RESUMEN
6-Thioguanine is an immunosuppressive drug, an analogue of guanine, applied to treat acute leukemia and inflammatory bowel disease. Excessive use of 6-thioguanine during clinical treatment may cause side effects. Moreover, providing a dose too low will be ineffective. Therefore, there is a critical need for a rapid, selective and routine approach to quantifying 6-thioguanine in body fluids to support a clinical application. A fully validated HPLC method has been developed to determine 6-thioguanine in whole blood samples using 5-bromouracil as an internal standard. 6-Thioguanine nucleotides were released from erythrocytes by perchloric acid, and then hydrolysed at 100 °C to the parent thiopurine, 6-thioguanine. The following validation parameters of the method were determined: specificity/selectivity, linearity range (479-17,118 ng/mL, R > 0.992), limits of detection (150 ng/mL) and quantification (479 ng/mL), accuracy (- 5.6 < Bias < 14.7), repeatability (CV 1.30-3.24%), intermediate precision (CV 4.19-5.78%), extraction recovery (79.1-103.6%) and carryover. Furthermore, the stability of the drug in whole blood samples under various storage conditions was investigated. The suggested method is suitable for determining 6-thioguanine in whole blood erythrocyte samples for drug level monitoring, thus correct dosing.
Asunto(s)
Líquidos Corporales , Tioguanina , Cromatografía Líquida de Alta Presión , Eritrocitos , BromouraciloRESUMEN
PURPOSE: To clarify the radiosensitization mechanism masking the Auger effect of the cells possessing brominated DNA, the electronic properties of DNA-related molecules containing Br were investigated by X-ray spectroscopy and specific heat measurement. MATERIALS AND METHODS: X-ray absorption near-edge structure (XANES) and X-ray photoemission spectroscopy (XPS) were used to measure the electronic properties of the nucleotides with and without Br. We determined the specific heat of 5-bromouracil crystals with thymine as a reference molecule at low temperatures of 3-48 K to calculate the microscopic state numbers. RESULTS: Obtained XANES and XPS spectra indicated that both the lowest unoccupied molecular orbital (LUMO) and the core-levels were not affected by the Br incorporation. The state numbers of 5-bromouracil calculated from the specific heats obtained around 25 K was about 1.5 times larger than that for thymine below 20 K, although the numbers were almost the same below 5 K. DISCUSSION: Our results suggest that the Br atom may not contribute substantially to the LUMO and core-level electronic states of the molecule, but rather to the microscopic states related to the excitation of lattice vibrations, which may be involved in valence electronic states.
Asunto(s)
Bromo , Timina , Bromouracilo , ADNRESUMEN
5-Halouracil, which is a DNA base analog in which the methyl group at the C5 position of thymine is replaced with a halogen atom, has been used in studies of DNA damage. In DNA strands, the uracil radical generated from 5-halouracil causes DNA damage via a hydrogen-abstraction reaction. We analyzed the photoreaction of 5-halouracil in various DNA structures and revealed that the reaction is DNA structure-dependent. In this review, we summarize the results of the analysis of the reactivity of 5-halouracil in various DNA local structures. Among the 5-halouracil molecules, 5-bromouracil has been used as a probe in the analysis of photoinduced electron transfer through DNA. The analysis of groove-binder/DNA and protein/DNA complexes using a 5-bromouracil-based electron transfer system is also described.
Asunto(s)
Bromouracilo , ADN , Bromouracilo/química , ADN/química , Timina/química , Uracilo/químicaRESUMEN
Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.
