Natural Guanine Research Articles

Natural guanine derivatives exert PARP-inhibitory and cytoprotective effects in a model of cardiomyocyte damage under oxidative stress

Inhibitors of human poly(ADP-ribose) polymerase (PARP) are considered as promising agents for the treatment of cardiovascular, neurological, and other diseases accompanied by inflammation and oxidative stress. Previously, the ability of the natural compounds 7-methylguanine (7mGua) and 8-hydroxy-7-methylguanine (8h7mGua) to suppress the activity of the recombinant PARP protein was demonstrated. In the present work, we have investigated the possibility of PARP-inhibitory and cytoprotective action of 7mGua and 8h7mGua against rat cardiomyoblast cultures (undifferentiated and differentiated H9c2). It was found that 7mGua and 8h7mGua rapidly penetrate into cells and effectively suppress H2O2-stimulated PARP activation (IC50 = 270 and 55 µM, respectively). The pronounced cytoprotective effects of 7mGua and 8h7mGua were shown in a cellular model of oxidative stress, and 8h7mGua exceeded the classic PARP inhibitor 3-aminobenzamide for effectiveness. The obtained data indicate the prospects for the development of PARP inhibitors based on guanine derivatives and their testing on models of ischemia-reperfusion tissue damage.

Natural Guanine Derivatives Exert PARP-Inhibitory and Cytoprotective Effects in a Model of Cardiomyocyte Damage under Oxidative Stress.

Inhibitors of human poly(ADP-ribose) polymerase (PARP) are considered as promising agents for treatment of cardiovascular, neurological, and other diseases accompanied by inflammation and oxidative stress. Previously, the ability of natural compounds 7-methylguanine (7mGua) and 8-hydroxy-7-methylguanine (8h7mGua) to suppress activity of the recombinant PARP protein was demonstrated. In the present work, we have investigated the possibility of PARP-inhibitory and cytoprotective action of 7mGua and 8h7mGua against the rat cardiomyoblast cultures (undifferentiated and differentiated H9c2). It was found that 7mGua and 8h7mGua rapidly penetrate into the cells and effectively suppress the H2O2-stimulated PARP activation (IC50 = 270 and 55 μM, respectively). The pronounced cytoprotective effects of 7mGua and 8h7mGua were shown in a cellular model of oxidative stress, and effectiveness of 8h7mGua exceeded the classic PARP inhibitor 3-aminobenzamide. The obtained data indicate promise for the development of PARP inhibitors based on guanine derivatives and their testing using the models of ischemia-reperfusion tissue damage.

Exploiting natural riboswitches for aptamer engineering and validation.

Over the past three decades, researchers have found that some engineered aptamers can be made to work well in test tubes but that these same aptamers might fail to function in cells. To help address this problem, we developed the 'Graftamer' approach, an experimental platform that exploits the architecture of a natural riboswitch to enhance in vitro aptamer selection and accelerate in vivo testing. Starting with combinatorial RNA pools that contain structural features of a guanine riboswitch aptamer interspersed with regions of random sequence, we performed multiplexed in vitro selection with a collection of small molecules. This effort yielded aptamers for quinine, guanine, and caffeine that appear to maintain structural features of the natural guanine riboswitch aptamer. Quinine and caffeine aptamers were each grafted onto a natural guanine riboswitch expression platform and reporter gene expression was monitored to determine that these aptamers function in cells. Additionally, we determined the secondary structure features and survival mechanism of a class of RNA sequences that evade the intended selection strategy, providing insight into improving this approach for future efforts. These results demonstrate that the Graftamer strategy described herein represents a convenient and straightforward approach to develop aptamers and validate their in vivo function.

Photoisomerization and ultrasensitive fluorescence detection of toxic deoxyguanosine adduct ANdG: a theoretical study

