Homology and enzymatic requirements of microhomology-dependent alternative end joining

Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.

Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing

DNA repair, one of the fundamental processes occurring in a cell, safeguards the genome and maintains its integrity. Among various DNA lesions, double-strand breaks are considered to be the most deleterious, as they can lead to potential loss of genetic information, if not repaired. Non-homologous end joining (NHEJ) and homologous recombination are two major double-strand break repair pathways. SCR7, a DNA ligase IV inhibitor, was recently identified and characterized as a potential anticancer compound. Interestingly, SCR7 was shown to have several applications, owing to its unique property as an NHEJ inhibitor. Here, we focus on three main areas of research in which SCR7 is actively being used, and discuss one of the applications, i.e. genome editing via CRISPR/Cas, in detail. In the past year, different studies have shown that SCR7 significantly increases the efficiency of precise genome editing by inhibiting NHEJ, and favouring the error-free homologous recombination pathway, both in vitro and in vivo. Overall, we discuss the current applications of SCR7 to shed light on the unique property of the small molecule of having distinct applications in normal and cancer cells, when used at different cellular concentrations.

Gene enrichment using antibodies to DNA/RNA hybrids : mapping the ribosomal DNA of slime mold and rat

A novel method for gene enrichment has been developed and applied to mapping the rRNA genes of two eucaryotic organisms. The method makes use of antibodies to DNA/RNA hybrids prepared by injecting rabbits with the synthetic hybrid poly(rA)•poly(dT). Antibodies which cross-react with non-hybrid nucleic acids were removed from the purified IgG fraction by adsorption on columns of DNA-Sepharose, oligo(dT)-cellulose, and poly(rA)-Sepharose. Subsequent purification of the specific DNA/RNA hybrid antibody was carried out on a column of oligo(dT)-cellulose to which poly(rA) was hybridized. Attachment of these antibodies to CNBr-activated Sepharose produced an affinity resin which specifically binds DNA/RNA hybrids.

In order to map the rDNA of the slime mold Dictyostelium discoideum, R-loops were formed using unsheared nuclear DNA and the 178 and 268 rRNAs of this organism. This mixture was passed through a column containing the affinity resin, and bound molecules containing R- loops were eluted by high salt. This purified rDN A was observed directly in the electron microscope. Evidence was obtained that there is a physical end to Dictyostelium rDN A molecules approximately 10 kilobase pairs (kbp) from the region which codes for the 268 rRNA. This finding is consistent with reports of other investigators that the rRNA genes exist as inverse repeats on extra-chromosomal molecules of DNA unattached to the remainder of the nuclear DNA in this organism.

The same general procedure was used to map the rRNA genes of the rat. Molecules of DNA which contained R-loops formed with the 188 and 288 rRNAs were enriched approximately 150- fold from total genomal rat DNA by two cycles of purification on the affinity column. Electron microscopic measurements of these molecules enabled the construction of an R-loop map of rat rDNA. Eleven of the observed molecules contained three or four R-loops or else two R-loops separated by a long spacer. These observations indicated that the rat rRNA genes are arranged as tandem repeats. The mean length of the repeating units was 37.2 kbp with a standard deviation of 1.3 kbp. These eleven molecules may represent repeating units of exactly the same length within the errors of the measurements, although a certain degree of length heterogeneity cannot be ruled out. If significantly shorter or longer repeating units exist, they are probably much less common than the 37.2 kbp unit.

The last section of the thesis describes the production of antibodies to non-histone chromosomal proteins which have been exposed to the ionic detergent sodium dodecyl sulfate (SDS). The presence of low concentrations of SDS did not seem to affect either production of antibodies or their general specificity. Also, a technique is described for the in situ immunofluorescent detection of protein antigens in polyacrylamide gels.

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

DNA-mediated oxidation of transcription factor p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the π-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

To determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]³⁺, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.

The chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were ¹³C₂D₂-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward ¹³C₂D₂-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

DNA-Mediated Charge Transport Devices for Protein Detection

Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the π-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.

First, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.

Using low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.

For effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 μL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.

With the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.

As differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.

Hardware paralelo reconfigurável para identificação de alinhamentos de sequências de DNA.