Asunto(s)
Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Microscopía Confocal/métodos , Proteínas Mitocondriales/metabolismo , Bromouracilo/análogos & derivados , Bromouracilo/química , Células Hep G2 , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas Mitocondriales/genética , ARN Mitocondrial/química , Imagen Individual de Molécula/métodos , Uridina/análogos & derivados , Uridina/químicaRESUMEN
Chlorpyrifos, Bromacil and Terbuthylazine are commonly used as insecticides and herbicides to control weeds and prevent non-desirable growth of algae, fungi and bacteria in many agricultural applications. Despite their highly negative effects on human health, environmental modeling of these pesticides in the vadose zone until they reach groundwater is still not being conducted on a regular basis. This work shows results obtained by version 5.08 of the Pesticide Root Zone Model (PRZM5) numerical model to simulate the fate and transport of Chlorpyrifos, Bromacil and Terbuthylazine between 2006 and 2018 inside the Buñol-Cheste aquifer in Spain. The model uses a whole set of parameters to solve a modified version of the mass transport equation considering the combined effect of advection, dispersion and reactive transport processes. The simulation process was designed for a set of twelve scenarios considering four application doses for each pesticide. Results show that the maximum concentration value for every scenario exceeds the current Spanish Maximum Concentration Limit (0.1 µg/L). Numerical simulations were able to reproduce concentration observations over time despite the limited amount of available data.
Asunto(s)
Cloropirifos , Agua Subterránea , Contaminantes Químicos del Agua , Bromouracilo/análogos & derivados , Humanos , España , Triazinas , Contaminantes Químicos del Agua/análisisRESUMEN
Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Bromouracilo/análogos & derivados , Clonación Molecular/métodos , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Trifluridina/metabolismo , Uridina/análogos & derivados , Esclerosis Amiotrófica Lateral/genética , Secuencia de Bases , Bromouracilo/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Pliegue de Proteína , Espectroscopía de Protones por Resonancia Magnética/métodos , Superóxido Dismutasa-1/química , Triptófano/metabolismo , Uridina/metabolismoRESUMEN
The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be observed in vitro by the incorporation of a 5-Bromouridine (BrU), which is subsequently visualized by fluorescent immunolabeling. Here, a combined protocol for the MRGs detection is detailed, consisting of BrU labeling and visualization of one of their bona fide protein components, Fas-activated serine-threonine kinase domain 2 (FASTKD2). Based on immunodetection, the half-life and kinetics of the MRGs under various experimental conditions can further be determined by chasing the BrU pulse with an excess of Uridine.
Asunto(s)
Bromouracilo/análogos & derivados , Inmunohistoquímica/métodos , Complejos Multiproteicos/metabolismo , ARN Mitocondrial/metabolismo , Ribonucleoproteínas/metabolismo , Uridina/análogos & derivados , Bromouracilo/metabolismo , ADN Mitocondrial/metabolismo , Semivida , Células HeLa , Humanos , Cinética , Complejos Multiproteicos/química , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleoproteínas/química , Transcripción Genética , Uridina/metabolismoRESUMEN
RNA turnover is an essential part of the gene expression pathway, and there are several experimental approaches for its determination. High-throughput measurement of global RNA turnover rates can provide valuable information about conditions or proteins that impact gene expression. Here, we present a protocol for mitochondrial RNA turnover analysis which involves metabolic labeling of RNA coupled with quantitative high-throughput fluorescent microscopy. This approach gives an excellent opportunity to discover new factors involved in mitochondrial gene regulation when combined with loss-of-function screening strategy.
Asunto(s)
Regulación de la Expresión Génica , Inmunohistoquímica/métodos , Mitocondrias/genética , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , Bromouracilo/análogos & derivados , Bromouracilo/química , Expresión Génica , Células HeLa , Humanos , Microscopía Fluorescente/métodos , Estabilidad del ARN , ARN Mitocondrial/química , ARN Interferente Pequeño/genética , Coloración y Etiquetado/métodos , Transcripción Genética , Transfección , Uridina/análogos & derivados , Uridina/químicaRESUMEN
Gene expression is determined by a balance between RNA synthesis and RNA degradation. To elucidate the underlying regulatory mechanisms and principles of this, simultaneous measurements of RNA synthesis and degradation are required. Here, we report the development of "Dyrec-seq," which uses 4-thiouridine and 5-bromouridine to simultaneously quantify RNA synthesis and degradation rates. Dyrec-seq enabled the quantification of RNA synthesis and degradation rates of 4702 genes in HeLa cells. Functional enrichment analysis showed that the RNA synthesis and degradation rates of genes are actually determined by the genes' biological functions. A comparison of theoretical and experimental analyses revealed that the amount of RNA is determined by the ratio of RNA synthesis to degradation rates, whereas the rapidity of responses to external stimuli is determined only by the degradation rate. This study emphasizes that not only RNA synthesis but also RNA degradation is important in shaping gene expression patterns.
