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1.
Phys Chem Chem Phys ; 22(14): 7381-7391, 2020 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-32211689

RESUMO

The thienoguanine nucleobase (thGb) is an isomorphic fluorescent analogue of guanine. In aqueous buffer at neutral pH, thGb exists as a mixture of two ground-state H1 and H3 keto-amino tautomers with distinct absorption and emission spectra and high quantum yield. In this work, we performed the first systematic photophysical characterization of thGb as a function of pH (2 to 12). Steady-state and time-resolved fluorescence spectroscopies, supplemented with theoretical calculations, enabled us to identify three additional thGb forms, resulting from pH-dependent ground-state and excited-state reactions. Moreover, a thorough analysis allowed us to retrieve their individual absorption and emission spectra as well as the equilibrium constants which govern their interconversion. From these data, the complete photoluminescence pathway of thGb in aqueous solution and its dependence as a function of pH was deduced. As the identified forms differ by their spectra and fluorescence lifetime, thGb could be used as a probe for sensing local pH changes under acidic conditions.


Assuntos
Corantes Fluorescentes/química , Guanina/análogos & derivados , Guanina/química , Concentração de Íons de Hidrogênio , Luminescência , Espectrometria de Fluorescência , Água/química
2.
BMC Bioinformatics ; 21(1): 40, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005130

RESUMO

BACKGROUND: Quadruplexes are specific structure motifs occurring, e.g., in telomeres and transcriptional regulatory regions. Recent discoveries confirmed their importance in biomedicine and led to an intensified examination of their properties. So far, the study of these motifs has focused mainly on the sequence and the tertiary structure, and concerned canonical structures only. Whereas, more and more non-canonical quadruplex motifs are being discovered. RESULTS: Here, we present ElTetrado, a software that identifies quadruplexes (composed of guanine- and other nucleobase-containing tetrads) in nucleic acid structures and classifies them according to the recently introduced ONZ taxonomy. The categorization is based on the secondary structure topology of quadruplexes and their component tetrads. It supports the analysis of canonical and non-canonical motifs. Besides the class recognition, ElTetrado prepares a dot-bracket and graphical representations of the secondary structure, which reflect the specificity of the quadruplex's structure topology. It is implemented as a freely available, standalone application, available at https://github.com/tzok/eltetrado. CONCLUSIONS: The proposed software tool allows to identify and classify tetrads and quadruplexes based on the topology of their secondary structures. It complements existing approaches focusing on the sequence and 3D structure.


Assuntos
Biologia Computacional/métodos , DNA/química , Quadruplex G , DNA/genética , Guanina/química , Guanina/metabolismo , Conformação de Ácido Nucleico , Software
3.
Chem Commun (Camb) ; 56(17): 2546-2549, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32040115

RESUMO

Oligonucleotides represent powerful DNA-recognition tools, but the formation of undesirable "self-duplexes" becomes more probable with increasing DNA affinity. Herein, we have developed a modified nucleobase with "self-avoiding" properties. Facile methylation of guanine yields a cationic N7-methylguanine, which suppresses the formation of self-duplexes whilst improving DNA affinity through electrostatic interaction.


Assuntos
DNA/química , Guanina/química , Sítios de Ligação , Cátions , Metilação , Ácidos Nucleicos Peptídicos/química , Eletricidade Estática
4.
Phys Chem Chem Phys ; 22(10): 5509-5522, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32104818

RESUMO

We compute at the anharmonic level the vibrational spectra of the Watson-Crick dimer formed by guanosine (G) and cytidine (C) in chloroform, together with those of G, C and the most populated GG dimer. The spectra for deuterated and partially deuterated GC are also computed. We use DFT calculations, with B3LYP and CAM-B3LYP as reference functionals. Solvent effects from chloroform are included via the Polarizable Continuum Model (PCM), and by performing tests on models including up two chloroform molecules. Both B3LYP and CAM-B3LYP calculations reproduce the shape of the experimental spectra well in the fingerprint region (1500-1700 cm-1) and in the N-H stretching region (2800-3600 cm-1), with B3LYP providing better quantitative agreement with experiments. According to our calculations, the N-H amido streching mode of G falls at ∼2900 cm-1, while the N-H amino of G and C falls at ∼3100 cm-1 when hydrogen-bonded, or ∼3500 cm-1 when free. Overtone and combination bands strongly contribute to the absorption band at ∼3300 cm-1. Inclusion of bulk solvent effects significantly increases the accuracy of the computed spectra, while solute-solvent interactions have a smaller, though still noticeable, effect. Some key aspects of the anharmonic treatment of strongly vibrationally coupled supermolecular systems and the related methodological issues are also discussed.


