Noncanonical DNA Cleavage by BamHI Endonuclease in Laterally Confined DNA Monolayers Is a Step Function of DNA Density and Sequence.

Cleavage of DNA at noncanonical recognition sequences by restriction endonucleases (star activity) in bulk solution can be promoted by global experimental parameters, including enzyme or substrate concentration, temperature, pH, or buffer composition. To study the effect of nanoscale confinement on the noncanonical behaviour of BamHI, which cleaves a single unique sequence of 6 bp, we used AFM nanografting to generate laterally confined DNA monolayers (LCDM) at different densities, either in the form of small patches, several microns in width, or complete monolayers of thiol-modified DNA on a gold surface. We focused on two 44-bp DNAs, each containing a noncanonical BamHI site differing by 2 bp from the cognate recognition sequence. Topographic AFM imaging was used to monitor end-point reactions by measuring the decrease in the LCDM height with respect to the surrounding reference surface. At low DNA densities, BamHI efficiently cleaves only its cognate sequence while at intermediate DNA densities, noncanonical sequence cleavage occurs, and can be controlled in a stepwise (on/off) fashion by varying the DNA density and restriction site sequence. This study shows that endonuclease action on noncanonical sites in confined nanoarchitectures can be modulated by varying local physical parameters, independent of global chemical parameters.

Rational design of an XNA ligase through docking of unbound nucleic acids to toroidal proteins.

Xenobiotic nucleic acids (XNA) are nucleic acid analogues not present in nature that can be used for the storage of genetic information. In vivo XNA applications could be developed into novel biocontainment strategies, but are currently limited by the challenge of developing XNA processing enzymes such as polymerases, ligases and nucleases. Here, we present a structure-guided modelling-based strategy for the rational design of those enzymes essential for the development of XNA molecular biology. Docking of protein domains to unbound double-stranded nucleic acids is used to generate a first approximation of the extensive interaction of nucleic acid processing enzymes with their substrate. Molecular dynamics is used to optimise that prediction allowing, for the first time, the accurate prediction of how proteins that form toroidal complexes with nucleic acids interact with their substrate. Using the Chlorella virus DNA ligase as a proof of principle, we recapitulate the ligase's substrate specificity and successfully predict how to convert it into an XNA-templated XNA ligase.

Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli

Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.

Signal-on electrochemical assay for label-free detection of TdT and BamHI activity based on grown DNA nanowire-templated copper nanoclusters.

Electrochemical methods allow fast and inexpensive analysis of enzymatic activity. Here, a simple and yet efficient "signal-on" electrochemical assay for sensitive, label-free detection of DNA-related enzyme activity was established on the basis of terminal deoxynucleotidyl transferase (TdT)-mediated extension strategy. TdT, which is a template-independent DNA polymerase, can catalyze the sequential addition of deoxythymidine triphosphate (dTTP) at the 3'-OH terminus of single-stranded DNA (ssDNA); then, the TdT-yield T-rich DNA nanowires can be employed as the synthetic template of copper nanoclusters (CuNCs). Grown DNA nanowires-templated CuNCs (noted as DNA-CuNCs) were attached onto graphene oxide (GO) surface and exhibited unique electrocatalytic activity to H2O2 reduction. Under optimal conditions, the proposed biosensor was utilized for quantitatively monitoring TdT activity, with the observed LOD of 0.1 U/mL. It also displayed high selectivity to TdT with excellent stability, and offered a facile, convenient electrochemical method for TdT-relevant inhibitors screening. Moreover, the proposed sensor was successfully used for BamHI activity detection, in which a new 3'-OH terminal was exposed by the digestion of a phosphate group. Ultimately, it has good prospects in DNA-related enzyme-based biochemical studies, disease diagnosis, and drug discovery. Graphical Abstract Extraordinary TdT-generated DNA-CuNCs are synthesized and act as a novel electrochemical sensing platform for sensitive detection of TdT and BamHI activity in biological environments.

