Guanine Residues Research Articles

Ataxia telangiectasia mutated- and Rad3-related kinase drives both the early and the late DNA-damage response to the monofunctional antitumour alkylator S23906

Numerous anticancer agents and environmental mutagens target DNA. Although all such compounds interfere with the progression of the replication fork and inhibit DNA synthesis, there are marked differences in the DNA-damage response pathways they trigger, and the relative impact of the proximal or the distal signal transducers on cell survival is mainly lesion-specific. Accordingly, checkpoint kinase inhibitors in current clinical development show synergistic activity with some DNA-targeting agents, but not with others. In the present study, we characterize the DNA-damage response to the antitumour acronycine derivative S23906, which forms monofunctional adducts with guanine residues in the minor groove of DNA. S23906 exposure is accompanied by specific recruitment of RPA (replication protein A) at replication sites and rapid Chk1 activation. In contrast, neither MRN (Mre11-Rad50-Nbs1) nor ATM (ataxia-telangiectasia mutated), contributes to the initial response to S23906. Interestingly, genetic attenuation of ATR (ATM- and Ras3-related) activity inhibits not only the early phosphorylation of histone H2AX and Chk1, but also interferes with the late phosphorylation of Chk2. Moreover, loss of ATR function or pharmacological inhibition of the checkpoint kinases by AZD7762 is accompanied by abrogation of the S-phase arrest and increased sensitivity towards S23906. These findings identify ATR as a central co-ordinator of the DNA-damage response to S23906, and provide a mechanistic rationale for combinations of S23906 and similar agents with checkpoint abrogators.

Mass spectrometry evidence for cisplatin as a protein cross-linking reagent.

Cisplatin is a potent anticancer drug, which functions by cross-linking adjacent DNA guanine residues. However within 1 day of injection, 65-98% of the platinum in the blood plasma is protein-bound. It is generally accepted that cisplatin binds to methionine and histidine residues, but what is often underappreciated is that platinum from cisplatin has a 2+ charge and can form up to four bonds. Thus, it has the potential to function as a cross-linker. In this report, the cross-linking ability of cisplatin is demonstrated by Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) with the use of standard peptides, the 16.8 kDa protein calmodulin (CaM), but was unsuccessful for the 64 kDa protein hemoglobin. The high resolution and mass accuracy of FTICR MS along with the high degree of fragmentation of large peptides afforded by collisionally activated dissociation (CAD) and electron capture dissociation (ECD) are shown to be a valuable means of characterizing cross-linking sites. Cisplatin is different from current cross-linking reagents by targeting new functional groups, thioethers, and imidazoles groups, which provides complementarity with existing cross-linkers. In addition, platinum(II) inherently has two positive charges which enhance the detection of cross-linked products. Higher charge states not only promote the detection of cross-linking products with less purification but result in more comprehensive MS/MS fragmentation and can assist in the assignment of modification sites. Moreover, the unique isotopic pattern of platinum flags cross-linking products and modification sites by mass spectrometry.

Cleavage of DNA fragments induced by UV nanosecond laser excitation at 193 nm

The cleavage of dsDNA fragments in aqueous solution after irradiation with UV laser pulses at 193 nm has been studied. Samples were investigated using polyacrylamide gel electrophoresis. The intensity of damage of particular phosphodiester bond after hot alkali treatment was shown to depend on the base pair sequence. It was established that the probability of cleavage is twice higher for sites of DNA containing two or more successively running guanine residues. A possible mechanism of damage to the DNA molecule connected with the migration of holes along the helix is discussed.

