DNA Sequence Selectivity Research Articles

The DNA sequence specificity of bleomycin cleavage in telomeric sequences in human cells

Bleomycin is an antibiotic drug that is widely used in cancer chemotherapy. Telomeres are located at the ends of chromosomes and comprise the tandemly repeated DNA sequence (GGGTTA)( n ) in humans. Since bleomycin cleaves DNA at 5'-GT dinucleotide sequences, telomeres are expected to be a major target for bleomycin cleavage. In this work, we determined the DNA sequence specificity of bleomycin cleavage in telomeric sequences in human cells. This was accomplished using a linear amplification procedure, a fluorescently labelled oligonucleotide primer and capillary gel electrophoresis with laser-induced fluorescence detection. This represents the first occasion that the DNA sequence specificity of bleomycin cleavage in telomeric DNA sequences in human cells has been reported. The bleomycin DNA sequence selectivity was mainly at 5'-GT dinucleotides, with lesser amounts at 5'-GG dinucleotides. The cellular bleomycin telomeric DNA damage was also compared with bleomycin telomeric damage in purified human genomic DNA and was found to be very similar. The implications of these results for the understanding of bleomycin's mechanism of action in human cells are discussed.

Greatly extended storage stability of electrochemical DNA biosensors using ternary thiolated self-assembled monolayers

While high storage stability of sequence-selective DNA biosensors is crucial towards their routine applications, commonly used electrochemical hybridization biosensors are characterized with limited storage stability. In this article we demonstrate that recently developed ternary thiolated monolayers impart dramatic improvement in the storage stability of DNA electrochemical biosensors. In particular, highly stable multicomponent interfaces are prepared by co-immobilizing the thiolated capture probe (SHCP) with 1,6-hexanedithiol (HDT) on gold substrates, followed by the incorporation of 6-mercapto-1-hexanol (MCH) diluent. The resulting (SHCP/HDT+MCH) DNA hybridization recognition platform offers substantially higher storage stability compared to conventional binary (SHCP+MCH) monolayers. The (SHCP/HDT+MCH) ternary monolayers maintain their initial signal (S)-to-noise (N) ratio (S/N) over a prolonged 3 months period upon storage at 4°C, compared to the rapid sensitivity loss observed using the common binary interfaces. This attractive stability performance promises the convenient usage of pre-prepared electrodes after prolonged time storage without any treatment. Such dramatic improvements in the storage stability have been achieved through a rational optimization of the concentration ratio of the SHCP and the other components of the ternary SAM. The improved storage stability of SHCP/HDT+MCH interfaces observed at higher concentrations of SHCP is attributed to a hindered displacement of SHCP by MCH in the resulting compact layers. The ability to design highly stable nucleic acid interfaces using common chemicals obviates the need of using specialized expensive reagents.

Abstract 1777: Antitumor effect of PM01183 in a human pancreatic adenocarcinoma orthotopic model

