DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in beverages

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA21) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH• radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADHmeasured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50nMof AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages.

Electrocatalytic evaluation of DNA damage by superoxide radical for antioxidant capacity assessment

The integrity of DNA purine bases was herein used to evaluate the antioxidant capacity. Unlike other DNA-based antioxidant sensors reported so far, the damaging agent chosen was the O 2 radical enzymatically generated by the xanthine/xanthine oxidase system. An adenine-rich oligonucleotide was adsorbed on carbon paste electrodes and subjected to radical damage in the presence/absence of several antioxidant compounds. As a result, partial damage on DNA was observed. A minor product of the radical oxidation was identified by cyclic voltammetry as a diimine adenine derivative also formed during the electrochemical oxidation of adenine/guanine bases. The protective efficiency of several antioxidant compounds was evaluated after electrochemical oxidation of the remaining unoxidized adenine bases, by measuring the electrocatalytic current of NADH mediated by the adsorbed catalyst species generated. A comparison between O 2 and OH radicals as a source of DNA lesions and the scavenging efficiency of various antioxidant compounds against both of them is discussed. Finally, the antioxidant capacity of beverages was evaluated and compared with the results obtained with an optical method.

BIiossensor Enzimático para avaliação da Capacidade Antioxidente

Os radicais livres formam-se naturalmente nos organismos vivos, pois a sua produção/geração está interligada com o processo de produção de energia (respiração), processos inflamatórios (fagocitose), regulação do crescimento celular, sinalização intercelular e síntese de substâncias biológicas relevantes. Estes também podem ser introduzidos por vias exógenas (poluição, radiação, tabaco, alimentação, etc). Os radicais livres têm capacidade de reagir com o material nucleico (ADN e ARN), proteínas e substâncias oxidáveis, causando danos oxidativos responsáveis pelo envelhecimento e originar doenças degenerativas, tais como, o cancro, arteriosclerose, artrite reumatoide, entre outras. De forma a combater os efeitos pejorativos provocados pelos radicais, os organismos vivos desenvolveram complexos sistemas de defesa antioxidante. Estes sistemas são constituídos por antioxidantes endógenos, produzidos pelos seres vivos, tais como enzimas ou por antioxidantes exógenos obtidos por via da alimentação (por exemplo o ácido ascórbico). Neste sentido, um antioxidante tem capacidade de eliminar ou reduzir a propagação da cadeia de geração de radicais livres. Neste trabalho foi desenvolvido um biossensor enzimático para a quantificação da capacidade antioxidante total de matrizes alimentares. A construção deste biossensor consistiu na eletroimobilização da adenina no elétrodo de pasta de carbono (EPC) ou na adsorção física da dA20 na superfície do EPC. O dano oxidativo foi induzido pelo radical hidroxilo gerado pela reação de Fenton. Nesta dissertação, foi estudada a capacidade de alguns antioxidantes em eliminar o efeito pejorativo dos radicais livres e combater a integridade das bases de adenina ou do dA20.Os antioxidantes estudados foram o ácido ascórbico e alguns ácidos fenólicos como o ácido hidroxibenzoico (ácido gálico) e ácidos hidroxicinâmicos (ácido cafeico e ácido cumárico). Estes antioxidantes têm a capacidade de neutralizar o radical hidroxilo e proteger a adenina/dA20 imobilizado na superfície do EPC. O comportamento da Lacase foi estudado na presença do ácido gálico e do ácido ascórbico. Os estudos eletroquímicos foram realizados através da voltametria de onda quadrada (VOQ), sendo que a interação entre a adenina/ou o dA20 imobilizada na superfície do EPC e os radicais livres na ausência e presença de antioxidantes foi avaliada por meio de mudanças no pico anódico produzido pela oxidação da adenina /dA20. Os resultados demonstraram que estes biossensores permitem a avaliação da capacidade antioxidante total em águas aromatizadas.

Estudos da lesão ao DNA por corantes têxteis e da capacidade protetora de flavonóides empregando biossensor eletroquímico: Carolina Venturini Uliana. -

Pós-graduação em Química - IQ

Anticancer activity of organotin compounds. 2. Interaction of diorganotin dihalides with nucleic acid bases and nucleosides; the synthesis of adenine, adenosine and 9-methyladenine adducts

The syntheses of the complexes formulated as SnMe2Cl2(Ad)2 (I), SnMe2Cl2(Ado)2 (II), SnMe2Cl2- (9-MeAd)2 (III) [Ad = adenine, Ado = adenosine, 9-MeAd = 9-methyladenine] as well as the more unexpected SnPhCl2(OH)(Ad)2·3H2O (IV) and SnPhCl3(Ado)2 (V) by reaction of SnMe2Cl2 or SnPh2Cl2 with the appropriate bases in methanol is described. 1H NMR studies suggest that coordination is through the N-7 position of the adenine base.