Introducing Recombinant Picornaviral Genomes into Cells

IntroductionPicornarviruses comprise a large group of small, nonenveloped, positive-sense, single-stranded RNA viruses. The picornavirus life cycle is usually rapid and exclusively cytoplasmic, without integration into the host cell’s genome or a nuclear phase. Due to their biology and genetic constraints, the utility of picornaviruses for general gene delivery purposes is limited. However, they may prove useful as vaccine vectors. Furthermore, picornavirus-driven expression of various reporter genes or foreign RNA elements is of interest to the picornavirus molecular virologist. Introduction of recombinant picornaviral genomes into the cell relies on the historic observation that the isolated virion RNA is infectious. This property extends to in-vitro-transcribed RNA as long as the authentic viral 5' end is preserved. That said, up to two additional 5'-terminal guanine residues (remnants from the T7 RNA polymerase-based in vitro transcription), although reducing infectivity, can be tolerated. Additional nucleotides at the 3' end are of far less consequence. Thus, any unique restriction site downstream from the poly(A) sequence (preferably as close as possible downstream) can be used to linearize the plasmid containing the viral genome before in vitro transcription.

Multimodal In Vivo Imaging and Blood Monitoring of Intrinsic and Extrinsic Apoptosis

Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer. We have developed an apoptosis biosensor by fusing green fluorescent protein (GFP) to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) or DEVD. We showed that this fusion is retained in the cytoplasm of cells in an inactive form. Upon apoptosis, the DEVD peptide is cleaved in response to caspase-3 activation, freeing ssGluc, which can now enter the secretory pathway where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time. Because Gluc is secreted from cells via conventional pathway through the endoplasmic reticulum (ER), Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging. This reporter provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models.

Highly fluorescent guanosine mimics for folding and energy transfer studies

Guanosines with substituents at the 8-position can provide useful fluorescent probes that effectively mimic guanine residues even in highly demanding model systems such as polymorphic G-quadruplexes and duplex DNA. Here, we report the synthesis and photophysical properties of a small family of 8-substituted-2′-deoxyguanosines that have been incorporated into the human telomeric repeat sequence using phosphoramidite chemistry. These include 8-(2-pyridyl)-2′-deoxyguanosine (2PyG), 8-(2-phenylethenyl)-2′-deoxyguanosine (StG) and 8-[2-(pyrid-4-yl)-ethenyl]-2′-deoxyguanosine (4PVG). On DNA folding and stability, 8-substituted guanosines can exhibit context-dependent effects but were better tolerated by G-quadruplex and duplex structures than pyrimidine mismatches. In contrast to previously reported fluorescent guanine analogs, 8-substituted guanosines exhibit similar or even higher quantum yields upon their incorporation into nucleic acids (Φ = 0.02–0.45). We have used these highly emissive probes to quantify energy transfer efficiencies from unmodified DNA nucleobases to 8-substituted guanosines. The resulting DNA-to-probe energy transfer efficiencies (ηt) are highly structure selective, with ηt(duplex) < ηt(single-strand) < ηt(G-quadruplex). These trends were independent of the exact structural features and thermal stabilities of the G-quadruplexes or duplexes containing them. The combination of efficient energy transfer, high probe quantum yield, and high molar extinction coefficient of the DNA provides a highly sensitive and reliable readout of G-quadruplex formation even in highly diluted sample solutions of 0.25 nM.

Organometallic Half-Sandwich Iridium Anticancer Complexes

The low-spin 5d(6) Ir(III) organometallic half-sandwich complexes [(η(5)-Cp(x))Ir(XY)Cl](0/+), Cp(x) = Cp*, tetramethyl(phenyl)cyclopentadienyl (Cp(xph)), or tetramethyl(biphenyl)cyclopentadienyl (Cp(xbiph)), XY = 1,10-phenanthroline (4-6), 2,2'-bipyridine (7-9), ethylenediamine (10 and 11), or picolinate (12-14), hydrolyze rapidly. Complexes with N,N-chelating ligands readily form adducts with 9-ethylguanine but not 9-ethyladenine; picolinate complexes bind to both purines. Cytotoxic potency toward A2780 human ovarian cancer cells increases with phenyl substitution on Cp*: Cp(xbiph) > Cp(xph) > Cp*; Cp(xbiph) complexes 6 and 9 have submicromolar activity. Guanine residues are preferential binding sites for 4-6 on plasmid DNA. Hydrophobicity (log P), cell and nucleus accumulation of Ir correlate with cytotoxicity, 6 > 5 > 4; they distribute similarly within cells. The ability to displace DNA intercalator ethidium bromide from DNA correlates with cytotoxicity and viscosity of Ir-DNA adducts. The hydrophobicity and intercalative ability of Cp(xph) and Cp(xbiph) make a major contribution to the anticancer potency of their Ir(III) complexes.