A rapid method for detecting protein-nucleic acid interactions by protein induced fluorescence enhancement.

Many fundamental biological processes depend on intricate networks of interactions between proteins and nucleic acids and a quantitative description of these interactions is important for understanding cellular mechanisms governing DNA replication, transcription, or translation. Here we present a versatile method for rapid and quantitative assessment of protein/nucleic acid (NA) interactions. This method is based on protein induced fluorescence enhancement (PIFE), a phenomenon whereby protein binding increases the fluorescence of Cy3-like dyes. PIFE has mainly been used in single molecule studies to detect protein association with DNA or RNA. Here we applied PIFE for steady state quantification of protein/NA interactions by using microwell plate fluorescence readers (mwPIFE). We demonstrate the general applicability of mwPIFE for examining various aspects of protein/DNA interactions with examples from the restriction enzyme BamHI, and the DNA repair complexes Ku and XPF/ERCC1. These include determination of sequence and structure binding specificities, dissociation constants, detection of weak interactions, and the ability of a protein to translocate along DNA. mwPIFE represents an easy and high throughput method that does not require protein labeling and can be applied to a wide range of applications involving protein/NA interactions.

Epstein-Barr Virus (EBV)-BamHI-A Rightward Transcript (BART)-6 and Cellular MicroRNA-142 Synergistically Compromise Immune Defense of Host Cells in EBV-Positive Burkitt Lymphoma.

BACKGROUND This study was designed to explore the molecular mechanism underlying the effect of cellular miRNAs and EBV miRNA upon the expression of targets such as PTEN, and their involvement in the pathogenesis of Burkitt lymphoma. MATERIAL AND METHODS In this study, we examined several differentially expressed cellular miRNAs in EBV-positive versus EBV-negative Burkett lymphoma tissue samples, and confirmed PTEN as targets of cellular miR-142 by using a bioinformatics tool, luciferase reporter system, oligo transfection, real-time PCR, and Western blot analysis. RESULTS We further confirmed the binding site of miR-142 in the 3'UTR of the target genes, and established the negative regulatory relationship between miRNA and mRNAs with luciferase activity assay. To verify the regulatory relationship between the miRNAs and PTEN, we evaluated the expression of PTEN in the tissue samples, and found that PTEN was downregulated in EBV- positive Burkett lymphoma. Additionally, lymphoma cells were transfected with EBV-BART-6-3p and miR-142 and we found that EBV-BART-6-3p and miR-142 synergistically reduced expression of IL-6R and PTEN. Furthermore, we also examined viability of the cells in each treatment group, and showed that EBV-BART-6-3p and miR-142 synergistically promoted proliferation of the cells. CONCLUSIONS These findings improve our knowledge about the role of miR-142/EBV-BART-6-3p and their target, PTEN, in the development of Burkett lymphoma; they could be novel therapeutic targets for the treatment of EBV-positive Burkett lymphoma.

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system.

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.

Host Gene Expression Is Regulated by Two Types of Noncoding RNAs Transcribed from the Epstein-Barr Virus BamHI A Rightward Transcript Region.