Abstract Background: Pancreatic ductal adenocarcinoma is the fourth cause of death in the Western world and, despite the advances in cancer therapies, limited therapeutic benefit (palliating symptoms, better quality of life) may be offered to patients: the overall 5-year survival rate remains < 5%. PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that binds to selected DNA sequences and promotes apoptosis by inducing double-strand breaks at nanomolar concentrations. Confirmed antitumor clinical activity was observed at the recommended dose level of PM01183 in the first-in-human Phase I study in patients with advanced/refractory solid tumors. Currently, PM01183 clinical development includes Phase II single agent trials (pancreas, breast and platinum-resistant/refractory ovarian) as well as Phase I trials in combination (with doxorubicin and gemcitabine) and in advanced acute leukemia. The in vivo antitumor activity of PM01183 was investigated in the human NP9 pancreatic orthotopic model Material and Methods: Athymic nude female mice were orthotopically implanted with luciferase-expressing NP9Luc tumors. Fifteen days after implantation, tumor bearing mice were randomly allocated (N=12 /group) into either PM01183 (at 0.18 mg/kg), gemcitabine (at 180 mg/kg) or control (placebo) groups. Treatments were initiated (Day 0) and administered for 3 consecutive weeks (q7dx3; days 0, 7, 14). Response to treatment was assessed on Day 7 (short-term) by bioluminescence imaging (BLI), and by survival evaluation (long-term): mice were sacrificed when their tumor diameters reached ca. 2 cm or when they became moribund. Statistical differences between groups were determined using two-tailed Mann-Whitney U test and by analysis of the Kaplan-Meier curve. Results: Results showed that PM01183 (q7dx3 at 0.18 mg/kg) induced statistically significant tumor reduction compared to placebo-treated animals by BLI analysis (P= 0.006) on Day 7. Reductions in tumor size were associated to changes in survival median times: PM01183 treatment significantly increased the survival time compared to either gemcitabine- (P= 0.003) or placebo-treated (P= 0.0001) animals. Conclusion: PM01183 demonstrated in vivo antitumor activity in a human pancreatic orthotopic model (NP9), as reflected by metabolic, size-related and survival rate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1777. doi:1538-7445.AM2012-1777

The Use of Automated Sequencing Techniques to Investigate the Sequence Selectivity of DNA‐Damaging Agents

In this review, the use of automated DNA sequencing techniques to determine the sequence specificity of compounds that interact with DNA is discussed. The sequence specificity of a DNA-damaging agent is an essential element in determining the cellular mechanism of action of a drug. A number of DNA-damaging compounds are mutagenic, carcinogenic, as well as being widely used as cancer chemotherapeutic agents. The distribution of lesions in a sequence of DNA can give vital clues in the determination of the precise mechanism of interaction of the agent with DNA. The DNA sequence specificity of a number of DNA-damaging agents has been delineated using automated DNA sequencing technology, and these studies are discussed in this review. The current state-of-the-art methodology involves capillary electrophoresis with laser-induced fluorescence detection usually on an Applied Biosystems ABI 3730 capillary sequencer. This current technique has higher resolution, greater sensitivity, higher precision, more rapid separation times, is safer and easier to perform than previous methods. The two main methods to determine the DNA sequence selectivity of compounds that interact with DNA are described: end labelling and the polymerase stop assay. The interaction of the antitumour drug, bleomycin, with DNA is utilized to illustrate the recent technological advances.

Electrochemical monitoring of indicator-free DNA hybridization by carbon nanotubes–chitosan modified disposable graphite sensors

Single walled carbon nanotubes (SWCNT)-chitosan (CHIT) modified pencil graphite electrodes (PGEs) were developed for monitoring of DNA hybridization. SWCNT-chitosan modified PGE (CNT–CHIT–PGE), Chitosan modified PGE (CHIT–PGE) and unmodified PGE (bare-PGE) were firstly characterized by using scanning electron microscopy (SEM), and their electrochemical behaviors were investigated using electrochemical impedance spectroscopy (EIS). The concentrations of CHIT, carbon nanotube (CNT) and also amino linked DNA probe etc. were respectively optimized in order to obtain the better working conditions of CNT–CHIT modified PGE in DNA analysis. The sequence selective DNA hybridization related to Hepatitis B virus (HBV) was then explored in the case of hybridization between amino linked HBV probe and its complementary (target), or noncomplementary (NC), or mismatch (MM) sequences, and also hybridization in mixture sample.