Electrochemical Oxidation of Native Double‐Stranded DNA on a Graphene‐Modified Glassy Carbon Electrode

AbstractThe electrochemistry of double‐stranded DNA (dsDNA) was investigated on a graphene‐modified glassy carbon electrode (GR/GCE). Electrochemical characterization showed that GR on GCE surface significantly enhanced the specific surface area and electrical conductivity of the sensing platform. On GR/GCE, the electrooxidation peak of guanine residues in dsDNA was obviously enhanced and the peak potential was significantly lowered as compared to bare GCE. The electrochemical parameters and influence factors for measurements were investigated. Under the optimal conditions, the peak current linearly increased with dsDNA concentrations in the range from 1 to 500 µg mL−1 with a detection limit of 0.35 µg mL−1.

Theoretical Study of Polaron Formation in Poly(G)−Poly(C) Cations

Polaron formation in poly(G)-poly(C) cations is investigated with density functional theory (DFT) and molecular mechanics (MM) employing a two-layer ONIOM method. In these calculations, the high layer, composed of all complementary base pairs, is treated by a DFT method, while the low layer, which includes the sugar-phosphate backbone, counterions and water molecules, is described by the AMBER force field. The high layer is the model system in which the charge transfer takes place. According to our calculations, three or four guanines move in a paddle-like fashion when an electron is removed from the neutral model system. In the cation model system, about 80% of the charge is delocalized onto the guanine residues, and the remaining charge is delocalized onto the cytosine residues. This happens because guanine has a lower ionization potential (IP) than cytosine. The counterions and water molecules in the low layer are important in the geometry optimization. The optimized geometry of the model system is closer to the standard B-form structure when counterions and water molecules are included than when they are omitted. Comparison of the optimized neutral and cationic model systems reveals a polaron in poly(G)-poly(C) cations extending from the first to the third guanine. It is demonstrated that the position of counterions and the number of surrounding water molecules can affect polaron formation.

The minor groove-binding agent ELB-21 forms multiple interstrand and intrastrand covalent cross-links with duplex DNA and displays potent bactericidal activity against methicillin-resistant Staphylococcus aureus.

ObjectivesThe antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple adducts with duplex DNA through covalent interactions with appropriately spaced guanine residues; it is now known to form interstrand and intrastrand adducts with oligonucleotide sequences of variable length. We determined the DNA sequence preferences of ELB-21 in relation to its capacity to exert a bactericidal effect by damaging DNA.MethodsFormation of adducts by ELB-21 and 12- to 14-mer DNA duplexes was investigated using ion-pair reversed phase liquid chromatography and mass spectrometry. Drug-induced changes in gene expression were measured in prophage-free Staphylococcus aureus RN4220 by microarray analysis.ResultsELB-21 preferentially formed intrastrand adducts with guanines separated by three nucleotide base pairs. Interstrand and intrastrand adducts were formed with duplexes both longer and shorter than the preferred target sequences. ELB-21 elicited rapid bactericidal effects against prophage-carrying and prophage-free S. aureus strains; cell lysis occurred following activation and release of resident prophages. Killing appeared to be due to irreparable damage to bacterial DNA and susceptibility to ELB-21 was governed by the capacity of staphylococci to repair DNA lesions through induction of the SOS DNA damage response mediated by the RecA-LexA pathway.ConclusionsThe data support the contention that ELB-21 arrests DNA replication, eliciting formation of ssDNA-RecA filaments that inactivate LexA, the SOS repressor, and phage repressors such as Cl, resulting in activation of the DNA damage response and de-repression of resident prophages. Above the MIC threshold, DNA repair is ineffective.

