A silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose functions as an efficient photosensitizer for photodynamic therapy.

We synthesized a new silyl porphyrin derivative conjugated with 6-deoxy-6-sulfo-α-d-glucopyranose (SGlc). Conjugation with SGlc improved A549 cellular uptake without significant changes in the photophysical and photochemical properties and subcellular localization. This improved cellular uptake led to enhanced photodynamic activity. Furthermore, conjugation with SGlc suppressed dark toxicity. These advantages were not observed for a conjugate with a glucose molecule. These results indicated that the conjugation with SGlc is a promising strategy for enhancing photodynamic efficacy.

A new fluorescence labeling method for molecular analysis of double-stranded DNA.

In this study, a double-stranded DNA (dsDNA) fluorescent labeling method was developed using the fusion proteins of fluorescent protein (FP), and 7 kDa DNA-binding family members including Sso7d from Sulfolobus solfataricus, Aho7c from Acidianus hospitalis, ATSV7 from Acidianus tailed spindle virus and Sto7 from Sulfolobus tokodaii. Using this fluorescent DNA labeling method, we succeeded in single-molecule imaging of bacteriophage λDNA molecules stretched on glass surfaces. The fluorescence of the λDNA with FP fusion proteins decayed 2.4- to 6.4-fold slower than that of the typical intercalating method with SYTOX Green (SxG). In addition, the dynamic behaviors of FP-fused Aho7c-λDNA were relaxed and stretched with and without buffer flow, respectively, in microflow channels and were similar to that with typical intercalating dye, such as YOYO-1 and SxG. this fluorescent DNA labeling method. This fluorescent DNA labeling method can solve the problem of rapid fluorescence decay due to the intercalating dyes and therefore can be expected as an alternative to compound-based fluorescent dye. Thus, this study establishes FP fusion proteins as useful fluorescent DNA probes at the single-molecule level.

Synthesis of 3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside (ODSG) as a lipase-resistant SQAP derivative.

Sulfoquynovosylacyl propanediol (SQAP; 1) has been developed as a radiosensitizer (anti-cancer agent) for solid tumors, but it was easily cleaved in vivo and had a problem of short residence time. We synthesized a novel compound of a SQAP derivative (3-octadecanoxypropyl 6-deoxy-6-sulfo-α-d-glucopyranoside: ODSG; 2) to solve these problems not easily cleaved by lipase. ODSG (2) cytotoxicity was investigated in vitro, resulting in low toxicity like SQAP (1).

DNA Manipulation and Single-Molecule Imaging.

DNA replication, repair, and recombination in the cell play a significant role in the regulation of the inheritance, maintenance, and transfer of genetic information. To elucidate the biomolecular mechanism in the cell, some molecular models of DNA replication, repair, and recombination have been proposed. These biological studies have been conducted using bulk assays, such as gel electrophoresis. Because in bulk assays, several millions of biomolecules are subjected to analysis, the results of the biological analysis only reveal the average behavior of a large number of biomolecules. Therefore, revealing the elementary biological processes of a protein acting on DNA (e.g., the binding of protein to DNA, DNA synthesis, the pause of DNA synthesis, and the release of protein from DNA) is difficult. Single-molecule imaging allows the analysis of the dynamic behaviors of individual biomolecules that are hidden during bulk experiments. Thus, the methods for single-molecule imaging have provided new insights into almost all of the aspects of the elementary processes of DNA replication, repair, and recombination. However, in an aqueous solution, DNA molecules are in a randomly coiled state. Thus, the manipulation of the physical form of the single DNA molecules is important. In this review, we provide an overview of the unique studies on DNA manipulation and single-molecule imaging to analyze the dynamic interaction between DNA and protein.

Distribution and metabolism of 14C-sulfoquinovosylacylpropanediol (14C-SQAP) after a single intravenous administration in tumor-bearing mice.