Using Nucleic Acid Microarrays To Perform Molecular Epidemiology and Detect Novel β-Lactamases: a Snapshot of Extended-Spectrum β-Lactamases throughout the World

The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008-2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla(ESBL) (bla(SHV), bla(TEM), and bla(CTX-M) genes of groups 1, 2, 9, and 8/25) and bla(KPC) genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla(ESBL) and bla(KPC) genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla(ESBL) gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla(TEM) (n = 5), bla(SHV) (n = 11), bla(CTX-M) (n = 19), and bla(KPC) (n = 3) were detected. A new bla(SHV) variant, bla(SHV-129), and a new bla(KPC) variant, bla(KPC-11), were also identified. The most common bla genes found in this study were bla(CTX-M-15), bla(CTX-M-14), and bla(SHV-12). Using nucleic acid microarrays, we obtained a "molecular snapshot" of bla(ESBL) genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants bla(SHV-129) and bla(KPC-11).

Fluorescent detection of silver(I) and cysteine using SYBR Green I and a silver(I)-specific oligonucleotide

We report on a novel method for the determination of silver ion (Ag+) and cysteine (Cys) by using the probe SYBR Green I (SGI) and an Ag+-specific cytosine-rich oligonucleotide (C-DNA). The fluorescence of SGI is very weak in the absence or presence of randomly coiled C-DNA. If, however, C-DNA interacts with Ag+ through the formation of cytosine-Ag+-cytosine (C-Ag+-C) base pairs, the randomly coiled C-DNA undergoes a structural changes to form a hairpin-like structure, thereby increasing the fluorescence of SGI. This fluorescence turn-on process allows the detection of Ag+ in the 10–600 nM concentration range, with a detection limit of 4.3 nM. Upon the reaction of Ag+ with Cys, Cys specifically removes Ag+ from the C-Ag+-C base pairs and destroys the hairpin-like structure. This, in turn, results in a decrease in fluorescence intensity. This fluorescence turn-off process enables the determination of Cys in the 8–550 nM concentration range, with a detection limit of 4.5 nM. The method reported here for the determination of either Ag+ or Cys is simple, sensitive, and affordable, and may be applied to other detection systems if appropriately selected DNA sequences are available.

Development of Aptamer Beacons for Rapid Presumptive Detection of Bacillus Spores

A library of 92 DNA aptamer sequences was developed against Bacillus anthracis (nonpathogenic Sterne strain) spores and anthrose sugar immobilized on magnetic beads. The selected DNA sequences were studied for similarities and potential binding pockets between the B. anthracis spore and anthrose aptamers. Several recurring loop structures were identified and tested for their potential to act as aptamer beacons when labeled with TYE 665 dye on their 5' ends and Iowa Black quencher on their 3' ends. Of these candidate sequences, two beacons designated BAS-6F and BAS-6R emerged which gave strong fluorescence responses at high spore concentrations (greater than 30,000 spores/ml). These aptamer beacons also detect B. cereus and B. thuringiensis spores with greater fluorescence intensity, but do not strongly detect vegetative cells from an array of other bacterial species. BAS-6F and 6R are also not capable of detecting pure anthrose, thereby probably ruling that epitope out as a spore surface target for these particular beacons. While not extremely sensitive, the BAS-6F and 6R aptamer beacons are potentially valuable for rapid presumptive detection of anthrax or Bacillus spores in suspect powders or bioterrorist activity where spore concentrations are anticipated to be high. The sequence similarities of these beacons to other published Bacillus spore aptamers are also discussed.

A resonance light scattering quenching system for studying DNA sequence recognition of actinomycin D

The DNA sequence recognition study of DNA-targeted anticancer drugs is a theoretical basis for improving the selectivity of anticancer drugs. With the high synergy effect of cocoamidopropyl hydroxy sulfobetaine (HSB), a resonance light scattering (RLS) quenching system for DNA sequence recognition studies of actinomycin D (ACTD) was developed in this contribution. By the strategy, DNA sequence selectivity as well as the recognition mechanisms of ACTD was systematically investigated. The results suggested that ACTD had the selectivity to single-stranded DNA (ssDNA) with an equilibrium constant (K(RLS)) of 12.4 mmol mg(-1). Also it had a preference for Guanine and Cytosine bases with a K(RLS) of 6.69 L mmol(-1). The selectivity mechanism between ACTD and DNA was also well discussed with the help of UV-Vis absorption spectroscopy. Compared with other methods, the RLS quenching system has the advantages of reliability and speediness, and it avoids complex modification processes and is a better bionic system for the above research. Results obtained from this work would supply a theoretical basis for improving anticancer activity and designing similar anticancer drugs.