Direct electrochemistry of double strand DNA on ionic liquid modified screen-printed graphite electrode

An ionic liquid modified screen-printed graphite electrode (SPE) was used for direct electrochemistry of herring sperm double strand DNA (dsDNA) by voltammetry. Due to the high conductivity of ionic liquid n-octylpyridinum hexafluorophosphate (OPPF), this electrode exhibited excellent electrochemical activity for the oxidation of dsDNA. Two irreversible oxidation peaks were obtained at the developed electrode, which corresponded to the oxidation of guanine and adenine residues present in the dsDNA. The basic electrochemical behavior of dsDNA at the OPPF modified SPE was carefully investigated. Combined with the differential pulse voltammetry, this electrode exhibited a good linear range from 20 μg mL −1 to 120 μg mL −1 with a detection limit of 5 μg mL −1 for the direct determination of dsDNA. Furthermore, the OPPF modified electrode displayed high reproducibility and stability for the dsDNA determination.

Fluorescence Studies of Local Environmental Heterogeneity Around Guanine Residues of the Telomeric DNA Quadruplex Conformation

Formation and stabilization of G-quadruplexed DNA formed by the guanine-rich human telomeric sequence d(TTAGGG)4 (HT4), has been shown to inhibit the abnormal activity of telomerase in tumor cells, making stabilization of this DNA secondary structure a target for potential cancer therapeutics. To investigate the effect of local environment on the guanine residues of the G-quadruplex, we previously designed several fluorescence analogs of the HT4 sequence with incorporation of 6-methyl-8-(2-deoxy-D-ribofuranosyl) isoxanthopterin (6MI) at varying guanine-tetrad positions: G1, G4, and G11, which show fluorescence sensitivity to quadruplex folding. In the current studies, we have collected decay-associated spectra (DAS) for the fluorescence lifetime components (τlong, τmedium, and τshort) resolved for each of the mono-labeled sequences. Multi-exponential decay profiles suggest local environmental heterogeneity surrounding the guanine residues in both the folded and unfolded states. Interestingly, the DAS for τlong observed for guanine positions G1 and G11, which are located at the 5’- and 3’-ends, respectively, of the quadruplex-forming sequence, showed significant (10nm) red-wavelength shifts on quadruplex folding. In contrast no DAS shifts are observed for the G4 position, located in the loop region of the folded quadruplex. Thus, in addition to base stacking of the guanine residues with neighboring bases, guanine positions located at G1 and G11, on average, may also be exposed to a more polar environment with quadruplex folding, possibly due to conformational heterogeneity as they are located near the termini of the telomeric sequence. The solvent accessibility of these positions may make them key docking sites for designing ligands that can stabilize the folded conformation of the G-quadruplex.This work was supported by NIH SCORE Grant S06-GM076168-01 (LD).

Thiol-Activated DNA Damage by α-Bromo-2-cyclopentenone

Some biologically active chemicals are relatively stable in the extracellular environment but, upon entering the cell, undergo biotransformation into reactive intermediates that covalently modify DNA. The diverse chemical reactions involved in the bioactivation of DNA-damaging agents are both fundamentally interesting and of practical importance in medicinal chemistry and toxicology. The work described here examines the bioactivation of α-haloacrolyl-containing molecules. The α-haloacrolyl moiety is found in a variety of cytotoxic natural products including clionastatin B, bromovulone III, discorahabdins A, B, and C, and trichodenone C, in mutagens such as 2-bromoacrolein and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), and in the anticancer drug candidates brostallicin and PNU-151807. Using α-bromo-2-cyclopentenone (1) as a model compound, the activation of α-haloacrolyl-containing molecules by biological thiols was explored. The results indicate that both low molecular weight and peptide thiols readily undergo conjugate addition to 1. The resulting products are consistent with a mechanism in which initial addition of thiols to 1 is followed by intramolecular displacement of bromide to yield a DNA-alkylating episulfonium ion intermediate. The reaction of thiol-activated 1 with DNA produces labile lesions at deoxyguanosine residues. The sequence specificity and salt dependence of this process is consistent with involvement of an episulfonium ion intermediate. The alkylated guanine residue resulting from the thiol-triggered reaction of 1 with duplex DNA was characterized using mass spectrometry. The results provide new insight regarding the mechanisms by which thiols can bioactivate small molecules and offer a more complete understanding of the molecular mechanisms underlying the biological activity of cytotoxic, mutagenic, and medicinal compounds containing the α-haloacrolyl group.

