG-3'/5'-CAG-3' triad, induced repeat contractions during DNA polymerase-mediated primer extension through the CAG repeat template. A thorough capillary electrophoresis and sequencing analysis revealed that the d(CAG)20 template gave shortened nascent strands mainly containing 3-6 CTG units in the presence of NA.