Sulfoquinovosylacylpropanediol (SQAP) is a novel potent radiosensitizer that inhibits angiogenesis in vivo and results in increased oxigenation and reduced tumor volume. We investigated the distribution, metabolism, and excretion of SQAP in male KSN-nude mice transplanted with a human pulmonary carcinoma, Lu65. For the metabolism analysis, a 2 mg (2.98 MBq)/kg of [glucose-U-14C]-SQAP (CP-3839) was intravenously injected. The injected SQAP was decomposed into a stearic acid and a sulfoquinovosylpropanediol (SQP) in the body. The degradation was relatively slow in the carcinoma tissue.1,3-propanediol[1-14C]-SQAP (CP-3635) was administered through intravenous injection of a 1 mg (3.48 MBq)/kg dose followed by whole body autoradiography of the mice. The autoradiography analysis demonstrated that SQAP rapidly distributed throughout the whole body and then quickly decreased within 4 hours except the tumor and excretion organs such as liver, kidney. Retention of SQAP was longer in tumor parts than in other tissues, as indicated by higher levels of radioactivity at 4 hours. The radioactivity around the tumor had also completely disappeared within 72 hours.

A New Method for Immobilization of His-Tagged Proteins with the Application of Low-Frequency AC Electric Field.

Continued advancement of protein array, bioelectrode, and biosensor technologies is necessary to develop methods for higher amount and highly oriented immobilization activity of proteins. In pursuit of these goals, we developed a new immobilization method by combining electrostatic transport and subsequent molecular diffusion of protein molecules. Our developed immobilization method is based on a model that transports proteins toward the substrate surface due to steep concentration gradient generated by low-frequency AC electric field. The immobilization of the maximum amounts can be obtained by the application of the AC voltage of 80 Vpp, 20 Hz both for His-tagged Green Fluorescent Protein (GFP) and Discosoma sp. Red Fluorescent Protein (DsRed), used as model proteins. The amounts of the immobilized His-tagged GFP and DsRed were approximately seven-fold higher than that in the absence of the application of low-frequency AC electric field. Furthermore, the positively and negatively charged His-tagged GFP at acidic and alkaline pH were immobilized by applying of low-frequency AC electric field, whereas the non-charged His-tagged GFP at the pH corresponding to its isoelectric point (pI) was not immobilized. Therefore, unless the pH is equal to pI, the immobilization of electrically charged proteins was strongly enhanced through electrostatic transport and subsequent molecular diffusion.

Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state.

Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.

Direct single-molecule observations of local denaturation of a DNA double helix under a negative supercoil state.

Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA.

Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel.

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.

A new direct single-molecule observation method for DNA synthesis reaction using fluorescent replication protein A.

Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (-flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (-flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (-flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (-flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3'-5'exo-) was promoted by DNA stretching with buffer flow.

Direct observation of fluorescently labeled single-stranded λDNA molecules in a micro-flow channel.

We developed two labeling methods for the direct observation of single-stranded DNA (ssDNA), using a ssDNA binding protein and a ssDNA recognition peptide. The first approach involved protein fusion between the 70-kDa ssDNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). The second method used the ssDNA binding peptide of Escherichia coli RecA labeled with Atto488 (ssBP-488; Atto488-IRMKIGVMFGNPETTTGGNALKFY). The labeled ssλDNA molecules were visualized over time in micro-flow channels. We report substantially different dynamics between these two labeling methods. When ssλDNA molecules were labeled with RPA-YFP, terminally bound fusion proteins were sheared from the free ends of the ssλDNA molecules unless 25-mer oligonucleotides were annealed to the free ends. RPA-YFP-ssλDNA complexes were dissociated by the addition of 0.2 M NaCl, although complex reassembly was possible with injection of additional RPA-YFP. In contrast to the flexible dynamics of RPA-YFP-ssλDNA complexes, the ssBP-488-ssλDNA complexes behaved as rigid rods and were not dissociated even in 2 M NaCl.

Direct observation method of individual single-stranded DNA molecules using fluorescent replication protein A.

Direct observation studies of single molecules have revealed molecular behaviors usually hidden in the ensemble and time-averaging of bulk experiments. Direct single DNA molecule analysis of DNA metabolism reactions such as DNA replication, repair, and recombination is necessary to fully understand these essential processes. Intercalation of fluorescent dyes such as YOYO-1 and SYTOX Orange has been the standard method for observing single molecules of double-stranded DNA (dsDNA), but effective fluorescent dyes for observing single molecules of single-stranded DNA (ssDNA) have not been found. To facilitate direct single-molecule observations of DNA metabolism reactions, it is necessary to establish methods for discriminating ssDNA and dsDNA. To observe ssDNA directly, we prepared a fusion protein consisting of the 70 kDa DNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). This fusion protein had ssDNA-binding activity. In our experiments, dsDNA was stained by SYTOX Orange and ssDNA by RPA-YFP, and we succeeded in staining ssDNA and dsDNA by using RPA-YFP and SYTOX Orange simultaneously.