An Effective Identification of Species from DNA Sequence: A Classification Technique by Integrating DM and ANN

Species classification from DNA sequences remains as an open challenge in the area of bioinformatics, which deals with the collection, processing and analysis of DNA and proteomic sequence. Though incorporation of data mining can guide the process to perform well, poor definition, and heterogeneous nature of gene sequence remains as a barrier. In this paper, an effective classification technique to identify the organism from its gene sequence is proposed. The proposed integrated technique is mainly based on pattern mining and neural network-based classification. In pattern mining, the technique mines nucleotide patterns and their support from selected DNA sequence. The high dimension of the mined dataset is reduced using Multilinear Principal Component Analysis (MPCA). In classification, a well-trained neural network classifies the selected gene sequence and so the organism is identified even from a part of the sequence. The proposed technique is evaluated by performing 10-fold cross validation, a statistical validation measure, and the obtained results prove the efficacy of the technique.

Single-walled carbon nanotubes-polymer modified graphite electrodes for DNA hybridization

Single-walled carbon nanotubes (SWCNT)-poly(vinylferrocenium) (PVF +) modified pencil graphite electrodes (PGEs) were developed in our study for the electrochemical monitoring of a sequence-selective DNA hybridization event. Firstly, SWCNT-PVF + modified PGE, PVF + modified PGE and unmodified PGE were characterized using scanning electron microscopy (SEM). The electrochemical behavior of these electrodes was then investigated using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). The SWCNT-PVF + modified PGEs were optimized for improved DNA sensing ability by measuring the guanine oxidation signal. In order to obtain the full coverage immobilization of the DNA probe following the optimum working conditions, the effect of amino-linked, thiol-linked and, bare oligonucleotides (ODNs), and the concentration of the DNA probe on the response of the modified electrode were examined. After optimization studies, the sequence-selective DNA hybridization was evaluated in the case of hybridization between an amino-linked probe and its complementary (target), a noncomplementary (NC) sequence, calf thymus double stranded DNA (dsDNA), and target/mismatch (MM) mixtures in the ratio of 1:1. SWCNT-PVF + modified PGEs presented very effective discrimination of DNA hybridization owing to their superior selectivity and sensitivity.

Active Role of the Interdomain Linker of AraC

Mutations in the interdomain linker of the gene for the AraC regulatory protein of Escherichia coli that severely interfere with the protein's ability either to repress or to activate transcription have been found. These mutations have relatively small effects on the dimerization domain's ability to bind arabinose or to dimerize the protein or on the DNA-binding domain's affinity for a single DNA half-site. The linker mutations, however, dramatically change the affinity of AraC for binding to two direct-repeat DNA half-sites. Less dramatically, the induction-deficient linker variants also display altered DNA sequence selectivity. These results show that changing the sequence of the interdomain linker can profoundly affect the dimerization domain-DNA-binding domain interactions in AraC. The smaller effects on the functions of the individual domains could be the direct result of the linker alterations but more likely are the indirect result of the altered dimerization domain-DNA-binding domain interactions. In summary, the linker does not simply function as a passive and flexible connector between the domains of AraC but, instead, is more directly involved in the protein's dimerization domain-DNA-binding domain interactions.