Amostras de DNA são encontradas em fragmentos, obtidos em vestígios de uma cena de crime, ou coletados de amostras de cabelo ou sangue, para testes genéticos ou de paternidade. Para identificar se esse fragmento pertence ou não a uma sequência de DNA, é necessário compará-los com uma sequência determinada, que pode estar armazenada em um banco de dados para, por exemplo, apontar um suspeito. Para tal, é preciso uma ferramenta eficiente para realizar o alinhamento da sequência de DNA encontrada com a armazenada no banco de dados. O alinhamento de sequências de DNA, em inglês DNA matching, é o campo da bioinformática que tenta entender a relação entre as sequências genéticas e suas relações funcionais e parentais. Essa tarefa é frequentemente realizada através de softwares que varrem clusters de base de dados, demandando alto poder computacional, o que encarece o custo de um projeto de alinhamento de sequências de DNA. Esta dissertação apresenta uma arquitetura de hardware paralela, para o algoritmo BLAST, que permite o alinhamento de um par de sequências de DNA. O algoritmo BLAST é um método heurístico e atualmente é o mais rápido. A estratégia do BLAST é dividir as sequências originais em subsequências menores de tamanho w. Após realizar as comparações nessas pequenas subsequências, as etapas do BLAST analisam apenas as subsequências que forem idênticas. Com isso, o algoritmo diminui o número de testes e combinações necessárias para realizar o alinhamento. Para cada sequência idêntica há três etapas, a serem realizadas pelo algoritmo: semeadura, extensão e avaliação. A solução proposta se inspira nas características do algoritmo para implementar um hardware totalmente paralelo e com pipeline entre as etapas básicas do BLAST. A arquitetura de hardware proposta foi implementada em FPGA e os resultados obtidos mostram a comparação entre área ocupada, número de ciclos e máxima frequência de operação permitida, em função dos parâmetros de alinhamento. O resultado é uma arquitetura de hardware em lógica reconfigurável, escalável, eficiente e de baixo custo, capaz de alinhar pares de sequências utilizando o algoritmo BLAST.

Saline-saturated DMSO-EDTA as a storage medium for microbial DNA analysis from coral mucus swab samples

The mucus surface layer of corals plays a number of integral roles in their overall health and fitness. This mucopolysaccharide coating serves as vehicle to capture food, a protective barrier against physical invasions and trauma, and serves as a medium to host a community of microorganisms distinct from the surrounding seawater. In healthy corals the associated microbial communities are known to provide antibiotics that contribute to the coral’s innate immunity and function metabolic activities such as biogeochemical cycling. Culture-dependent (Ducklow and Mitchell, 1979; Ritchie, 2006) and culture-independent methods (Rohwer, et al., 2001; Rohwer et al., 2002; Sekar et al., 2006; Hansson et al., 2009; Kellogg et al., 2009) have shown that coral mucus-associated microbial communities can change with changes in the environment and health condition of the coral. These changes may suggest that changes in the microbial associates not only reflect health status but also may assist corals in acclimating to changing environmental conditions. With the increasing availability of molecular biology tools, culture-independent methods are being used more frequently for evaluating the health of the animal host. Although culture-independent methods are able to provide more in-depth insights into the constituents of the coral surface mucus layer’s microbial community, their reliability and reproducibility rely on the initial sample collection maintaining sample integrity. In general, a sample of mucus is collected from a coral colony, either by sterile syringe or swab method (Woodley, et al., 2008), and immediately placed in a cryovial. In the case of a syringe sample, the mucus is decanted into the cryovial and the sealed tube is immediately flash-frozen in a liquid nitrogen vapor shipper (a.k.a., dry shipper). Swabs with mucus are placed in a cryovial, and the end of the swab is broken off before sealing and placing the vial in the dry shipper. The samples are then sent to a laboratory for analysis. After the initial collection and preservation of the sample, the duration of the sample voyage to a recipient laboratory is often another critical part of the sampling process, as unanticipated delays may exceed the length of time a dry shipper can remain cold, or mishandling of the shipper can cause it to exhaust prematurely. In remote areas, service by international shipping companies may be non-existent, which requires the use of an alternative preservation medium. Other methods for preserving environmental samples for microbial DNA analysis include drying on various matrices (DNA cards, swabs), or placing samples in liquid preservatives (e.g., chloroform/phenol/isoamyl alcohol, TRIzol reagent, ethanol). These methodologies eliminate the need for cold storage, however, they add expense and permitting requirements for hazardous liquid components, and the retrieval of intact microbial DNA often can be inconsistent (Dawson, et al., 1998; Rissanen et al., 2010). A method to preserve coral mucus samples without cold storage or use of hazardous solvents, while maintaining microbial DNA integrity, would be an invaluable tool for coral biologists, especially those in remote areas. Saline-saturated dimethylsulfoxide-ethylenediaminetetraacetic acid (20% DMSO-0.25M EDTA, pH 8.0), or SSDE, is a solution that has been reported to be a means of storing tissue of marine invertebrates at ambient temperatures without significant loss of nucleic acid integrity (Dawson et al., 1998, Concepcion et al., 2007). While this methodology would be a facile and inexpensive way to transport coral tissue samples, it is unclear whether the coral microbiota DNA would be adversely affected by this storage medium either by degradation of the DNA, or a bias in the DNA recovered during the extraction process created by variations in extraction efficiencies among the various community members. Tests to determine the efficacy of SSDE as an ambient temperature storage medium for coral mucus samples are presented here.