A new DNA combing method for biochemical analysis.

A simple molecular combing method for analysis of biochemical reactions, called the moving droplet method, has been developed. In this method, small droplets containing DNA molecules run down a sloped glass substrate, and this creates a moving interface among the air, droplet, and substrate that stretches the DNA molecules. This method requires a much smaller volume of sample solution than other established combing methods, allowing wider application in various fields. Using this method, lambdaDNA molecules were stretched and absorbed to a glass substrate, and single-molecule analysis of DNA synthesis by DNA polymerases was performed.

Biochemical characterization of a SET and transposase fusion protein, Metnase: its DNA binding and DNA cleavage activity.

Metnase (SETMAR) is a SET and transposase fusion protein that promotes in vivo end joining activity and mediates genomic integration of foreign DNA. Recent studies showed that Metnase retained most of the transposase activities, including 5'-terminal inverted repeat (TIR)-specific binding and assembly of a paired end complex, and cleavage of the 5'-end of the TIR element. Here we show that R432 within the helix-turn-helix motif is critical for sequence-specific recognition, as the R432A mutation abolishes its TIR-specific DNA binding activity. Metnase possesses a unique DNA nicking and/or endonuclease activity that mediates cleavage of duplex DNA in the absence of the TIR sequence. While the HTH motif is essential for the Metnase-TIR interaction, it is not required for its DNA cleavage activity. The DDE-like motif is crucial for its DNA cleavage action as a point mutation at this motif (D483A) abolished its DNA cleavage activity. Together, our results suggest that Metnase's DNA cleavage activity, unlike those of other eukaryotic transposases, is not coupled to its sequence-specific DNA binding.

Characterization of a second proliferating cell nuclear antigen (PCNA2) from Drosophila melanogaster.

The eukaryotic DNA polymerase processivity factor, proliferating cell nuclear antigen, is an essential component in the DNA replication and repair machinery. In Drosophila melanogaster, we cloned a second PCNA cDNA that differs from that encoded by the gene mus209 (for convenience called DmPCNA1 in this article). The second PCNA cDNA (DmPCNA2) encoded a 255 amino acid protein with 51.7% identity to DmPCNA1, and was ubiquitously expressed during Drosophila development. DmPCNA2 was localized in nuclei as a homotrimeric complex and associated with Drosophila DNA polymerase delta and epsilonin vivo. Treatment of cells with methyl methanesulfonate or hydrogen peroxide increased the amount of both DmPCNA2 and DmPCNA1 associating with chromatin, whereas exposure to UV light increased the level of association of only DmPCNA1. Our observations suggest that DmPCNA2 may function as an independent sliding clamp of DmPCNA1 when DNA repair occurs.

The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair.

The molecular mechanism by which foreign DNA integrates into the human genome is poorly understood yet critical to many disease processes, including retroviral infection and carcinogenesis, and to gene therapy. We hypothesized that the mechanism of genomic integration may be similar to transposition in lower organisms. We identified a protein, termed Metnase, that has a SET domain and a transposase/nuclease domain. Metnase methylates histone H3 lysines 4 and 36, which are associated with open chromatin. Metnase increases resistance to ionizing radiation and increases nonhomologous end-joining repair of DNA doublestrand breaks. Most significantly, Metnase promotes integration of exogenous DNA into the genomes of host cells. Therefore, Metnase is a nonhomologous end-joining repair protein that regulates genomic integration of exogenous DNA and establishes a relationship among histone modification, DNA repair, and integration. The data suggest a model wherein Metnase promotes integration of exogenous DNA by opening chromatin and facilitating joining of DNA ends. This study demonstrates that eukaryotic transposase domains can have important cell functions beyond transposition of genetic elements.

Selective inhibitors of terminal deoxyribonucleotidyltransferase (TdT): baicalin and genistin.