The toxic deoxyguanosine adduct (ANdG) is one of the most representative carcinogenic DNA adducts, resulting from human exposure to aromatic amine (AA). Accordingly, the ultrasensitive detection of AA-derived adduct in DNA with minimal disturbance to the native structures is key to elucidating carcinogenesis mechanisms and mitigating cancer risk. Considering the lack of adequate intrinsic optical emission in two obtained rotamers (ANG1 and ANG2), a set of quasi-intrinsic fluorescent probes based on the complementary cytosine are proposed to identify covalent G-adduct. It is found that the expanded C-analogues in solution could bring red-shifted absorption maxima and enhanced photoluminescence due to the additional π-conjugation. In particular, because PhpC possesses large Stokes shift (98 nm) and the highest fluorescence intensity (f flu. = 0.546) in emission, it is proposed as the biosensor to monitor the optical changes in the presence and absence of the considered ANG. Compared with native C base, the absorption maximum of PhpC is red-shifted by 145 nm, which contributes to the selective excitation after incorporating into the nucleic acids. More importantly, the fluorescence is insensitive to base pairing with natural guanine, while the efficient fluorescence quenching is observed after pairing with ANG1/ANG2 as a result of the obvious excited state intermolecular charge transfer. To evaluate the direct application of the bright C-analogues with a high selectivity for deoxyguanosine adduct ANdG in DNA, we further examined the effect of linking deoxyribose on absorption and fluorescence emission, which are consistent with the experimental data.

Structure and mechanism of the methyltransferase ribozyme MTR1.

RNA-catalysed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyses the site-specific synthesis of 1-methyladenosine (m1A) in RNA, using O6-methylguanine (m6G) as methyl group donor. Here we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2'-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution.

Modified Guanines as Constituents of Smart Ligands for Nucleic Acid Quadruplexes.

Repetitive guanine-rich nucleic acid sequences play a crucial role in maintaining genome stability and the cell life cycle and represent potential targets for regulatory drugs. Recently, it has been demonstrated that guanine-based ligands with a porphyrin core can be used as markers of G-quadruplex assemblies in cell tissues. Herein, model systems of guanine-based ligands are explored by DFT methods. The energies of formation of modified guanine tetrads and those of modified tetrads stacked on the top of natural guanine tetrads have been calculated. The interaction energy has been decomposed into contributions from hydrogen bonding, stacking, and ion coordination and a twist-rise potential energy scan has been performed to find the individual local minima. Energy decomposition analysis reveals the impact of various substituents (F, Cl, Br, I, Me, NMe2 ) on individual energy terms. In addition, cooperative reinforcement in forming the modified and stacked tetrads, as well as the frontier orbitals participating in the hydrogen-bonding framework involving the HOMO-LUMO gap between the occupied σHOMO on the proton-accepting C=O and =N- groups and unoccupied σLUMO on the N-H groups, has been studied. The investigated systems are demonstrated to have a potential in ligand development, mainly due to stacking enhancement compared with natural guanine, which is used as a reference.

Electrochemical behavior of 7-deazaguanine- and 7-deazaadenine-modified DNA at the hanging mercury drop electrode

DNA modification with synthetic analogs of natural nucleotides and/or their conjugates with external redox active groups is applied in the development of electrochemical DNA sensors or assay for DNA hybridization, SNP typing, DNA damage and so forth. 7-Deazapurines (Pu*) are analogs of natural purine bases in which N7 atom is replaced by CH group. The Pu* bases retain Watson–Crick base pairing of their parent purines (and the ability to form duplex DNA) but are incapable of Hoogsteen pairing (and thus cannot be involved in triplex or quadruples DNA structures). Previously, we studied electrochemical oxidation of Pu* residues in DNA fragments (prepared by PCR in the presence of Pu* deoxynucleoside triphosphates) at a carbon electrode and reported on significantly lower potentials of oxidation of both 7-deazaguanine (G*) and 7-deazaadenine (A*), compared to natural guanine (G) and adenine (A), respectively. In this work, we studied faradaic and tensammetric responses of G*- or A*-modified DNA on the hanging mercury drop electrode (HMDE). While A* was reduced at the HMDE, giving rise to a similar irreversible cathodic peak as the natural A, G* did not yield any peak analogous to the peak G due to guanine, in agreement with a loss of corresponding redox site in G*. Responses of DNA modified with A* were relatively similar to those of unmodified DNA (albeit we observed certain differences in tensammetric peak currents). Effects of G substitution by G* were more pronounced, being reflected in diminution of peak due to guanine, decrease of the peak CA (due to cytosine and adenine reduction) and in significantly changed shape of tensammetric DNA signals, indicating altered adsorption/desorption processes. While substitution of A by A* resulted in certain destabilization of the DNA duplex at the negatively charged HMDE surface (in qualitative agreement with significantly decreased melting temperature of the same DNA duplexes in solution), G*-modified duplex DNA displayed apparently lower susceptibility to surface denaturation.

Improved bioactivity of G-rich triplex-forming oligonucleotides containing modified guanine bases.

Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-rich TFOs partially substituted with 8-aza-7-deaza-guanine (PPG) have improved target site binding in potassium compared with TFOs containing the natural guanine base. We designed PPG-substituted TFOs to bind to a polypurine sequence in the supFG1 reporter gene. The binding efficiency of PPG-substituted TFOs to the target sequence was analyzed using electrophoresis mobility gel shift assays. We have determined that in the presence of potassium, the non-substituted TFO, AG30 did not bind to its target sequence, however binding was observed with the PPG-substituted AG30 under conditions with up to 140 mM KCl. The PPG-TFOs were able to maintain their ability to induce genomic modifications as measured by an assay for gene-targeted mutagenesis. In addition, these compounds were capable of triplex-induced DNA double strand breaks, which resulted in activation of apoptosis.

Direct Voltammetric Analysis of DNA Modified with Enzymatically Incorporated 7-Deazapurines

Nucleic acids studies use 7-deazaguanine (G*) and 7-deazaadenine (A*) as analogues of natural purine bases incapable of forming Hoogsteen base pairs, which prevents them from being involved in DNA triplexes and tetraplexes. Reduced propensity of the G*- and/or A*-modified DNA to form alternative DNA structures is utilized, for example, in PCR amplification of guanine-rich sequences. Both G* and A* exhibit significantly lower potentials of their oxidation, compared to the respective natural nucleobases. At carbon electrodes, A* yields an oxidation peak which is by about 200-250 mV less positive than the peak due to adenine, but coincides with oxidation peak produced by natural guanine residues. On the other hand, oxidation signal of G* occurs at a potential by about 300 mV less positive than the peak due to guanine, being well separated from electrochemical signals of any natural DNA component. We show that enzymatic incorporation of G* and A* can easily be monitored by simple ex situ voltammetric analysis of the modified DNA at carbon electrodes. Particularly G* is shown as an attractive electroactive marker for DNA, efficiently incorporable by PCR. While densely G*-modified DNA fragments exhibit strong quenching of fluorescence of SYBR dyes, commonly used as fluorescent indicators in both gel staining and real time PCR applications, the electrochemical detection provides G*-specific signal suitable for the quantitation of the amplified DNA as well as for the determination of the DNA modification extent. Determination of DNA amplicons based on the measurement of peak G*(ox) is not affected by signals produced by residual oligonucleotide primers or primary templates containing natural purines.

Hetero-Ring-Expansion Design for Adenine-Based DNA Motifs: Evidence from DFT Calculations and Molecular Dynamics Simulations

The design of new DNA motifs is at present a very interesting topic. Recent progress indicates that the hetero-ring-expanded guanine (G) analogues possess enhanced properties compared with natural guanine. In this work, a series of hetero-ring-expanded adenine (A) analogues are designed, and their structures and electronic properties are investigated by means of density functional calculations and molecular dynamics simulations. The results indicate that the designed A-analogues can form stable base pairs with natural counterpart, and the pairing energetics for the Watson-Crick hydrogen-bonded dimers between the expanded A-analogues and natural T exhibit similarity to natural AT. Their tautomeric preferences are close to natural A, too. Furthermore, compared with natural ones, most size-expanded adenines and corresponding base pairs have smaller ionization potentials. In particular, several designed A analogues have ionization potentials even lower than natural G. The electron affinities of these modified A are comparable with that of natural A. The HOMO-LUMO gaps also behave with sensible trends. Most of A-analogues and their interrelated base pairs possess smaller gaps than the corresponding natural base and base pairs. Further, molecular dynamics simulations show the sufficient stabilities of the DNA analogues (dnA.dT)(12) (where nA represents the size-expanded A-analogues designed here) when forming duplexes as the natural one does. Clearly, these observations imply their promising applications as molecular wires and new DNA motifs.

Solid-phase synthesis of positively charged deoxynucleic guanidine (DNG) oligonucleotide incorporating 7-deazaguanine bases

DNG nucleotides represent a positively charged DNA analog in which the negatively charged phosphodiester linkages of DNA are replaced by positively charged guanidinium linkages. We report herein the synthesis of 3′-end, middle, and 5′-end monomers required for the synthesis of a DNG sequence in which the natural guanine base is replaced by 7-deazaguanine (c 7G). 7-Deazaguanine nucleobase was chosen because of their unique glycoside bond stability and their ability to prevent G-quartet formation. A facile and high yield two-step synthesis of xylo-7-deazaguanine 7, a key intermediate for introducing 3′-amino functionality, is carried out under Mitsunobu conditions. Subsequently, the 3′-Fmoc-protected thiourea monomers 13 and 19 were prepared from 7 via their corresponding 3′-amino-7-deazaguanines 11 and 18, respectively. The smooth coupling of these thiourea monomers with monomethoxytrityl (MMTr)-protected 3′-end monomer 25, prepared from 5, occurred on solid phase in 3′ → 5′ direction. The resultant trimeric HO-c 7Ggc 7Ggc 7G-OH ( 1) has been designed to be included into DNA using standard DNA synthesis technology. The combination of C-c 7G base pairing and electrostatic association of phosphodiester and guanidinium backbone allows the small synthesized DNG trimer 1 to form 1:1 complex with DNA-C pentamer.