Asunto(s)
ARN/metabolismo , Bromouracilo/análogos & derivados , Células HeLa , Humanos , ARN/biosíntesis , ARN/química , Análisis de Secuencia de ARN , Tiouridina , Uridina/análogos & derivadosRESUMEN
Identifying new binding forces between electron donor and acceptor entities is key to properly understanding molecular recognition and aggregation phenomena, which are of inmense importance to biology. For decades, the halogenation of DNA/RNA bases has been routinely carried out to solve solid state structures of nucleic acids (NA). However, the effects of this modification might be deeper than just a simple atom substitution since halogens are also known to undergo noncovalent binding (halogen bonding). Herein we show that halogenated NAs with either Br or I atoms are able to establish halogen bonds with properly disposed protein residues. An inspection of the Protein Data Bank (PDB) reveals several examples involving 5-iodo/5-bromouracil, 8-bromoadenine, and 5-iodocytosine bases that are consistent with the halogen bond geometry features. Computations reveal the favorable and moderately strong nature of this interaction, thus confirming the ability of halogenated bases to actively participate in protein-NA binding.
Asunto(s)
Halógenos/química , Ácidos Nucleicos/química , Proteínas/química , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Bromouracilo/química , Bromouracilo/metabolismo , Citosina/análogos & derivados , Citosina/química , Citosina/metabolismo , Bases de Datos de Proteínas , Halógenos/metabolismo , Transferasas Intramoleculares/química , Transferasas Intramoleculares/metabolismo , FN-kappa B/química , FN-kappa B/metabolismo , Ácidos Nucleicos/metabolismo , Proteínas/metabolismo , Electricidad Estática , TermodinámicaRESUMEN
In this study, we report experimental (Protein Data Bank (PDB) search) and theoretical (RI-MP2/def2-TZVP level of theory) evidence of the nature, stability, and directionality properties of intramolecular halogen bonding interactions (HaBs) between 5-bromo/5-iodoracil bases and backbone phosphate groups in nucleic acids (NAs). A PDB survey revealed relevant examples where intramolecular HaBs are undertaken and serve as a structural source of stability in RNA and DNA molecules. In order to develop suitable energy predictors, we started this investigation by calculating the interaction energy values and both the potential V(r) and kinetic G(r) energy densities (using Bader's "atoms in molecules" theory) of several halogen bond complexes involving 5-bromo/5-iodoracil molecules and biologically relevant electron donors. Once the energy predictors based on V(r)/G(r) energy densities were developed, we analyzed the HaBs observed in the biological examples retrieved from the PDB search in order to estimate the strength of the interaction. As far as our knowledge extends, intramolecular halogen bonds in NAs have not been previously quantified in the literature using this methodology and may be of great importance in understanding their structural properties as well as in the construction of molecular materials with DNA and other biological macromolecules.
Asunto(s)
Bromouracilo/química , ADN Cruciforme/química , ARN/química , Electricidad Estática , Uracilo/análogos & derivados , Bromo/química , Bromouracilo/metabolismo , ADN Cruciforme/metabolismo , Bases de Datos de Proteínas , Escherichia coli/química , Exodesoxirribonucleasa V/metabolismo , Humanos , Yodo/química , Modelos Químicos , Unión Proteica , ARN/metabolismo , Factor de Empalme U2AF/metabolismo , Termodinámica , Uracilo/química , Uracilo/metabolismoRESUMEN
Laser illuminated gold nanoparticles (AuNPs) efficiently absorb light and heat up the surrounding medium, leading to versatile applications ranging from plasmonic catalysis to cancer photothermal therapy. Therefore, an in-depth understanding of the thermal, optical, and electron induced reaction pathways is required. Here, the electrophilic DNA nucleobase analog 5-Bromouracil (BrU) has been used as a model compound to study its decomposition in the vicinity of AuNPs illuminated with intense ns laser pulses under various conditions. The plasmonic response of the AuNPs and the concentration of BrU and resulting photoproducts have been tracked by ultraviolet and visible (UV-Vis) spectroscopy as a function of the irradiation time. A kinetic model has been developed to determine the reaction rates of two parallel fragmentation pathways of BrU, and their dependency on laser fluence and adsorption on the AuNP have been evaluated. In addition, the size and the electric field enhancement of the decomposed AuNPs have been determined by atomic force microscopy and finite domain time difference calculations, respectively. A minor influence of the direct photoreaction and a strong effect of the heating of the AuNPs have been revealed. However, due to the size reduction of the irradiated AuNPs, a trade-off between laser fluence and plasmonic response of the AuNPs has been observed. Hence, the decomposition of the AuNPs might be limiting the achievable temperatures under irradiation with several laser pulses. These findings need to be considered for an efficient design of catalytic plasmonic systems.