UNLABELLED: In Epstein-Barr virus-infected epithelial cancers, the alternatively spliced BamHI A rightward transcripts (BARTs) are the most abundant viral polyadenylated RNA. The BART introns form the template for the production of 44 microRNAs (miRNAs), and the spliced and polyadenylated exons form nuclear non-protein-coding RNAs. Analysis of host cell transcription by RNA-seq during latency in AGS cells identified a large number of reproducibly changed genes. Genes that were downregulated were enriched for BART miRNA targets. Bioinformatics analysis predicted activation of the myc pathway and downregulation of XBP1 as likely mediators of the host transcriptional changes. Effects on XBP1 activity were not detected in these cells; however, myc activation was confirmed through use of a myc-responsive luciferase reporter. To identify potential regulatory properties of the spliced, polyadenylated BART RNAs, a full-length cDNA clone of one of the BART isoforms was obtained and expressed in the Epstein-Barr virus (EBV)-negative AGS cells. The BART cDNA transcript remained primarily nuclear yet induced considerable and consistent changes in cellular transcription, as profiled by RNA-seq. These transcriptional changes significantly overlapped the transcriptional changes induced during latent EBV infection of these same cells, where the BARTs are exclusively nuclear and do not encode proteins. These data suggest that the nuclear BART RNAs are functional long noncoding RNAs (lncRNAs). The abundant expression of multiple forms of noncoding RNAs that contribute to growth regulation without expression of immunogenic proteins would be an important mechanism for viral oncogenesis in the presence of a functional immune system. IMPORTANCE: Infection with Epstein-Barr virus (EBV) is nearly ubiquitous in the human population; however, it does contribute to the formation of multiple types of cancer. In immunocompromised patients, EBV causes multiple types of lymphomas by expressing viral oncogenes that promote growth and survival of infected B lymphocytes. EBV-positive gastric carcinoma does not require immune suppression, and the viral oncoproteins that are frequent targets for an immunological response are not expressed. This study demonstrates using transcriptional analysis that the expression of various classes of viral non-protein-coding RNAs likely contribute to the considerable changes in the host transcriptional profile in the AGS gastric cancer cell line. This is the first report to show that the highly expressed polyadenylated BamHI A rightward transcripts (BART) viral transcript in gastric carcinoma is in fact a functional viral long noncoding RNA. These studies provide new insight into how EBV can promote transformation in the absence of viral protein expression.

Graphene quantums dots combined with endonuclease cleavage and bidentate chelation for highly sensitive electrochemiluminescent DNA biosensing.

A novel strategy for highly sensitive electrochemiluminescence (ECL) detection of DNA was proposed based on site-specific cleavage of BamHI endonuclease combined with the excellent ECL activity of graphene quantum dots (GQDs) and bidentate chelation of the dithiocarbamate DNA (DTC-DNA) probe assembly. The difference between photoluminescence and ECL spectral peaks suggested that a negligible defect existed on the GQDs surface for generation of an ECL signal. The formed DTC-DNA was directly attached to the gold surface by bidentate anchoring (S-Au-S bonds), which conferred a strong affinity between the ligands and the gold surface, increasing the robustness of DNA immobilization on the gold surface. BamHI endonuclease site-specifically recognized and cleaved the duplex symmetrical sequence, which made the double-stranded DNA fragments and GQDs break off from the electrode surface, inducing a decrease of the ECL signal. Using hepatitis C virus-1b genotype complementary DNA (HCV-1b cDNA) as a model, a novel signal-off ECL DNA biosensor was developed based on variation of the ECL intensity before and after digestion of the DNA hybrid. Electrochemical impedance spectroscopy confirmed the successful fabrication of the ECL DNA biosensor. This ECL biosensor for HCV-1b cDNA determination exhibited a linear range from 5 fM to 100 pM with a detection limit of 0.45 fM at a signal-to-noise ratio of 3 and showed satisfactory selectivity and good stability, which validated the feasibility of the designed strategy. The proposed strategy may be conveniently combined with other specific biological recognition events for expansion of the biosensing application, especially in clinical diagnoses.

Synthesis of a monofunctional platinum compound and its activity alone and in combination with phytochemicals in ovarian tumor models.