1030. Pyrazolo[3,4-d]Pyrimidine Guanine Base Substitution in Triplex Forming Oligonucleotides Improves Target Specificity and Chromosomal Mutagenesis

Top of pageAbstract Triplex forming oligonucleotides (TFOs) can induce site-specific damage, repair and recombination in episomal and chromosomal targets. However, several limitations preclude their effective use for gene targeting at the present time. For example, purine-rich TFOs have reduced binding in physiologic levels of monovalent cations, especially K+, apparently due to self-association at the guanine N7. We have substituted a 30-mer purine-rich TFO with the base analog 7-deaza-8-aza-guanine (PPG), in which the N7 and C8 positions of the guanine ring are exchanged. We show here that the PPG- substituted TFOs have increased binding affinity to a polypurine target site in vitro, even in the presence of physiologic [K+]. As well, the PPG-substituted TFOs are more efficient at forming covalent crosslinks at a chromosomally integrated target site. Notably, TFOs containing PPG substitutions at every third guanine induced almost a four-fold increase in mutation frequency in a chromosomal reporter gene in mammalian cells, almost twice the rate as a TFO containing natural guanine. In addition, we are interested in using modified TFOs to target disease-related genes in mammalian cells; preliminary results indicate that PPG-substituted TFOs can target an endogenous intronic sequence of the beta-globin locus to promote gene correction. PPG modifications, therefore, can augment triplex specificity and stability, resulting in enhanced gene targeting and correction.

Enzymatic synthesis of 2'-deoxyguanosine with nucleoside deoxyribosyltransferase-II.

Nucleoside deoxyribosyltransferase-II (NdRT-II) of Lactobacillus helveticus, which catalyzes the transfer of a glycosyl residue from a donor deoxyribonucleoside to an acceptor base, has a broad specificity for the acceptor bases. Six-substituted purines were found to be substrates as acceptor bases for NdRT-II. Using this property of the enzyme, we established a practical procedure for enzymatic synthesis of 2'-deoxyguanosine (dGuo), consisting of the transglycosylation from thymidine to 6-substituted purine (2-amino-6-chloropurine; ACP) instead of natural guanine and the conversion of 2-amino-6-chloropurine-2'-deoxyriboside (ACPdR) to dGuo with bacterial adenosine deaminase. Through the successive reactions, dGuo was synthesized in high yield.

Effect of chemical modifications of cytosine and guanine in a cpg-motif of oligonucleotides: structure–immunostimulatory activity relationships

Oligodeoxynucleotides containing unmethylated CpG-motifs stimulate the innate immune system, including inducing B-cell proliferation and cytokine production. However, the mechanism of immunostimulation by CpG-oligonucleotides and the precise structural requirements and specific functional groups of cytosine and guanine necessary for recognition of and interaction with protein/receptor factors that are responsible for immune stimulation have not been elucidated. We sought to understand the critical role of each functional group of the cytosine and guanine moieties in a CpG-motif in inducing immunostimulatory activity. To this end, we examined structure–immunostimulatory activity relationships of phosphorothioate oligodeoxynucleotides (PS-oligos) containing YpG- and CpR-motifs (Y and R stand for pyrimidine and purine analogues, respectively). The PS-oligos containing a YpG-motif in which the natural deoxycytidine was replaced with deoxy-5-hydroxycytidine or deoxy-N4-ethylcytidine showed immunostimulatory activity. Substitution of deoxycytidine with a deoxy-5-methylisocytidine, deoxyuridine, or deoxy-P-base-nucleoside in the YpG-motif completely abolished the immunostimulatory activity, similar to the results observed with deoxy-5-methylcytidine. In the case of PS-oligos containing a CpR-motif, 7-deazaguanine substitution for natural guanine showed immunostimulatory activity similar to that of a parent PS-oligo. These studies suggest that the 2-keto, 3-imino and 4-amino groups of cytosine, and the 1-imino, 2-amino and 6-keto groups of guanine in a CpG-motif are important for the immunostimulatory activity of CpG-PS-oligos. The absence of N7 on guanine of the CpG-motif does not affect immunostimulatory activity significantly. These studies suggest that it is possible to develop YpG- and CpR-motifs as an alternative to CpG-motifs in PS-oligos for immunostimulatory studies.