Asunto(s)
Bromouracilo/química , Nanopartículas del Metal/química , Oro/química , Oro/efectos de la radiación , Cinética , Rayos Láser , Luz , Nanopartículas del Metal/efectos de la radiaciónRESUMEN
Two injectable reactive and sorption-active particle types were evaluated for their applicability in permeable reaction zones for in-situ removal of herbicides ("nanoremediation"). As model substances, atrazine and bromacil were used, two herbicides frequently occurring in groundwater. In order to provide recommendations for best use, particle performance was assessed regarding herbicide degradation and detoxification. For chemical reduction, Carbo-Iron® was studied, a composite material consisting of zerovalent iron and colloidal activated carbon. Carbo-Iron reduced bromacil with increased activity compared to nanoscale zerovalent iron (nZVI). The sole reaction product, 3-sec-butyl-6-methyluracil, showed 500-fold increase in half-maximal-effect concentration (EC50) towards the chlorophyte Scendesmus vacuolatus compared to the parent compound. The detoxification based on dehalogenation confirmed the dependency of the specific mode-of-action on the carbon-halide bond. For atrazine, neither nZVI nor Carbo-Iron showed significant degradation under the conditions applied. As novel subsurface treatment option, Trap-Ox® zeolite FeBEA35 was studied for generation of in-situ permeable oxidation barriers. Both adsorbed atrazine and bromacil underwent fast unselective oxidation. The transformation products of the Fenton-like reaction were identified, and oxidation pathways derived. For atrazine, a 300-fold increase in EC50 for S. vacuolatus was found over the duration of the reaction, and a loss of phytotoxicity to non-detectable levels for bromacil.
Asunto(s)
Atrazina/química , Bromouracilo/análogos & derivados , Carbono/química , Herbicidas/química , Hierro/química , Nanopartículas/química , Contaminantes Químicos del Agua/química , Zeolitas/química , Adsorción , Atrazina/toxicidad , Bromouracilo/química , Bromouracilo/toxicidad , Restauración y Remediación Ambiental , Estudios de Factibilidad , Agua Subterránea/química , Herbicidas/toxicidad , Oxidación-Reducción , Scenedesmus/crecimiento & desarrollo , Contaminantes Químicos del Agua/toxicidadRESUMEN
This study focuses on the effects of the bromine atom on the molecular structure parameters in the main tautomeric forms of 5-bromouracil (5BrU), and as well, its effect on hydration and on the Watson-Crick (WC) pairs as compared to uracil molecule. The influence of the bromine atom was studied in several environments. The hydration effect on the molecular structure and energies of the main tautomeric forms of 5BrU was analyzed by considering a variable number of water molecules in explicit form up to 30 to simulate the first and second hydration shells. The 'mutagenic' 2-hydroxy-4-oxo (U2) enol tautomer of 5BrU, but not of uracil, was absolutely favored over the keto form in clusters with more than 20 water molecules. For all calculations, B3LYP and M06-2X Methods were used. The effect of the bromine atom when it was inserted into the natural and reverse WC pairs uridine-adenosine was also determined, and counterpoise (CP) corrected interaction energies were calculated. The effect of the bromine atom was analyzed in several DNA:RNA hybrid microhelices. Different backbone and helical parameters were calculated and compared. The bromine atom destabilizes its base pair, with a remarkable increase in the rise parameter (Dz) corresponding to the microhelix, and to a slight increase in the diameter (d). Molecular docking calculations were also carried out with 5BrU for targeted proteins associated with diabetes, hepatocellular carcinoma and breast and lung cancers. The molecular docking analysis confirms that the 5BrU molecule may play an important role as a promising inhibitor against breast cancer.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Bromouracilo , ADN , Simulación del Acoplamiento Molecular , ARN , Bromo , Bromouracilo/química , HumanosRESUMEN