Assuntos
Clorofórmio/química , Citosina/química , Guanina/química , Vibração , Química Computacional
5.
J Phys Chem Lett ; 11(4): 1305-1309, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31967478

RESUMO

G-Quadruplexes are formed by guanine rich DNA/RNA sequences in the presence of metal ions, which occupy the central cavity of these four-stranded structures. We show that these metal ions have a significant effect on the photogeneration and the reactivity of guanine radicals. Transient absorption experiments on G-quadruplexes formed by association of four TGGGGT strands in the presence of K+ reveal that the quantum yield of one-photon ionization at 266 nm (8.1 × 10-3) is twice as high as that determined in the presence of Na+. Replacement of Na+ with K+ also suppresses one reaction path involving deprotonated radicals, (G-H2)• → (G-H1)• tautomerization. Such behavior shows that the underlying mechanisms are governed by dynamical processes, controlled by the mobility of metal ions, which is higher for Na+ than for K+. These findings may contribute to our understanding of the ultraviolet-induced DNA damage and optimize optoelectronic devices based on four-stranded structures, beyond DNA.


Assuntos
Quadruplex G , Guanina/química , Potássio/química , Dano ao DNA/efeitos da radiação , Radicais Livres/química , Radicais Livres/metabolismo , Íons/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fótons , Teoria Quântica , Raios Ultravioleta
6.
Bioelectrochemistry ; 132: 107416, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31981968

RESUMO

A hybrid bilayer lipid membrane (hBLM), constructed with a 1-hexadecanethiol self-assembled interior leaflet and a 1,2-dipalmitoyl-sn-glycero-3-cytidine nucleolipid exterior leaflet, was deposited at the surface of a gold (111) electrode. This system was used to investigate the molecular recognition reaction between the cytosine moieties of the lipid head group with guanine molecules in the bulk electrolyte solution. Electrochemical measurements and photon polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) were employed to characterize the system and determine the extent of the molecular recognition reaction. The capacitance of the hBLM-covered gold electrode was very low (~1 µF cm-2), therefore the charge density at the gold surface was small. Changing the electrode potential had a minimal effect on the complexation between the cytosine moieties and guanine molecules due to small changes in the static electric field across the membrane. This behavior favored the formation of the guanine-cytosine complex.


Assuntos
Citosina/química , Eletrodos , Ouro/química , Guanina/química , Bicamadas Lipídicas/química , Espectrofotometria Infravermelho
7.
Chem Commun (Camb) ; 56(12): 1839-1842, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31950946

RESUMO

Oxidative damage of guanine to 8-oxoguanine triggers a partial and variable loss of G-quadruplex/hemin DNAzyme activity and provides clues to the mechanistic origins of DNAzyme deactivation, which originates from an interplay between decreased G-quadruplex stability, lower hemin affinity and a modification of the nature of hemin binding sites.


Assuntos
DNA Catalítico/metabolismo , DNA Catalítico/química , Quadruplex G , Guanina/química , Guanina/metabolismo , Estrutura Molecular , Oxirredução
8.
Phys Chem Chem Phys ; 22(5): 2999-3007, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31957771

RESUMO

Infrared multiple photon dissociation (IRMPD) spectroscopy has been used to probe the structures of the three protonated base-pair mismatches containing 9-ethylguanine (9eG) in the gas phase. Computational chemistry has been used to determine the relative energies and compute the infrared spectra of these complexes. By comparing the IRMPD spectra with the computed spectra, in all cases, it was possible to deduce that what was observed experimentally were the lowest energy computed structures. The protonated complex between 9eG and 1-methylthymine (1mT) is protonated at N7 of 9eG-the most basic site of all three bases in this study-and bound in a Hoogsteen type structure with two hydrogen bonds. The experimental IRMPD spectrum for the protonated complex between 9eG and 9-methyladenine (9mA) is described as arising from a combination of the two lowest energy structures, both which are protonated at N1 of adenine and each containing two hydrogen bonds with 9eG being the acceptor of both. The protonated dimer of 9eG is protonated at N7 with an N7-H+-N7 ionic hydrogen bond. It also contains an interaction between a C-H of protonated guanine and the O6 carbonyl of neutral guanine which is manifested in a slight red shift of that carbonyl stretch. The protonated 9eG/9mA structures have been previously identified by X-ray crystallography in RNA and are contained within the protein database.