Few constraints limit the design of quinone methide-oligonucleotide self-adducts for directing DNA alkylation.

Nucleotide sequences minimally containing adenosine, cytosine or guanosine are sufficient to form intrastrand oligonucleotide quinone methide self-adducts reversibly for subsequent alkylation of complementary DNA. The general lack of sequence restrictions should now allow for alkylation of most any target of interest although reaction is most efficient when the self-adducts contain guanine residues and do not form hairpin structures.

Cation-Mediated Energy Transfer in G-Quadruplexes Revealed by an Internal Fluorescent Probe

A single pyridine unit incorporated into G-quadruplex DNA has revealed efficient energy transfer reactions in cation-containing G-quadruplexes. 8-(2-Pyridyl)-2'-deoxyguanosine, "2PyG", is a highly sensitive internal fluorescent probe of G-quadruplex folding and energy transfer. 2PyG was minimally disruptive to G-quadruplex folding and exhibited intense fluorescence, even when it was base-stacked with other guanine residues. Using 2PyG we have quantified energy transfer efficiencies within G-quadruplex structures prepared under conditions of excess Na(+)/K(+) (110 mM) or in 40% polyethylene glycol (PEG) under salt deficient conditions. G-quadruplex structures containing coordinated cations exhibited efficient DNA-to-probe energy transfer reactions (η(t) = 0.11-0.41), while PEG-folded G-quadruplexes exhibited very little energy transfer (η(t) = 0.02-0.07). Experiments conducted using unmodified G-quadruplexes suggest that cation coordination at the O(6) position of guanine residues results in enhanced quantum yields of G-quadruplex nucleobases that, in turn, serve as efficient energy donors to 2PyG. Given the growing interest in G-quadruplex-based devices and materials, these results will provide important design principles toward harnessing the potentially useful photophysical properties of G-quadruplex wires and other G-rich structures.

Urinary 8-hydroxydeoxyguanosine correlate with hallucinations rather than motor symptoms in Parkinson’s disease

BackgroundOxidative stress is causally associated with the pathogenesis of Parkinson’s disease (PD). Oxygen generates a large amount of reactive oxygen species (ROS). ROS including hydroxyl radicals and H2O2 react with guanine residues in DNA and produce 8-hydroxydeoxyguanosine (8-OHdG). 8-OHdG serves as a biomarker for oxidative stress in various diseases. MethodWe investigated urinary 8-OHdG levels in 61 PD patients and 28 normal subjects to evaluate the correlation with various clinical features. We quantified disease severity using the Unified Parkinson’s Disease Rating Scale for motor symptoms (UPDRS part 3), the Mini-Mental State Examination (MMSE) for mental function, and the Tottori University Hallucination Rating Scale (TUHARS) for quantifying hallucinations. ResultsThere were significant correlations between 8-OHdG and all the examined parameters, but the partial correlation coefficients excluding contributions of all the other parameters showed that only TUHARS and UPDRS part 3 are significantly related to 8-OHdG. In particular, TUHARS correlates best with urinary 8-OHdG levels. ConclusionThe significant correlation between urinary 8-OHdG levels and hallucinations but not with dementia suggests that hallucinations are likely to have unique but unidentified mechanisms that lead to excessive production of 8-OHdG.