This work focusses on the production of hydrogen peroxide and in the removal of bromacil by the electro-Fenton process using two different electrochemical cells: mixed tank cell (MTC) and flow-through cell (FTC). Both cells use boron doped diamond (BDD) as anode and carbon felt as cathode to promote the formation of hydrogen peroxide. In the case of the MTC, two surface area ratios, Acathode/Aanode, have been used. Results show that the H2O2 produced by MTC and FTCPSC increases with the time until a stabilization state. For the FTCPSC, the average hydrogen peroxide concentration produced increases progressively with the current, while for MTC the maximum values are found in applying very low current densities. In addition, the FTCPSC provides higher concentrations of hydrogen peroxide for the same current density applied. Regarding the MTC, it can be stated that the higher the area of the cathode, the higher is the amount of H2O2 produced and the lower is the cell voltage (because of a more efficient current lines distribution). The initial oxidation of bromacil is very efficiently attained being rapidly depleted from wastewater. However, the higher production of hydrogen peroxide obtained by the FTCPSC cell does not reflect on a better performance of the electro-Fenton process. Thus, bromacil is better mineralized using the MTC cell with the lowest cathode area. This observation has been explained because larger concentrations of produced hydrogen peroxide seems to benefit the oxidation of intermediates and not the mineralization.
Asunto(s)
Bromouracilo/análogos & derivados , Diamante/química , Peróxido de Hidrógeno/síntesis química , Aguas Residuales/química , Contaminantes Químicos del Agua/análisis , Boro/química , Bromouracilo/análisis , Carbono/química , Conductividad Eléctrica , Hierro/química , Oxidación-ReducciónRESUMEN
We studied the adsorption ability and tolerance of the thermophilic filamentous cyanobacteria Letolyngbya 7M towards Paraquat and Bromacil. Adsorption isotherms at pHâ¯=â¯7.0 showed an adsorption capacity of 24.4â¯mg/g and 66.8â¯mg/g, respectively, and a good fit to the Langmuir model (R2â¯=â¯0.97 and 0.99, respectively). To evaluate the effect of both herbicides on photosynthetic pigments and viability of cyanobacteria, cell autoflorescence and esterase activity was determined using flow cytometry. Autofluorescence was less sensitive to changes in cell viability, as it was only slightly reduced at high Paraquat and Bromacil concentrations. Herbicide effect on esterase activity is dose-dependent. Bromacil did not cause a significant effect on either chlorophyll a content or cell viability. This study demonstrates the potential of Leptolyngbya 7M to remove Paraquat and Bromacil herbicides from aqueous solution under laboratory conditions.
Asunto(s)
Cianobacterias , Herbicidas , Adsorción , Bromouracilo/análogos & derivados , Clorofila A , ParaquatRESUMEN
Low-energy electrons (LEEs) of energies ≤30 eV are generated in large quantities by ionizing radiation. These electrons can damage DNA; particularly, they can induce the more detrimental clustered lesions in cells. This type of lesions, which are responsible for a large portion of the genotoxic stress generated by ionizing radiation, is described in the Introduction. The reactions initiated by the collisions of 0.5-30 eV electrons with oligonucleotides, duplex DNA, and DNA bound to chemotherapeutic platinum drugs are explained and reviewed in the subsequent sections. The experimental methods of LEE irradiation and DNA damage analysis are described with an emphasis on the detection of cluster lesions, which are considerably enhanced in DNA-Pt-drug complexes. Based on the energy dependence of damage yields and cross-sections, a mechanism responsible for the clustered lesions can be attributed to the capture of a single electron by the electron affinity of an excited state of a base, leading to the formation of transient anions at 6 and 10 eV. The initial capture is followed by electronic excitation of the base and dissociative attachment-at other DNA sites-of the electron reemitted from the temporary base anion. The mechanism is expected to be universal in the cellular environment and plays an important role in the formation of clustered lesions.