Assuntos
Gases/química , Guanina/análogos & derivados , Espectrofotometria Infravermelho , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Pareamento Incorreto de Bases , Cristalografia por Raios X , Guanina/química , Guanina/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Fótons , Timina/análogos & derivados , Timina/química , Timina/metabolismo
9.
Chemistry ; 26(3): 568, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31696988

RESUMO

Invited for the cover of this issue is the group of Yingfu Li at McMaster University. The image depicts a molecular switch for ultra-specific detection of DNA utilizing a guanine-quadruplex (up-right structure) resistant to denaturation by urea (ball-and-stick structures). Read the full text of the article at 10.1002/chem.201903536.


Assuntos
DNA/genética , Guanina/química , Lítio/química , Polimorfismo de Nucleotídeo Único/genética , DNA/química , Guanina/análise
10.
Biomed Chromatogr ; 34(2): e4750, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31743472

RESUMO

O6 -benzylguanine (O6 BG) is an inhibitor of O6 -alkylguanine-DNA alkyltransferase (AGT). It binds to AGT by transferring its benzyl moiety to the cysteine residue at the active site of the enzyme. O6 BG synergizes the cytotoxic effects of alkylating agents by halting AGT-mediated DNA repair. O6 -benzyl-8-oxoguanine (8-oxo-O6 BG) is a metabolite of O6 BG, which is an equally potent inhibitor of AGT. In this work, we report the development and validation of an LC-MS/MS method for simultaneous determination of O6 BG and 8-oxo-O6 BG in human plasma. O6 BG and 8-oxo-O6 BG along with the analog internal standard, pCl-O6 BG, were extracted from alkalinized human plasma by liquid-liquid extraction using ethyl acetate, dried under nitrogen and reconstituted in the mobile phase. Reverse-phase chromatographic separation was achieved using isocratic elution with a mobile phase containing 80% acetonitrile and 0.05% formic acid in water at a flow rate of 0.600 ml/min. Quantification was performed using multiple-reaction-monitoring mode with positive ion-spray ionization. The linear calibration ranges of the method for O6 BG and 8-oxo-O6 BG were 1.25-250 ng/ml and 5.00-1.00 × 103 ng/ml, respectively, with acceptable assay accuracy, precision, recovery and matrix factor. This method was applied to the measurement of O6 BG and 8-oxo-O6 BG in patient plasma samples from a prior phase I clinical trial.


Assuntos
Cromatografia Líquida/métodos , Guanina/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Guanina/sangue , Guanina/química , Guanina/farmacocinética , Humanos , Limite de Detecção , Modelos Lineares , Extração Líquido-Líquido , Reprodutibilidade dos Testes
11.
Phys Chem Chem Phys ; 22(2): 642-646, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31822872

RESUMO

Redox equilibrium between the low potential aniline radical cation and the guanine in the GC base pair of duplex DNA has been established using pulse radiolysis. We relate the measurement of a radical one-electron reduction potential, E0', of 1.01 ± 0.03 V to the perturbation of the GC base pair to accommodate the neutral guanyl radical in an energetically more stable 'slipped' structure. The formation of the 'slipped' structure is exothermic by -11.4 kcal mol-1 as calculated by DFT, which is inline with our experimental results.