A Multiphasic Typing Approach to Subtype Streptococcus Equi Subspecies Equi

The objective of the present investigation was to differentiate between strains of Streptococcus equi subspecies equi implicated in abscess formation in vaccinated horses. Streptococcus equi isolates recovered from clinical specimens associated with equine strangles cases submitted to the University of Illinois Veterinary Diagnostic Laboratory were compared with S. equi isolates representing at least 12 lots of a commercial modified live vaccine (MLV) to determine whether the isolates obtained from the abscesses were vaccine or wild type. Genotyping techniques evaluated included enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR), repetitive extragenic palindrome PCR, BOX element PCR, ribotyping, and pulsed-field gel electrophoresis (PFGE). Phenotypic evaluations were performed using the Biolog GP2 Microplate (hereafter, Biolog). In cases where Biolog and PFGE results did not coincide, a single nucleotide polymorphism located in the upstream regulatory region of szp gene was used to identify the S. equi strains. PFGE and Biolog successfully differentiated wild-type S. equi strains isolated from clinical submissions from isolates of the MLV. PFGE genotyping enabled further subtyping of the wild-type strains, whereas Biolog combined with szp sequencing was useful in differentiating the MLV strain from its wild-type progenitor. Deletion of a single guanine residue located in the upstream regulatory region of the szp gene appears to be conserved among vaccine isolates, and shows a 98.5% correlation to Biolog identification. This multiphasic approach can be used to answer specific diagnostic questions pertaining to the source of infection and/or outbreak, or to address quarantine concerns.

Evidence of the formation of G-quadruplex structures in the promoter region of the human vascular endothelial growth factor gene

The polypurine/polypyrimidine (pPu/pPy) tract of the human vascular endothelial growth factor (VEGF) gene is proposed to be structurally dynamic and to have potential to adopt non-B DNA structures. In the present study, we further provide evidence for the existence of the G-quadruplex structure within this tract both in vitro and in vivo using the dimethyl sulfate (DMS) footprinting technique and nucleolin as a structural probe specifically recognizing G-quadruplex structures. We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments. We also demonstrated that nucleolin, which is known to bind to G-quadruplex structures, is able to bind specifically to the G-rich sequence of this region in negatively supercoiled DNA. Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo. Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates.

New Anthracenedione Derivatives with Improved Biological Activity by Virtue of Stable Drug−DNA Adduct Formation

Mitoxantrone is an anticancer agent that acts as a topoisomerase II poison, however, it can also be activated by formaldehyde to form DNA adducts. Pixantrone, a 2-aza-anthracenedione with terminal primary amino groups in its side chains, forms formaldehyde-mediated adducts with DNA more efficiently than mitoxantrone. Molecular modeling studies indicated that extension of the "linker" region of anthracenedione side arms would allow the terminal primary amino greater flexibility and thus access to the guanine residues on the opposite DNA strand. New derivatives based on the pixantrone and mitoxantrone backbones were synthesized, and these incorporated primary amino groups as well as extended side chains. The stability of DNA adducts increased with increasing side chain length of the derivatives. A mitoxantrone derivative bearing extended side chains (7) formed the most stable adducts with ∼100-fold enhanced stability compared to mitoxantrone. This finding is of great interest because long-lived drug-DNA adducts are expected to perturb DNA-dependent functions at all stages of the cell cycle.

ChemInform Abstract: Development of New Protecting Groups for Guanine Residue in Oligodeoxyribonucleotide Synthesis.

Attempts were made to develop new protecting groups for 1,6-lactam function of 2-N-acyl guanine in oligodeoxyribonucleotide synthesis. Several acyl groups, aryl groups, and carbamoyl groups were tested. Dimethylcarbamoyl and phenylacetyl groups are shown to be a good combination for guanine residue. 6-O-Di-methylcarbamoyl-2-N-pheylacetyl-2'-deoxy guanosine have been successfully used in the synthesis of d[AAGCTT], which is Hind Ⅲ recognition sequence.