Species identification and authentication of some of the most important seafood products in Iranian markets using biotechnological methods based on DNA

This study describes the molecular identification of sixteen fish species present in processed products imported into Iran for human consumption. DNA barcoding using direct sequencing of about 650 bp of the mitochondrial Cytochrome Oxidase subunit I gene revealed incorrect labeling (31.25%). Substitution of fish species constitutes serious economic fraud, and our results increase concern regarding the trading of processed fish products in Iran from both health and conservation points of view.

Construction and characterization of a Lipotes vexillifer genomic DNA BAC library

We constructed a genomic DNA library for Lipotes vexillifer (L. vexillifer), the Baiji or Yangtze River dolphin, one of the most endangered mammals in the world. The library consists of 149,000 BAC clones, with an average insert size of 83 kb, representing approximately 3.4 haploid genome equivalents. PCR amplification of four known L. vexillifer genes yielded two to four positive clones each. To demonstrate the utility of this library, we isolated and sequenced the L. vexillifer alpha lactalbumin gene, which is a gene specific to mammals and one which has been widely used as molecular tool in phylogenetic analysis. We also end-sequenced 20 randomly selected clones, resulting in the identification of at least five new L. vexilliter genes, five SSR loci, and one SINE locus. These results suggest that this library is a valuable resource for candidate gene cloning, physical mapping, and genome sequencing of this important and threatened species.

Is the genus Digramma synonymous to the genus Ligula (Cestoda : Pseudophyllidea)? Evidence from ITS and 5 ' end 28S rDNA sequences

The genus Digramma (Cestoda: Pseudophyllidea) described by Cholodkovsky in 1915 differs from the genus Ligula only by the number of the reproductive organs per proglottis. However, the occurrence of transitional forms in Digramma raises much confusion concerning its generic validity. In the present study, cestodes previously designated as Digramma and Ligula were collected from lakes in the lower and middle reaches of the Yangtze River, and also from Qinghai Lake on Qingzang plateau, China. The entire internal transcribed spacer of the ribosomal DNA (ITS rDNA) and 5' end of 28S rDNA were compared between the Digramma and Ligula specimens. The low level of nucleotide variation between the two genera may imply that cestodes in the genus Digramma are paraphyletic to the Ligula genus, and Digramma is a synonym of Ligula. However, whether previously identified Digramma cestodes represent different species in the genus Ligula requires further investigation.

Chromosomal in situ hybridization of Cyprinus carpio genomic DNA repetitive sequence CR1

Common carp Cyprinus carpio genomic DNA repetitive sequence CR1 has been DIG-labeled and hybridized in situ against chromosomes of red common carp (Cyprinus carpio L. Xingguo red var.). It is found that the repetitive sequence CR1 is mainly localized at the centromeric regions of chromosomes of the red common carp, The application of the chromosomal in situ hybridization technique on fish and the relationship between CR1 repetitive sequence distribution and its function have been discussed.