Assuntos
Citosina/química , DNA/química , Elétrons , Guanina/química , Pareamento de Bases , Oxirredução
12.
Biosens Bioelectron ; 147: 111735, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31634803

RESUMO

Design of suitable nanocomposites with tailored structures was significant in the fabrication of effective and reliable electrochemical sensors. Herein, the copper-nickel@nitrogen, boron-doped reduced graphene oxide (Cu-Ni@N,B-rGO) was successfully synthesized, which exhibited superior electrocatalytic performance towards guanine (G) and adenine (A) oxidation. The Cu-Ni NPs were sequentially decorated on N,B-rGO substrate via an environmentally friendly reduction strategy, which utilized glucose as reducer and stabilizing agent. The nanocomposites with large specific surface area, remarkable conductivity and high catalytic activity showed prominent synergistic effect owning to the uniform dispersion of Cu-Ni NPs on the surface of N,B-rGO. When applied to analysis of G and A using DPV, the wide linear ranges of 1.0-160.0 µM and 1.0-120.0 µM with the determination limits of 0.118 µM and 0.134 µM were obtained, respectively. The sensor was successfully applied to the detection of G and A in calf-thymus DNA with G/A ratio of 0.80. The facile preparation process and attractive sensing properties of the Cu-Ni@N,B-rGO nanocomposites made it a promising candidate for the development of advanced electrochemical sensor.


Assuntos
Adenina/isolamento & purificação , Técnicas Biossensoriais , Técnicas Eletroquímicas , Guanina/isolamento & purificação , Adenina/química , Animais , Catálise , Bovinos , Cobre/química , DNA/química , DNA/genética , Glucose/química , Grafite/química , Guanina/química , Limite de Detecção , Nanosferas/química , Níquel/química , Oxirredução
13.
Phys Chem Chem Phys ; 22(2): 838-853, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31840715

RESUMO

The five fundamental units of the genetic code: uracil (U), thymine (T), cytosine (C), adenine (A) and guanine (G) are known for extremely low vapor pressure and low thermal stability at elevated temperatures. Therefore, application of conventional techniques for the determination of sublimation enthalpies and vapor pressures fails to provide accurate results. Recently, a Fast Scanning Calorimetry method (FSC) for vapor pressure determination was developed for investigation of extremely low volatile, as well as for thermally unstable molecular and ionic molecules. This success has encouraged application of the FSC method for determination of vapor pressures and sublimation enthalpies of the five nucleobases, where available literature data are in disarray. The thermodynamic data of the nucleobases available in the literature were collected, evaluated, and combined with our experimental results to reconcile available experimental data. The set of evaluated thermochemical data on the five nucleobases was recommended as the benchmark properties for these thermally labile compounds.


Assuntos
Adenina/química , Calorimetria , Citosina/química , Guanina/química , Termodinâmica , Timina/química , Uracila/química , Pressão , Volatilização
14.
Genes (Basel) ; 11(1)2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861576

RESUMO

The FANCJ helicase unfolds G-quadruplexes (G4s) in human cells to support DNA replication. This action is coupled to the recruitment of REV1 polymerase to synthesize DNA across from a guanine template. The precise mechanisms of these reactions remain unclear. While FANCJ binds to G4s with an AKKQ motif, it is not known whether this site recognizes damaged G4 structures. FANCJ also has a PIP-like (PCNA Interacting Protein) region that may recruit REV1 to G4s either directly or through interactions mediated by PCNA protein. In this work, we measured the affinities of a FANCJ AKKQ peptide for G4s formed by (TTAGGG)4 and (GGGT)4 using fluorescence spectroscopy and biolayer interferometry (BLI). The effects of 8-oxoguanine (8oxoG) on these interactions were tested at different positions. BLI assays were then performed with a FANCJ PIP to examine its recruitment of REV1 and PCNA. FANCJ AKKQ bound tightly to a TTA loop and was sequestered away from the 8oxoG. Reducing the loop length between guanine tetrads increased the affinity of the peptide for 8oxoG4s. FANCJ PIP targeted both REV1 and PCNA but favored interactions with the REV1 polymerase. The impact of these results on the remodeling of damaged G4 DNA is discussed herein.


Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Guanina/análogos & derivados , Nucleotidiltransferases/genética , Antígeno Nuclear de Célula em Proliferação/genética , RNA Helicases/química , RNA Helicases/metabolismo , Motivos de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Dicroísmo Circular , Quadruplex G , Guanina/química , Humanos , Modelos Moleculares , Nucleotidiltransferases/química , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Domínios Proteicos , Espectrometria de Fluorescência
15.
J Phys Chem Lett ; 10(21): 6771-6779, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31609632

RESUMO

Strand hybridization is not only a fundamental molecular mechanism underlying the biological functions of nucleic acids but is also a key step in the design of efficient nanodevices. Despite recent efforts, the microscopic rules governing the hybridization mechanisms remain largely unknown. In this study, we exploit the energy landscape framework to assess how sequence-specificity modulates the hybridization mechanisms in DNA. We find that GG-tracts hybridize much more rapidly compared to GC-tracts, via either zippering or slithering pathways. For the hybridization of GG-tracts, both zippering and slithering mechanisms appear to be kinetically relevant. In contrast, for the GC-tracts, the zippering mechanism is dominant. Our work reveals that even for the relatively small systems considered, the energy landscapes feature multiple metastable states and kinetic traps, which is at odds with the conventional "all-or-nothing" model of DNA hybridization formulated on the basis of thermodynamic arguments alone. Interestingly, entropic effects are found to play an important role in determining the thermal stability of competing conformational ensembles and in determining the preferred hybridization pathways.


Assuntos
Oligonucleotídeos/química , Guanina/química , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligonucleotídeos/metabolismo , Termodinâmica
16.
Phys Chem Chem Phys ; 21(42): 23418-23424, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31624816

RESUMO

DNA-protein cross-links constitute bulky DNA lesions that interfere with the cellular machinery. Amongst these stable covalently tethered adducts, the efficient nucleophilic addition of the free amino group of lysines onto the guanine radical cation has been evidenced. In vitro addition of a trilysine peptide onto a guanine radical cation generated in a TGT oligonucleotide is so efficient that competitive addition of a water molecule, giving rise to 8-oxo-7,8-dihydroguanine, is not observed. This suggests a spatial proximity between guanine and lysine for the stabilization of the prereactive complex. We report all-atom microsecond scale molecular dynamics simulations that probe the structure and interactions of the trilysine peptide (KKK) with two oligonucleotides. Our simulations reveal a strong, electrostatically driven yet dynamic interaction, spanning several association modes. Furthermore, the presence of neighbouring cytosines has been identified as a factor favoring KKK binding. Relying on ab initio molecular dynamics on a model system constituted of guanine and methylammonium, we also corroborate a mechanistic pathway involving fast deprotonation of the guanine radical cation followed by hydrogen transfer from ammonium leaving as a result a nitrogen reactive species that can subsequently cross-link with guanine. Our study sheds new light on a ubiquitous mechanism for DNA-protein cross-links also stressing out possible sequence dependences.


Assuntos
Simulação de Dinâmica Molecular , Oligonucleotídeos/química , Oligopeptídeos/química , Sítios de Ligação , Guanina/química , Lisina/química , Teoria Quântica , Termodinâmica
17.
Phys Chem Chem Phys ; 21(41): 22857-22868, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31599896

RESUMO

Three low-energy isomers of 9-methylguanine, the amino-oxo (AO) form and two amino-hydroxy (AH1 and AH2) conformers, were trapped from the gas phase into low-temperature argon matrices. The AH1 and AH2 isomers, differing in the orientation of the OH group, were found to transform into each other upon excitation with near-IR light. The population of the AO form of the compound was not changed upon any near-IR irradiation of the matrix samples. Using monochromatic near-IR light, generated by a frequency-tunable laser source, it was possible to selectively induce the AH1 → AH2 or AH2 → AH1 conversion. Photoreversibility of this conformational transformation was then demonstrated. Exposure of matrix-isolated monomers of 9-methylguanine to broadband near-IR light also led to conformational conversions within the amino-hydroxy tautomeric form; the final stage of this process was always the same photostationary state independent of the initial ratio of AH1 and AH2 populations. Spontaneous conformational conversion, transforming the higher-energy AH2 form into the lower-energy AH1 isomer, was observed for matrix-isolated monomers of 9-methylguanine kept in the dark. The mechanism of this process must rely on quantum tunneling of the light hydrogen atom. Irradiation of matrix-isolated 9-methylguanine with UV laser light at λ = 288 or 285 nm led to a substantial consumption of the two AH forms, while the amount of AO isomer remained unchanged. On the other hand, a decrease in the population of the AO isomer occurred upon excitations at shorter wavelengths, λ = 280 or 275 nm. The spectral changes observed after UV-irradiation suggest the generation (and stabilization in the matrix) of a radical species, resulting from the photocleavage of the O-H or N1-H bonds, in the AH or AO isomer, respectively.