Preparation and characterization of zinc oxide nanoparticles and their sensor applications for electrochemical monitoring of nucleic acid hybridization

In this study, ZnO nanoparticles (ZNP) of approximately 30 nm in size were synthesized by the hydrothermal method and characterized by X-ray diffraction (XRD), Braun-Emmet-Teller (BET) N2 adsorption analysis and transmission electron microscopy (TEM). ZnO nanoparticles enriched with poly(vinylferrocenium) (PVF+) modified single-use graphite electrodes were then developed for the electrochemical monitoring of nucleic acid hybridization related to the Hepatitis B Virus (HBV). Firstly, the surfaces of polymer modified and polymer-ZnO nanoparticle modified single-use pencil graphite electrodes (PGEs) were characterized using scanning electron microscopy (SEM). The electrochemical behavior of these electrodes was also investigated using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). Subsequently, the polymer-ZnO nanoparticle modified PGEs were evaluated for the electrochemical detection of DNA based on the changes at the guanine oxidation signals. Various modifications in DNA oligonucleotides and probe concentrations were examined in order to optimize the electrochemical signals that were generated by means of nucleic acid hybridization. After the optimization studies, the sequence-selective DNA hybridization was investigated in the case of a complementary amino linked probe (target), or noncomplementary (NC) sequences, or target and mismatch (MM) mixture in the ratio of (1:1).

Deglycobleomycin A 6 analogues modified in the methylvalerate moiety

Previous studies have indicated that the methylvalerate subunit of bleomycin (BLM) plays an important role in facilitating DNA cleavage by BLM and deglycoBLM. Eleven methylvalerate analogues have been synthesized and incorporated into deglycoBLM congeners by the use of solid-phase synthesis. The effect of the valerate moiety in the deglycoBLM analogues has been studied by comparison with the parent deglycoBLM A 5 using supercoiled DNA relaxation and sequence-selective DNA cleavage assays. All of the deglycoBLM analogues were found to effect the relaxation of the plasmid DNA. Those analogues having aromatic C4-substituents exhibited cleavage efficiency comparable to that of deglycoBLM A 5. Some, but not all, of the deglycoBLM analogues were also capable of mediating sequence-selective DNA cleavage.

Abstract 2536: Investigation of the effect of the sequence selective DNA minor groove cross-linking agent SG2000 (SJG-136) on canine tumor cells in vitro and in vivo

Abstract SG2000 (SJG-136) is a novel sequence selective DNA minor groove cross-linking agent that causes minimal distortion of the helical structure of DNA, such that interstrand cross-links (ICLs) persist and are not easily repaired. It has potent antitumor activity against rodent and human tumour cells in vitro and in vivo and is currently in Phase II clinical trials in humans. The aim of this study was to evaluate whether SG2000 is effective against canine cancer cells in vitro and in vivo. Fourteen canine tumour cell lines, representing the main canine cancers, and two canine normal cell lines were screened for their IC50 values following SG2000 treatment using the SRB and MTT assays. A large differential activity was observed (0.33nM to >100nM following a 1 hour exposure) as previously reported in human tumour cells. Formation and persistence of ICLs were measured with the modified single cell gel electrophoresis (comet) assay in six canine tumour (two mast cell and four melanoma) cell lines. DNA damage response to SG2000-induced ICLs was also studied by measuring γH2AX foci formation in two melanoma cell lines. IC50 values were related to the peak levels of ICLs and γH2AX foci/cell. ICLs and γH2AX foci persisted for 48 hours following a 1 hour exposure. SG2000 anti-tumour and pharmacodynamic effects (ICL and γH2AX formation) were measured in vivo in athymic mice carrying a canine oral melanoma xenograft (LMeC). These were treated with 0.15 mg/Kg and 0.30 mg/Kg i.v. either as a single dose or as a weekly dose for 3 consecutive weeks. Dose-dependent tumour growth delay was observed following the single dose treatment. Greater activity was observed in the repeated dose schedule resulting in a 40 day median tumour growth delay in the 0.30 mg/Kg group. Dose-dependent ICLs were detected in both lymphocytes and tumour at 2 hours following SG2000 treatment and were still evident at 24 hours. The level of ICLs measured in peripheral lymphocytes was lower than that measured in the target tumour tissue, indicating a selective ICL formation in vivo. An increasing number of ICLs was observed in lymphocytes following repeated doses of SG2000. Other pharmacodynamic endpoints, including γH2AX (DNA double-strand breaks), caspase 3 (apoptosis), CD34 (blood vessels), Hoechst 33342 (perfusion) and pimonidazole (hypoxia) staining of tumours were also measured. Together these data suggests that SG2000 might be a valuable antitumor agent for the treatment of canine neoplasia and early phase clinical trials against solid tumours in dogs are planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2536. doi:10.1158/1538-7445.AM2011-2536