Asunto(s)
Antineoplásicos/química , ADN/efectos de la radiación , Electrones , Fármacos Sensibilizantes a Radiaciones/química , Bromouracilo/química , Carboplatino/química , Cisplatino/química , ADN/química , Roturas del ADN de Doble Cadena/efectos de la radiación , Células Eucariotas/química , Células Eucariotas/efectos de la radiación , Humanos , Oligonucleótidos/química , Oligonucleótidos/efectos de la radiación , Oxaliplatino/química , Plásmidos/química , Plásmidos/efectos de la radiación , Radiación IonizanteRESUMEN
The understanding of the 5-bromouracil (BrU) based photosensitization mechanism of DNA damage is of large interest due to the potential applications in photodynamic therapy. Photoinduced electron transfer (ET) in BrU labeled duplexes comprising the 5'-GBrU or 5'-ABrU sequence showed that a much lower reactivity was found for the 5'-GBrU pattern. Since the ionization potential of G is lower than that of A, this sequence selectivity has been dubbed a contrathermodynamic one. In the current work, we employ the Marcus and Marcus-Levich-Jortner theory of ET in order to shed light on the observed effect. By using a combination of Density Functional Theory (DFT) and solvation continuum models, we calculated the electronic couplings, reorganization energies, and thermodynamic stimuli for electron transfer which enabled the rates of forward and back ET to be estimated for the two considered sequences. The calculated rates show that the photoreaction could not be efficient if the ET process proceeded within the considered dimers. Only after introducing additional adenines between G and BrU, which accelerates the forward and slows down the back ET, is a significant amount of photodamage expected.
Asunto(s)
Bromouracilo/química , ADN/efectos de la radiación , Modelos Moleculares , Procesos Fotoquímicos , Adenina/química , Transporte de Electrón/efectos de la radiación , Electrones , Guanina/química , Cinética , Luz , Conformación de Ácido Nucleico , Relación Estructura-Actividad , TermodinámicaRESUMEN
Radiation therapy is a basic part of cancer treatment. To increase the DNA damage in carcinogenic cells and preserve healthy tissue at the same time, radiosensitizing molecules such as halogenated nucleobase analogs can be incorporated into the DNA during the cell reproduction cycle. In the present study 8.44 eV photon irradiation induced single strand breaks (SSB) in DNA sequences modified with the radiosensitizer 5-bromouracil (5BrU) and 8-bromoadenine (8BrA) are investigated. 5BrU was incorporated in the 13mer oligonucleotide flanked by different nucleobases. It was demonstrated that the highest SSB cross sections were reached, when cytosine and thymine were adjacent to 5BrU, whereas guanine as a neighboring nucleobase decreases the activity of 5BrU indicating that competing reaction mechanisms are active. This was further investigated with respect to the distance of guanine to 5BrU separated by an increasing number of adenine nucleotides. It was observed that the SSB cross sections were decreasing with an increasing number of adenine spacers between guanine and 5BrU until the SSB cross sections almost reached the level of a non-modified DNA sequence, which demonstrates the high sequence dependence of the sensitizing effect of 5BrU. 8BrA was incorporated in a 13mer oligonucleotide as well and the strand breaks were quantified upon 8.44 eV photon irradiation in direct comparison to a non-modified DNA sequence of the same composition. No clear enhancement of the SSB yield of the modified in comparison to the non-modified DNA sequence could be observed. Additionally, secondary electrons with a maximum energy of 3.6 eV were generated when using Si as a substrate giving rise to further DNA damage. A clear enhancement in the SSB yield can be ascertained, but to the same degree for both the non-modified DNA sequence and the DNA sequence modified with 8BrA.
Asunto(s)
Adenina/análogos & derivados , Bromouracilo , Daño del ADN/efectos de la radiación , Fármacos Sensibilizantes a Radiaciones , Adenina/química , Bromouracilo/química , Daño del ADN/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/química , Rayos Ultravioleta , VacioRESUMEN
Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. This review focuses on the methodology used to specifically measure RNA decay at a global level. By comparing and contrasting approaches and describing the experimental protocols in a modular manner, we intend to provide both experienced and new researchers to the field the ability to combine aspects of various protocols to fit the unique needs of biological questions not addressed by current methods.