Tricyclic “oxadiazaphosphorine oxide” guanosines: A new strategy to the protection of guanine bases during RNA synthesis

A novel strategyfor simultaneously protecting the lactam and N2-aminofunctions of guanine nucleosides is reported N2-benzoylated guanosines were readily converted into tricyclic “oxadiazaphosphorine oxide” guanosines upon reaction with N,N-diisopropylphosphoramidous dichloride followed by oxidation with t-butylhydroperoxide. the tricyclic guanine derivative is reconverted readily to the natural guanine aglycone upon treatment with ethylenediamine/ethanol (1:4, 1 min, rt.).

Rapid kinetics of alpha 2-adrenergic inhibition of adenylate cyclase. Evidence for a distal rate-limiting step.

Activation and inhibition of adenylate cyclase in the presence of GTP, the natural guanine nucleotide regulator, are too fast to study by standard biochemical methods. In order to identify the rate-limiting steps in adenylate cyclase regulation, we measured the kinetics of stimulation and inhibition of the enzyme on a subsecond to second time scale using a novel rapid-mix quench technique. Even using our rapid-mix quench method, activation by PGE1 and forskolin was instantaneous (cAMP accumulation was linear between 0.5 and 30 s). In contrast, we found a lag period of 1.2-10 s for epinephrine-mediated inhibition. The length of the lag depended on the concentration of GTP and monovalent cations present. In the absence of NaCl, the rate constant for the onset of inhibition (kinh) increased only slightly with GTP concentration saturating at a value of 0.16 s-1 (t1/2 4.3 s) at 1 microM GTP. In the presence of 100 mM NaCl, kinh was strongly dependent on GTP concentration, reaching a maximum value of 0.57 s-1 (t1/2 1.2 s) at 100 microM GTP. Thus, activation of both Gi and Gs in intact platelet membranes is much faster (t1/2 less than 5 s) than previously reported for reconstituted systems. Also, the strong dependence of the rate of adenylate cyclase inhibition on GTP concentration implies that the rate-limiting step in inhibition is distal to GTP binding. The effect of NaCl to increase the maximal rate of inhibition is specific for sodium since KCl has no effect on kinh.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased membrane-associated nucleoside diphosphate kinase activity as a possible basis for enhanced guanine nucleotide-dependent adenylate cyclase activity induced by picolinic acid treatment of simian virus 40-transformed normal rat kidney cells.

A biochemical analysis of an increase in guanine nucleotide-dependent adenylate cyclase activity induced by treatment of cultured SV40-transformed normal rat kidney cells with picolinic acid is described. In purified membranes from drug-treated cells with an ATP regenerating system in assay, GTP- and GTP plus hormone-stimulated adenylate cyclase activities were increased, whereas basal and NaF-stimulated cyclase activities, and steady state rate with guanosine 5'-(beta, gamma-imino)triphosphate were essentially unaltered by drug treatment. In assay systems devoid of ATP regenerating system, the drug-induced increase in cyclase activity was seen with GDP as well as with GTP, it being larger with GDP than with GTP in terms of activity ratio, whereas such an increase was not observed with their analogs, guanosine 5'-O-(2-thiodiphosphate) or guanosine 5'-(beta, gamma-imino)triphosphate. Guanosine 5'-(beta, gamma-imino)triphosphate-stimulated from drug-treated membranes became less sensitive to the inhibition by GDP as shown by a rightward shift in inhibition curve, but this shift could not be reproduced with guanosine 5'-O-(2-thiodiphosphate). From these results, it was concluded that altered guanine nucleotide metabolism in membranes was involved. Neither the amount of guanine nucleotide-binding protein nor its related functions including GTPase activity were changed by drug treatment. However, we observed in the drug-treated cell membranes, an increase in activity of nucleoside diphosphate kinase, an additional factor which has been proposed to play a role in regulating adenylate cyclase by replenishing GTP near the guanine nucleotide binding site (Kimura, N., and Shimada, N. (1983) J. Biol. Chem. 258, 2278-2283). The altered features of adenylate cyclase with the natural guanine nucleotides induced by drug treatment were explained as a result of this enhanced nucleoside diphosphate kinase activity associated with the membranes.