Abstract 3538: In vivo combination studies of PM01183 with alkylating, antimetabolites, DNA-topoisomerase inhibitors and tubulin binding agents

Abstract PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that binds to selected DNA sequences and promotes apoptosis by inducing double-strand breaks at nanomolar concentrations. PM01183 displays significant antitumor activity in several murine models of human cancer. PM01183 is currently in phase I/II clinical development for cancer treatment. Nonclinical combination studies are increasingly relevant during the development of any anticancer compound. As such, PM01183 was assayed in combination (two-drugs) with representatives of alkylating agents (dacarbazine, temozolomide and cisplatin), antimetabolites (5-FU and gemcitabine), DNA-topoisomerase inhibitors (irinotecan and doxorubicin) and tubulin binding agents (paclitaxel and vinorelbine). Different models were used depending on the combination explored: STS (HT1080) for dacarbazine; glioma (U-87 MG) for temozolomide; gastric (HGC-27) for cisplatin and 5-FU; pancreas (SW1990) for gemcitabine; colon (HT-29) and NSCLC (H460) for irinotecan; and ovarian (A2780) for doxorubicin, paclitaxel and vinorelbine. Athymic nu/nu mice bearing tumors (ca. 150 mm3) were randomly allocated to 13 treatment groups: i) placebo; ii) PM01183 at 4 different dose levels, namely MTD (0.180 mg/kg), 0.75MTD, 0.5MTD and 0.25MTD; iii) Compound to be combined, at 4 different dose levels MTD, 0.75MTD, 0.5MTD and 0.25MTD; and, iv) PM01183 plus the combined compound, administered with the combination at (1+1), (0.75+0.75), (0.50+0.50) and (0.25+0.25) of MTD ratios. The antitumor activity was followed by T/C, (change in tumor volume for each treated -T- and placebo -C- groups during placebo-treated survival period). Then, the fraction affected (Fa) by the treatment was calculated (1-T/C) and, the combination index (CI) by the CI-isobol method determined. Synergism (CI ≤ 1 at Fa > 0.8) was recorded when PM01183 was combined with dacarbazine (HT1080), cisplatin (HGC-27), 5-fluororacil (HGC-27), gemcitabine (SW1990), irinotecan (H460), doxorubicin (A2780), paclitaxel (A2780) and vinorelbine (A2780). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3538. doi:10.1158/1538-7445.AM2011-3538

The sequence selectivity of DNA-targeted 9-aminoacridine cisplatin analogues in a telomere-containing DNA sequence

In this study, the detailed DNA sequence specificity of four acridine Pt complexes was examined and compared with that of cisplatin. The DNA sequence specificity was determined in a telomere-containing DNA sequence using a polymerase stop assay, with a fluorescent primer and an automated capillary DNA sequencer. The Pt compounds included an acridine intercalating moiety that was modified to give a 9-aminoacridine derivative, a 7-methoxy-9-aminoacridine derivative, a 7-fluoro-9-aminoacridine derivative and a 9-ethanolamine-acridine derivative. Compared with cisplatin, the DNA sequence specificity was most altered for the 7-methoxy-9-aminoacridine compound, followed by the 9-aminoacridine derivative, the 7-fluoro-9-aminoacridine compound and the 9-ethanolamine-acridine derivative. The DNA sequence selectivity for the four acridine Pt complexes was shifted away from runs of consecutive guanines towards single guanine bases, especially 5'-GA dinucleotides and sequences that contained 5'-CG. The sequence specificity was examined in telomeric and non-telomeric DNA sequences. Although it was found that telomeric DNA sequences were extensively damaged by the four acridine Pt complexes, there was no extra preference for telomeric sequences.