Determination of urinary oxidative DNA damage marker 8-hydroxy-2 '-deoxyguanosine and the association with cigarette smoking

8-hydroxy-2'-deoxyguanosine (8OHdG) has been widely used as a biomarker of oxidative DNA damage in both animal models and human studies. To evaluate the effect of cigarette smoking on oxidative stress, we studied the levels of urinary 8OHdG from smokers and non-smokers and investigated the association with cigarette smoking. The urinary 8OHdG concentrations were determinated by capillary electrophoresis with end-column amprometric detection (CE-AD) after a single-step solid phase extraction (SPE), and then quantitatively expressed as a function of creatinine excretion. To increase the concentration sensitivity, a dynamic pH junction was used and the focusing effect was obvious when using 30 mM phosphate (pH 6.50) as sample matrix. The limit of detection is 4.3 nM (signal-to-noise ratio S/N = 3). The relative standard deviation (R.S.D.) was 1.1% for peak current, and 2.3% for migration time. Based on the selected CE-AD method, it was found that the mean value of urinary 8OHdG levels in the smokers significantly higher than that in non-smokers (31.4 +/- 18.9 nM versus 14.4 +/- 7.6 nM, P = 0.0004; 23.5 +/- 21.3 mug g(-1) creatinine versus 12.6 +/- 13.2 mug g(-1) creatinine, P = 0.028). (C) 2004 Elsevier B.V. All rights reserved.

Study of urinary 8-hydroxydeoxyguanosine as a biomarker of oxidative DNA damage in diabetic nephropathy patients

Increased oxidative stress induced by hyperglycemia may contribute to the pathogenesis of diabetic complications. Urinary 8-hydroxydeoxyguanosine (8-OHdG) has been reported to serve as a sensitive biomarker of oxidative DNA damage and also of oxidative stress. This article studied oxidative DNA damage in patients with diabetic nephropathy and in healthy control subjects by urinary 8-OHdG evaluations. Contents of 8-OHdG in urine were analyzed by capillary electrophoresis with end-column amperometric detection (CE-AD) after a single-step solid-phase extraction (SPE). Levels of urinary 8-OHdG in diabetic nephropathy patients with macroalbuminuria was significant higher than in control subjects (5.72 +/- 6.89 mumol/mol creatinine versus 2.33 +/- 2.83 mumol/mol creatinine, P = 0.018). A significant difference of 24 h urinary 8-OHdG excretions exists between the patients with macroalbuminuria and the patients with nonnoalbuminuria (19.2 +/- 16.8 mug/24 h versus 8.1 +/- 1.7 mug/24 h, P = 0.015). There was a positive correlation between urinary excretion of 8-OHdG and glycosylated hemoglobin (HbA(1)c) (r = 0.287, P = 0.022). A weak correlation exists between the levels of 8-OHdG and triglyceride (r = 0.230, P = 0.074). However, the urinary 8-OHdG contents are not correlated with blood pressure and total cholesterol. The increased excretion of urinary 8-OHdG is seen as indicating an increased systemic level of oxidative DNA damage in diabetic nephropathy patients. (C) 2004 Elsevier B.V. All rights reserved.

CE with electrochemical detection for investigation of label-free recognition of amino acid amides by guanine-rich DNA aptamers

In this work, we report a simple and effective investigation into adaptive interactions between guanine-rich DNA aptamers and amino acid amides by CE with electrochemical (EC) detection. Argininamide (Arm) and tyrosinamide (Tym) were chosen as model molecules. On a copper electrode, Arm generated a good EC signal in 60 mM NaOH at 0.7 V (vs Ag/ AgCl), while Tym. was detected well on a platinum electrode at 1. 3 V in 20 mM phosphate of pH 7.0. Based on their EC properties, the ligands themselves were used as indicators for the adaptive interactions investigated by CE-EC, making any step of labeling and/or modification of aptamers with indicators exempted. Hydrophilic ionic liquid was used as an additive in running buffer of CE to improve the sensitivity of Arm detection, whereas the additive was not used for Tym. detection due to its negative effect. Two guanine-rich DNA aptamers were used for molecular recognition of Arm and Tym. When the aptamers were incubated with ligands, they bound the model molecules with high affinity and specificity, reflected by obvious decreases in the signals of ligands but no changes in those of the control molecules. However, the ligands were hardly affected by the control ssDNAs after incubation. The results revealed the specific recognition of Arm and Tym. by the aptamers.