Currently used platinum drugs fail to provide long-term cure for ovarian cancer mainly because of acquired drug resistance. In this study, a new monofunctional planaramineplatinum(II) complex, namely tris(8-hydroxyquinoline)monochloroplatinum(II) chloride (coded as LH3), was synthesised and investigated for its activity against human ovarian A2780, cisplatin-resistant A2780 (A2780(cisR)) and ZD0473-resistant A2780 (A2780(ZD0473R)) cancer cell lines, alone and in combination with the phytochemicals curcumin, genistein and resveratrol. Cellular levels of glutathione in A2780 and A2780(cisR) cell lines before and after treatment with LH3 and its combinations with genistein and curcumin were also determined. Interaction of the compounds with salmon sperm DNA, pBR322 plasmid DNA and damage to DNA in A2780 and A2780(cisR) cells due to interaction with LH3-alone and in combination with phytochemicals were also investigated. LH3 was found to be much more active than cisplatin against the resistant tumor models and greatest synergism in activity was observed when combinations of LH3 with genistein and curcumin were administered as a bolus. For combinations of LH3 with the phytochemicals, platinum accumulation and the level of Pt-DNA binding were found to be greater in the resistant A2780(cisR) cell line than in the parental A2780 cell line. Greater activity of LH3 than cisplatin against the resistant ovarian cell lines indicates that it may have the potential for development as a novel anticancer drug and that its combination with phytochemicals can serve to further enhance drug efficacy.

2'-O-methyl nucleotide modified DNA substrates influence the cleavage efficiencies of BamHI and BglII.

Induction of endonucleolytic DNA cleavage is an essential event that links the initiating stimuli to the final effects of cells. The cleavage efficiency and thus the final yield could be affected by many factors, including structures of DNA substrates, composite structures of enzymes-substrates or enzymes-nucleic analogs and so on. However, it is not clear whether a nucleotide derivative-substituted in DNA substrates can influence the efficiency of enzymatic cleavage. To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2'-O-methyl modified nucleotides (OMeN) to modify DNA substrates of isocaudemers BamHI and BglII in this study, and used FRET assay as an efficient method for analysis of enzyme cleavage. Experimental results demonstrated that OMeN-substituted recognition sequences influenced the cleavage rates significantly in a position-dependent manner. OMeN substitutions can reduce the cleavage as expected. Surprisingly, OMeN substitutions can also enhance the cleavage rates. The kinetics parameters of Vmax and Km have been obtained by fitting the Michaelis-Menten kinetic equation. These 2'- OMe nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property could enrich our understanding about the endonuclease cleavage mechanism and enhance our ability to regulate the enzymatic cleavage efficiency for applications in synthetic biology.

Novel function of the Fanconi anemia group J or RECQ1 helicase to disrupt protein-DNA complexes in a replication protein A-stimulated manner.

Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.

Expression, purification and characterization of galectin-1 in Escherichia coli.

As a member of beta-galactoside-binding proteins family, Galectin-1 (Gal-1) contains a single carbohydrate recognition domain, by means of which it can bind glycans both as a monomer and as a homodimer. Gal-1 is implicated in modulating cell-cell and cell-matrix interactions and may act as an autocrine negative growth factor that regulates cell proliferation. Besides, it can also suppress TH1 and TH17 cells by regulating dendritic cell differentiation or suppress inflammation via IL-10 and IL-27. In the present study, Gal-1 monomer and concatemer (Gal-1②), which can resemble Gal-1 homodimer, were expressed in Escherichia coli and their bioactivities were analyzed. The results of this indicate that both Gal-1 and Gal-1② were expressed in E. coli in soluble forms with a purity of over 95% after purifying with ion-exchange chromatography. Clearly, both Gal-1 and Gal-1② can effectively promote erythrocyte agglutination in hemagglutinating activity assays and inhibit Jurkat cell proliferation in MTT assays. All these data demonstrate that bacterially-expressed Gal-1 and Gal-1② have activities similar to those of wild type human Gal-1 whereas the bioactivity of concatemer Gal-1② was stronger than those of the bacterially-expressed and wild type human Gal.

Electrochemical detection of the MT-ND6 gene and its enzymatic digestion: application in human genomic sample.