Nucleic-Acid Analogs with Restricted Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 5. Synthesis, Characterization, and Pairing Properties of Oligo-αlpha-D-(bicyclodeoxynucleotides) of the Bases Adenine and Thymine (αlpha-Bicyclo-DNA)

10.1002/hlca.19950780816.abs A conformational analysis of the (3′S,5′R)-2′-deoxy-3′,5′-ethano-α-D-ribonucleosides (a-D-bicyclodeoxynucleosides) based on the X-ray analysis of N4-benzoyl-α-D-(bicyclodeoxycytidine) 6 and on 1H-NMR analysis of the α-D-bicyclodeoxynucleoside derivatives 1-7 reveals a rigid sugar structure with the furanose units in the l′-exo/2′-endo conformation and the secondary OH groups on the carbocyclic ring in the pseudoequatorial orientation. Oligonucleotides consisting of α-D-bicyclothymidine and α-D-bicyclodeoxyadenosine were successfully synthesized from the corresponding nucleosides by phosphoramidite methodology on a DNA synthesizer. An evaluation of their pairing properties with complementary natural RNA and DNA by means of UV/melting curves and CD spectroscopy show the following characteristics: i) α-bcd(A10) and α-bcd(T10) (α = short form of α-D)efficiently form complexes with complementary natural DNA and RNA. The stability of these hybrids is comparable or slightly lower as those with natural β-d(A10) or β-d(T10)( β = short form ofβ-D). ii) The strand orientation in α-bicyclo-DNA/β-DNA duplexes is parallel as was deduced from UV/melting curves of decamers with nonsymmetric base sequences. iii) CD Spectroscopy shows significant structural differences between α-bicyclo-DNA/β-DNA duplexes compared to α-DNA/β-DNA duplexes. Furthermore, α-bicyclo-DNA is ca. 100-fold more resistant to the enzyme snake-venom phosphodiesterase with respect to β-DNA and about equally resistant as α-DNA.

Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism

Adenine phosphoribosyltransferase (APRT) is an important enzyme component of the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of purine bases rendering this biosynthetic pathway an attractive target for antiparasitic drug design. The recombinant human adenine phosphoribosyltransferase (hAPRT) structure was resolved in the presence of AMP in the active site to 1.76 angstrom resolution and with the substrates PRPP and adenine simultaneously bound to the catalytic site to 1.83 angstrom resolution. An additional structure was solved containing one subunit of the dimer in the apo-form to 2.10 angstrom resolution. Comparisons of these three hAPRT structures with other `type I` PRTases revealed several important features of this class of enzymes. Our data indicate that the flexible loop structure adopts an open conformation before and after binding of both substrates adenine and PRPR Comparative analyses presented here provide structural evidence to propose the role of Glu 104 as the residue that abstracts the proton of adenine N9 atom before its nucleophilic attack on the PRPP anomeric carbon. This work leads to new insights to the understanding of the APRT catalytic mechanism.

Electrochemical determination of antioxidant capacities in flavored waters by guanine and adenine biosensors

The immobilization and electro-oxidation of guanine and adenine asDNA bases on glassy carbon electrode are evaluated by square wave voltammetric analysis. The influence of electrochemical pretreatments, nature of supporting electrolyte, pH, accumulation time and composition of DNA nucleotides on the immobilization effect and the electrochemical mechanism are discussed. Trace levels of either guanine or adenine can be readily detected following short accumulation time with detection limits of 35 and 40 ngmL−1 for guanine and adenine, respectively. The biosensors of guanine and adenine were employed for the voltammetric detection of antioxidant capacity in flavored water samples. The method relies on monitoring the changes of the intrinsic anodic response of the surface-confined guanine and adenine species, resulting from its interaction with free radicals from Fenton-type reaction in absence and presence of antioxidant. Ascorbic acid was used as standard to evaluate antioxidant capacities of samples. Analytical data was compared with that of FRAP method.

High-resolution crystal structure of the intramolecular d(TpA) thymine-adenine photoadduct and its mechanistic implications

A high-resolution crystal structure is reported for d(TpA)*, the intramolecular thymine-adenine photoadduct that is produced by direct ultraviolet excitation of the dinucleoside monophosphate d(TpA). It confirms the presence of a central 1,3-diazacyclooctatriene ring linking the remnants of the T and A bases, as previously deduced from heteronuclear NMR measurements by Zhao et al. (The structure of d(TpA)*, the major photoproduct of thymidylyl-(3'-5')-deoxyadenosine. Nucleic Acids Res., 1996, 24, 1554-1560). Within the crystal, the d(TpA)* molecules exist as zwitterions with a protonated amidine fragment of the eight-membered ring neutralizing the charge of the internucleotide phosphate monoanion. The absolute configuration at the original thymine C5 and C6 atoms is determined as 5S,6R. This is consistent with d(TpA)* arising by valence isomerization of a precursor cyclobutane photoproduct with cis-syn stereochemistry that is generated by [2 + 2] photoaddition of the thymine 5,6-double bond across the C6 and C5 positions of adenine. This mode of photoaddition should be favoured by the stacked conformation of adjacent T and A bases in B-form DNA. It is probable that the primary photoreaction is mechanistically analogous to pyrimidine dimerization despite having a much lower quantum yield.