Assuntos
Argônio/química , Temperatura Baixa , Guanina/análogos & derivados , Raios Infravermelhos , Raios Ultravioleta , Guanina/química , Isomerismo , Conformação Molecular/efeitos da radiação
18.
Molecules ; 24(19)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31569643

RESUMO

Chronic inflammation is closely associated with cancer development. One possible mechanism for inflammation-induced carcinogenesis is DNA damage caused by reactive halogen species, such as hypochlorous acid, which is released by myeloperoxidase to kill pathogens. Hypochlorous acid can attack genomic DNA to produce 8-chloro-2'-deoxyguanosine (ClG) as a major lesion. It has been postulated that ClG promotes mutagenic replication using its syn conformer; yet, the structural basis for ClG-induced mutagenesis is unknown. We obtained crystal structures and kinetics data for nucleotide incorporation past a templating ClG using human DNA polymerase ß (polß) as a model enzyme for high-fidelity DNA polymerases. The structures showed that ClG formed base pairs with incoming dCTP and dGTP using its anti and syn conformers, respectively. Kinetic studies showed that polß incorporated dGTP only 15-fold less efficiently than dCTP, suggesting that replication across ClG is promutagenic. Two hydrogen bonds between syn-ClG and anti-dGTP and a water-mediated hydrogen bond appeared to facilitate mutagenic replication opposite the major halogenated guanine lesion. These results suggest that ClG in DNA promotes G to C transversion mutations by forming Hoogsteen base pairing between syn-ClG and anti-G during DNA synthesis.


Assuntos
Dano ao DNA/efeitos dos fármacos , DNA/química , Guanina/análogos & derivados , Mutagênicos/farmacologia , DNA Polimerase beta/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/química , Guanina/farmacologia , Halogenação , Humanos , Ligação de Hidrogênio , Cinética , Modelos Biológicos , Conformação Molecular , Mutagênicos/química
19.
J Chem Theory Comput ; 15(12): 6984-6991, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31665604

RESUMO

A double proton transfer reaction in a guanine-cytosine (GC) base pair has been proposed as a possible mechanism for rare tautomer (G*C*) formation and thus a source of spontaneous mutations. We analyze this system with free energy calculations based on extensive Quantum Mechanics/Molecular Mechanics simulations to properly consider the influence of the DNA biomolecular environment. We find that, although the G*C* rare tautomer is metastable in the gas phase, it is completely unstable in the conditions found in cells. Thus, our calculations show that a double proton reaction cannot be the source of spontaneous point mutations. We have also analyzed the intrabase H transfer reactions in guanine. Our results show that the DNA environment gives rise to a large free energy difference between the rare and canonical tautomers. These results show the key role of the DNA biological environment for the stability of the genetic code.


Assuntos
Pareamento de Bases , Citosina/química , DNA de Forma B/química , Guanina/química , Prótons , Teoria Quântica
20.
Mol Cell ; 76(5): 797-810.e10, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31606272

RESUMO

Protein silencing represents an essential tool in biomedical research. Targeted protein degradation (TPD) strategies exemplified by PROTACs are rapidly emerging as modalities in drug discovery. However, the scope of current TPD techniques is limited because many intracellular materials are not substrates of proteasomal clearance. Here, we described a novel targeted-clearance strategy (autophagy-targeting chimera [AUTAC]) that contains a degradation tag (guanine derivatives) and a warhead to provide target specificity. As expected from the substrate scope of autophagy, AUTAC degraded fragmented mitochondria as well as proteins. Mitochondria-targeted AUTAC accelerated both the removal of dysfunctional fragmented mitochondria and the biogenesis of functionally normal mitochondria in patient-derived fibroblast cells. Cytoprotective effects against acute mitochondrial injuries were also seen. Canonical autophagy is viewed as a nonselective bulk decomposition system, and none of the available autophagy-inducing agents exhibit useful cargo selectivity. With its target specificity, AUTAC provides a new modality for research on autophagy-based drugs.


Assuntos
Autofagia/fisiologia , Guanina/química , Proteólise/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Guanina/fisiologia , Humanos , Mitocôndrias/metabolismo , Engenharia de Proteínas/métodos , Proteínas Quinases/metabolismo , Estabilidade Proteica
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