A simple electrochemical biosensor was developed for the detection of the mitochondrial NADH dehydrogenase 6 gene (MT-ND6) and its enzymatic digestion by BamHI enzyme. This biosensor was fabricated by modification of a glassy carbon electrode with gold nanoparticles (AuNPs/GCE) and a probe oligonucleotide (ssDNA/AuNPs/GCE). The probe, which is a thiolated segment of the MT-ND6 gene, was deposited by self-assembling immobilization on AuNPs/GCE. Two indicators including methylene blue (MB) and neutral red (NR) were used as the electroactive indicators and the electrochemical response of the modified electrode was measured by differential pulse voltammetry. The proposed biosensor can detect the complementary sequences of the MT-ND6 gene. Also the modified electrode was used for the detection of an enzymatic digestion process by BamHI enzyme. The electrochemical biosensor can detect the MT-ND6 gene and its enzymatic digestion in polymerase chain reaction (PCR)-amplified DNA extracted from human blood. Also the biosensor was used directly for detection of the MT-ND6 gene in all of the human genome.

Probing the role of interfacial waters in protein-DNA recognition using a hybrid implicit/explicit solvation model.

When proteins bind to their DNA target sites, ordered water molecules are often present at the protein-DNA interface bridging protein and DNA through hydrogen bonds. What is the role of these ordered interfacial waters? Are they important determinants of the specificity of DNA sequence recognition, or do they act in binding in a primarily nonspecific manner, by improving packing of the interface, shielding unfavorable electrostatic interactions, and solvating unsatisfied polar groups that are inaccessible to bulk solvent? When modeling details of structure and binding preferences, can fully implicit solvent models be fruitfully applied to protein-DNA interfaces, or must the individualistic properties of these interfacial waters be accounted for? To address these questions, we have developed a hybrid implicit/explicit solvation model that specifically accounts for the locations and orientations of small numbers of DNA-bound water molecules, while treating the majority of the solvent implicitly. Comparing the performance of this model with that of its fully implicit counterpart, we find that explicit treatment of interfacial waters results in a modest but significant improvement in protein side-chain placement and DNA sequence recovery. Base-by-base comparison of the performance of the two models highlights DNA sequence positions whose recognition may be dependent on interfacial water. Our study offers large-scale statistical evidence for the role of ordered water for protein-DNA recognition, together with detailed examination of several well-characterized systems. In addition, our approach provides a template for modeling explicit water molecules at interfaces that should be extensible to other systems.

Endonuclease-responsive aptamer-functionalized hydrogel coating for sequential catch and release of cancer cells.

Rare circulating tumor cells are a promising biomarker for the detection, diagnosis, and monitoring of cancer. However, it remains a challenge to develop biomedical devices for specific catch and nondestructive release of circulating tumor cells. The purpose of this study was to explore a unique system for cell catch and release by using aptamer-functionalized hydrogels and restriction endonucleases. The results show that the hydrogel coating was highly resistant to nonspecific cell binding with ~5-15 cells/mm(2) on the hydrogel surface. In contrast, under the same condition, the aptamer-functionalized hydrogel coating could catch target cancer cells with a density over 1000 cells/mm(2). When the hydrogel coating was further treated with the restriction endonucleases, the bound cells were released from the hydrogel coating because of the endonuclease-mediated sequence-specific hydrolysis of the aptamer sequences. The release efficiency reached ~99%. Importantly, ~98% of the released cells maintained viability. Taken together, this study demonstrates that it is promising to apply endonuclease-responsive aptamer-functionalized hydrogels as a coating material to develop medical devices for specific catch and nondestructive release of rare circulating tumor cells.

Toward detection of DNA-bound proteins using solid-state nanopores: insights from computer simulations.