Photophysics and photostability of adenine in aqueous solution: A theoretical study

The sequential Monte Carlo/CASPT2 approach was employed to investigate deactivation and emission processes from the lowest-lying pi pi * and n pi * excited states of 9H-adenine in aqueous solution. It is found that conical intersections connecting the pi pi* and n pi* states with the ground state are also present in solution, whereas the barriers for the deactivation paths are significantly smaller on solvated conditions. The large destabilization of the n pi* state found in solution possibly prevents its involvement in the deactivation photophysics and explains the change from a bi- to a mono-exponential decay for the molecule in the gas phase and solution, respectively. (C) 2010 Elsevier B.V. All rights reserved.

A New Family of High-Affinity Transporters for Adenine, Cytosine, and Purine Derivatives in Arabidopsis

In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant Ki = 20 to 35 μM), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives.

Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases

A universal base that is capable of substituting for any of the four natural bases in DNA would be of great utility in both mutagenesis and recombinant DNA experiments. This paper describes the properties of oligonucleotides incorporating two degenerate bases, the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one and the purine base N6-methoxy-2,6-diaminopurine, designated P and K, respectively. An equimolar mixture of the analogues P and K (called M) acts, in primers, as a universal base. The thermal stability of oligonucleotide duplexes were only slightly reduced when natural bases were replaced by P or K. Templates containing the modified bases were copied by Taq polymerase; P behaved as thymine in 60% of copying events and as cytosine in 40%, whereas K behaved as if it were guanine (13%) or adenine (87%). The dUTPase gene of Caenorhabditis elegans, which we have found to contain three nonidentical homologous repeats, was used as a model system to test the use of these bases in primers for DNA synthesis. A pair of oligodeoxyribonucleotides, each 20 residues long and containing an equimolar mixture of P and K at six positions, primed with high specificity both T7 DNA polymerase in sequencing reactions and Taq polymerase in PCRs; no nonspecific amplification was obtained on genomic DNA of C. elegans. Use of P and K can significantly reduce the complexity of degenerate oligonucleotide mixtures, and when used together, P and K can act as a universal base.

Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases.

We have synthesized 13 hammerhead ribozyme variants, each containing an abasic residue at a specific position of the catalytic core. The activity of each of the variants is significantly reduced. In four cases, however, activity can be rescued by exogenous addition of the missing base. For one variant, the rescue is 300-fold; for another, the rescue is to the wild-type level. This latter abasic variant (G10.1X) has been characterized in detail. Activation is specific for guanine, the base initially removed. In addition, the specificity for guanine versus adenine is substantially altered by replacing C with U in the opposite strand of the ribozyme. These results show that a binding site for a small, noncharged ligand can be created in a preexisting ribozyme structure. This has implications for structure-function analysis of RNA, and leads to speculations about evolution in an "RNA world" and about the potential therapeutic use of ribozymes.

Synthesis And Antitumor Activity Of Novel 1-substituted Phenyl 3-(2-oxo-1,3,4-oxadiazol-5-yl) β-carbolines And Their Mannich Bases.

A series of novel 1-(substituted phenyl)-3-(2-oxo-1,3,4-oxadiazol-5-yl) β-carbolines (4a-e) and the corresponding Mannich bases 5-9(a-c) were synthesized and evaluated for their in vitro antitumor activity against seven human cancer cell lines. Compounds of 4a-e series showed a broad spectrum of antitumor activity, with GI50 values lower than 15μM for five cell lines. The derivative 4b, having the N,N-dimethylaminophenyl group at C-1, displayed the highest activity with GI50 in the range of 0.67-3.20μM. A high selectivity and potent activity were observed for some Mannich bases, particularly towards resistant ovarian (NCI-ADR/RES) cell lines (5a, 5b, 6a, 6c and 9b), and ovarian (OVCAR-03) cell lines (5b, 6a, 6c, 9a, 9b and 9c). In addition, the interaction of compound 4b with DNA was investigated by using UV and fluorescence spectroscopic analysis. These studies indicated that 4b interact with ctDNA by intercalation binding.