Effect of base sequence on the DNA cross-linking properties of pyrrolobenzodiazepine (PBD) dimers

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective DNA minor-groove cross-linking agents that possess two electrophilic imine moieties (or their equivalent) capable of forming covalent aminal linkages with guanine C2-NH2 functionalities. The PBD dimer SJG-136, which has a C8–O–(CH2)3–O–C8′′ central linker joining the two PBD moieties, is currently undergoing phase II clinical trials and current research is focused on developing analogues of SJG-136 with different linker lengths and substitution patterns. Using a reversed-phase ion pair HPLC/MS method to evaluate interaction with oligonucleotides of varying length and sequence, we recently reported (JACS, 2009, 131, 13 756) that SJG-136 can form three different types of adducts: inter- and intrastrand cross-linked adducts, and mono-alkylated adducts. These studies have now been extended to include PBD dimers with a longer central linker (C8–O–(CH2)5–O–C8′), demonstrating that the type and distribution of adducts appear to depend on (i) the length of the C8/C8′-linker connecting the two PBD units, (ii) the positioning of the two reactive guanine bases on the same or opposite strands, and (iii) their separation (i.e. the number of base pairs, usually ATs, between them). Based on these data, a set of rules are emerging that can be used to predict the DNA–interaction behaviour of a PBD dimer of particular C8–C8′ linker length towards a given DNA sequence. These observations suggest that it may be possible to design PBD dimers to target specific DNA sequences.

Expanding DNA Sequence Recognition with Minor Groove Binders

Heterocyclic cations are taken up into cells and can target the DNA minor groove and they offer a potential method to control cellular gene expression. A limitation of these compounds is their limited DNA sequence recognition capability and a key goal of our research has been to expand their ability to target selected DNA sequences. The results show that formation of minor groove stacked-dimer complexes is an effective way to enhance the specificity and expand the range of applications of the compounds. The discovery of the diamidine DB293 (phenyl-furan-benzimidazole), the first dication to recognize DNA as a stacked dimer in the minor groove, provided an opening to this effort. This approach, filling the minor groove with two stacked heterocyclic cations, provides an opportunity to interact with wider minor grooves which are difficult to recognize by a single molecule. Dimer recognition of DNA by these compounds is highly sensitive to compound structure as well as the target sequence. DB1242 (phenyl-pyrimidine-phenyl), a linear dication, is able to stack as a dimer in a GC rich sequence forming a curved complex which matches to the shape of minor groove. This is a example of how a linear compound with chemical functionalities in an optimum position can form intra as well as intermolecular interactions with DNA. Our finding that TTAA sequences are difficult to target, can be selectively recognized by stacked minor groove dimmers opens a new way to interact with that sequence. These investigations indicate that compound design tools can be used to obtain new types of specific binding to DNA. Supported by NIAID grant AI64200.

Challenges of Microarray Applications for Microbial Detection and Gene Expression Profiling in Food

Microarrays are a high throughput technology used in molecular biology. The different types of microarrays have been applied to clinical and environmental microbiology, microbial ecology, human, veterinary, and plant diagnostics. Since multiple genes can be analyzed simultaneously, this technology has been extended to food microbiology for detection and gene expression analyses of food-borne pathogens. Although this technique has multiple applications, microarray technology presently has low sensitivity and also suffers from problems such as reproducibility and reliability. This paper focuses on microarray applications for microbial detection and gene expression profiling of food-borne pathogens including some of the challenges and key issues such as target nucleic acid isolation and selection of target DNA sequences.