Through all-atom molecular dynamics simulations, we explore the use of nanopores in thin synthetic membranes for detection and identification of DNA binding proteins. Reproducing the setup of a typical experiment, we simulate electric field driven transport of DNA-bound proteins through nanopores smaller in diameter than the proteins. As model systems, we use restriction enzymes EcoRI and BamHI specifically and nonspecifically bound to a fragment of dsDNA, and streptavidin and NeutrAvidin proteins bound to dsDNA and ssDNA via a biotin linker. Our simulations elucidate the molecular mechanics of nanopore-induced rupture of a protein-DNA complex, the effective force applied to the DNA-protein bond by the electrophoretic force in a nanopore, and the role of DNA-surface interactions in the rupture process. We evaluate the ability of the nanopore ionic current and the local electrostatic potential measured by an embedded electrode to report capture of DNA, capture of a DNA-bound protein, and rupture of the DNA-protein bond. We find that changes in the strain on dsDNA can reveal the rupture of a protein-DNA complex by altering both the nanopore ionic current and the potential of the embedded electrode. Based on the results of our simulations, we suggest a new method for detection of DNA binding proteins that utilizes peeling of a nicked double strand under the electrophoretic force in a nanopore.

Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site.

Lentiviral vectors (LVs) are powerful tools for transgene expression in vivo and in vitro. However, the construction of LVs is of low efficiency, due to the large sizes and lack of proper clone sites. Therefore, it is critical to develop efficient strategies for cloning LVs. Here, we reported a combinatorial strategy to efficiently construct LVs using EGFP, hPlk2 wild type (WT) and mutant genes as inserts. Firstly, site-directed mutagenesis (SDM) was performed to create BamH I site for the inserts; secondly, pWPI LV was dephosphorylated after BamH I digestion; finally, the amounts and ratios of the insert and vector DNA were optimized to increase monomeric ligation. Our results showed that the total percentage of positive clones was approximately 48%±7.6%. Using this method, almost all the vectors could be constructed through two or three minipreps. Therefore, our study provided an efficient method for constructing large-size vectors.

Cytotoxicity and DNA interactions of some platinum(II) complexes with substituted benzimidazole ligands.

In the present study, four Pt(II) complexes with 2-ethyl (1)/or benzyl (2)/or p-chlorobenzyl (3)/or 2-phenoxymethyl (4) benzimidazole carrier ligands were evaluated for their in vitro cytotoxic activities against the human HeLa cervix, oestrogen receptor-positive MCF-7 breast, and oestrogen receptor-negative MDA-MB 231 breast cancer cell lines. The plasmid DNA interactions and inhibition of the BamHI restriction enzyme activities of the complexes were also studied. Complex 3 was found to be more active than carboplatin for all examined cell lines and comparable with cisplatin, except for the HeLa cell line.

Intermolecular and intramolecular quencher based quantum dot nanoprobes for multiplexed detection of endonuclease activity and inhibition.

DNA cleavage by endonucleases plays an important role in many biological events such as DNA replication, recombination, and repair and is used as a powerful tool in medicinal chemistry. However, conventional methods for assaying endonuclease activity and inhibition by gel electrophoresis and chromatography techniques are time-consuming, laborious, not sensitive, or costly. Herein, we combine the high specificity of DNA cleavage reactions with the benefits of quantum dots (QDs) and ultrahigh quenching abilities of inter- and intramolecular quenchers to develop highly sensitive and specific nanoprobes for multiplexed detection of endonucleases. The nanoprobe was prepared by conjugating two sets of DNA substrates carrying quenchers onto the surface of aminated QDs through direct assembly and DNA hybridization. With this new design, the background fluorescence was significantly suppressed by introducing inter- and intramolecular quenchers. When these nanoprobes are exposed to the targeted endonucleases, specific DNA cleavages occur and pieces of DNA fragments are released from the QD surface along with the quenchers, resulting in fluorescence recovery. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. The detection was accomplished with a single excitation light. Multiplexed detection was demonstrated by simultaneously assaying EcoRI and BamHI (as model analytes) using two different emissions of QDs. The limits of detection were 4.0 × 10(-4) U/mL for EcoRI and 8.0 × 10(-4) U/mL for BamHI, which were at least 100 times more sensitive than traditional gel electrophoresis and chromatography assays. Moreover, the potential application of the proposed method for screening endonuclease inhibitors has also been demonstrated. The assay protocol presented here proved to be simple, sensitive, effective, and easy to carry out.