Studies of mammalian terminal deoxyribonucleotidyltransferase (TdT) are facilitated by use of inhibitors that selectively knock down the activity of the enzyme. We have screened for selective inhibitors of TdT and identified a natural compound with this property in the Japanese vegetable, Arctium lappa. The compound has little effect on the activities of mammalian DNA polymerases, such as alpha, beta, delta or lambda polymerase, and prokaryotic DNA polymerases, such as Taq DNA polymerase, T4 DNA polymerase and Klenow fragment. H1- and C13-NMR spectroscopic analyses showed the compound to be baicalin, a compound previously reported as an anti-inflammatory or antipyretic agent. The IC50 value of baicalin to TdT was 18.6 microM. We also found that genistin, a baicalin derivative known to be antimutagenic, more selectively inhibited TdT activity than baicalin, although its IC50 value was weaker (28.7 microM). Genistin and baicalin also inhibited the activity of truncated TdT (the so-called pol beta core domain) in which the BRCT motif was deleted in its N-terminal region. In kinetic analyses, inhibition by either genistin or baicalin was competitive with the primer and non-competitive with the dNTP substrate. The compounds may, therefore, bind directly to the primer-binding site of TdT and simultaneously disturb dNTP substrate incorporation into the primer. Genistin and baicalin should prove to be useful agents for studying TdT.

Structural analysis of epolactaene derivatives as DNA polymerase inhibitors and anti-inflammatory compounds.

Epolactaene (compound 1), a neuritogenic compound found in human neuroblastoma cells, was found to show anti-inflammatory activity in vivo in this study. DNA polymerases and DNA topoisomerase II (topo II) were some of the major molecular targets of compound 1. Since the agent seems to be a potential pharmaceutical medicine, we synthesized derivatives chemically and obtained seven compounds, 1 to 7 to screen clinically more efficient epolactaene derivatives. A comparison of its structural derivatives revealed that the long alkyl side chain seemed to have an important role in the inhibitory effect. Notably, C18-alkyl chain conjugated epolactaene (compound 5) was the strongest inhibitor of DNA polymerase alpha, beta, lambda (pol alpha, beta, lambda) and topo II, with IC50 values of 13, 135, 4.4 and 5 microM, respectively, and 500 microg of compound 5 caused a marked reduction in TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 65.0%). Compound 5 did not influence the activities of plant or prokaryotic DNA polymerases, or of other DNA metabolic enzymes such as telomerase, RNA polymerase and deoxyribonuclease I. Based on these results, the relationship among the three-dimensional structure of epolactaene derivatives and the inhibition of polymerases and topo II, and anti-inflammation is discussed.

Inhibitory effect of conjugated eicosapentaenoic acid on human DNA topoisomerases I and II.

DNA topoisomerases (topos) and DNA polymerases (pols) are involved in many aspects of DNA metabolism such as replication reactions. We reported previously that long chain unsaturated fatty acids such as polyunsaturated fatty acids (PUFA) (i.e., eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA)) inhibited the activities of eukaryotic pols in vitro. In the present study, we found that PUFA also inhibited human topos I and II activities, and the inhibitory effect of conjugated fatty acids converted from EPA and DHA (cEPA and cDHA) on pols and topos was stronger than that of normal EPA and DHA. cEPA and cDHA inhibited the activities of mammalian pols and human topos, but did not affect the activities of plant and prokaryotic pols or other DNA metabolic enzymes tested. cEPA was a stronger inhibitor than cDHA with IC(50) values for mammalian pols and human topos of 11.0-31.8 and 0.5-2.5 microM, respectively. Therefore, the inhibitory effect of cEPA on topos was stronger than that on pols. Preincubation analysis suggested that cEPA directly bound both topos I and II, but did not bind or interact with substrate DNA. This is the first report that conjugated PUFA such as cEPA act as inhibitors of pols and topos. The results support the therapeutic potential of cEPA as a leading anti-cancer compound that poisons pols and topos.

Affinity capture of a mammalian DNA polymerase beta by inhibitors immobilized to resins used in solid-phase organic synthesis.

The application of resins normally used in solid-phase organic synthesis to the affinity capture of a mammalian DNA polymerase beta (pol beta) is reported. Lithocholic acid (LCA), an inhibitor of pol beta, was immobilized on various solid supports, and the batch affinity purification of pol beta from a mixture of proteins using these LCA-immobilized resins was examined. Of the resins tested, TentaGel was the most effective at purifying pol beta and at resisting nonspecific absorption of proteins. The immobilized LCA recognized pol beta specifically, which resulted in pol beta binding to the resin. Using the LCA-immobilized resin, it was possible to purify pol beta from a mixture of proteins. Furthermore, it was possible to concentrate pol beta from a crude nuclear extract of human T lymphoma Molt4 cells. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The pol beta inhibitors LCA, C18-beta-SQDG, and epolactaene were immobilized on the photoaffinity beads by photoreaction. The batch affinity purification of pol beta from a protein mixture could be also achieved with these beads.