Synthetic pyrrole-imidazole polyamide inhibits expression of the human transforming growth factor-beta1 gene.
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Pyrrole-imidazole (Py-Im) polyamides can bind to the predetermined base pairs in the minor groove of double-helical DNA with high affinity. These synthetic small molecules can interfere with transcription factor-DNA interaction and inhibit or activate the transcription of corresponding genes. In the present study, we designed and synthesized a Py-Im polyamide to target -545 to -539 base pairs of human transforming growth factor-beta1 (hTGF-beta1) promoter adjacent to the fat-specific element 2 (FSE2) to inhibit the expression of the gene. Gel mobility shift assay showed that the synthetic Py-Im polyamide binds to its corresponding double-strand oligonucleotides, whereas the mismatch polyamides did not bind. Fluorescein isothiocyanate-labeled Py-Im polyamide was detected in the nuclei of human vascular smooth muscle cells (VSMCs) after 2- to 48-h incubation. Py-Im polyamide significantly decreased the promoter activity of hTGF-beta1 determined by in vitro transcription experiments and luciferase assay. In cultured human VSMCs, Py-Im polyamide targeting hTGF-beta1 promoter significantly inhibited expressions of hTGF-beta1 mRNA and protein. These results indicate that the synthetic Py-Im polyamide designed to bind hTGF-beta1 promoter inhibited hTGF-beta1 gene and protein expression successfully. This novel agent will be used for the TGF-beta-related diseases as a gene therapy. (+info)
Investigation of noncovalent complexes between beta-cyclodextrin and polyamide acids containing N-methylpyrrole and N-methylimidazole by electrospray ionization mass spectrometry.
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Electrospray ionization (ESI) mass spectrometry was utilized to investigate noncovalent complexes between beta-cyclodextrin (beta-CD) and five novel polyamide acids containing N-methylpyrrole and N-methylimidazole. The 1:1 binding mode was specified by examining the binding stoichiometry from ESI mass spectra. It found that polyamide acids with beta-CD have binding affinities in the order: ImImImbetaCOOH > ImPyImbetaCOOH > ImPyPybetaCOOH > PyPyPybetaCOOH > NO(2)PyPyPybetaCOOH. The method gives, simultaneously, the binding constants between beta-CD and polyamide acids based on a novel linear equation. (+info)
Development of gene silencing pyrrole-imidazole polyamide targeting the TGF-beta1 promoter for treatment of progressive renal diseases.
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Pyrrole-imidazole (Py-Im) polyamides are nuclease-resistant novel compounds that inhibit gene expression by binding to the minor groove of DNA. A Py-Im polyamide that targets rat TGF-beta1 was designed as a gene-silencing agent for progressive renal diseases, and the distribution and the effects of this polyamide on renal injury were examined in Dahl-salt sensitive (Dahl-S) rats. For identification of transcription factor binding elements for activation of the rat TGF-beta1 gene, recombinant TGF-beta1 reporter plasmids were transfected into HEK-293 cells, and promoter activity was measured. Py-Im polyamide was designed to the activator protein-1 binding site of the rat TGF-beta1 promoter. This Py-Im polyamide showed strong, fast, and specific binding to the target DNA in gel mobility shift and Biacore assays. Py-Im polyamide significantly inhibited TGF-beta1 promoter activity and expression of TGF-beta1 mRNA and protein in rat mesangial cells. Intravenously administered fluorescein-labeled polyamide distributed to the kidney of rats. Py-Im polyamide significantly inhibited expression of TGF-beta1 mRNA and protein in the renal cortex of Dahl-S rats and reduced the increase in urinary protein and albumin in Dahl-S rats independent of changes in blood pressure. These results indicate that Py-Im polyamide that targets TGF-beta1 will be a novel gene-silencing agent for the TGF-beta1-associated diseases, including progressive renal diseases. (+info)
Defining the sequence-recognition profile of DNA-binding molecules.
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Determining the sequence-recognition properties of DNA-binding proteins and small molecules remains a major challenge. To address this need, we have developed a high-throughput approach that provides a comprehensive profile of the binding properties of DNA-binding molecules. The approach is based on displaying every permutation of a duplex DNA sequence (up to 10 positional variants) on a microfabricated array. The entire sequence space is interrogated simultaneously, and the affinity of a DNA-binding molecule for every sequence is obtained in a rapid, unbiased, and unsupervised manner. Using this platform, we have determined the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor. The approach also yielded unique insights into the altered sequence-recognition landscapes as a result of cooperative assembly of DNA-binding molecules in a ternary complex. Solution studies strongly corroborated the sequence preferences identified by the array analysis. (+info)
Alkylation of template strand of coding region causes effective gene silencing.
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We recently developed a new type of pyrrole (Py)-imidazole (Im) polyamide-tetrahydrocyclopropabenzindolone (CBI) conjugate with an indole linker as a stable sequence-specific alkylating agent. In this study, we investigated the gene silencing activities of polyamides A, B and C, which selectively alkylate specific sequences in the promoter region, non-coding strand and coding strand, respectively, of the green fluorescent protein (GFP) gene. GFP vectors were transfected into human colon carcinoma cells (HCT116), and the cells were treated with 100 nM of the polyamides for 24 h. Fluorescence microscopy indicated that a significant reduction of GFP fluorescence was only observed in the cells that were treated with polyamide C. In clear contrast, polyamides A and B did not show such activity. Moreover, real-time PCR demonstrated selective reduction of the expression of GFP mRNA following treatment with polyamide C. These results suggest that alkylating Py-Im polyamides that target the coding strand represent a novel approach for sequence-specific gene silencing. (+info)
Crystallization and X-ray diffraction analysis of 6-aminohexanoate-dimer hydrolase from Arthrobacter sp. KI72.
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To investigate the structure-function relationship between 6-aminohexanoate-dimer hydrolase (EII) from Arthrobacter sp. and a cryptic protein (EII') which shows 88% sequence identity to EII, a hybrid protein (named Hyb-24) of EII and EII' was overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in MES buffer pH 6.5. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 96.37, c = 113.09 A. Diffraction data were collected from native and methylmercuric chloride derivative crystals to resolutions of 1.75 and 1.80 A, respectively. (+info)
Anticancer efficacies of cisplatin-releasing pH-responsive nanoparticles.
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The objective of these investigations was to test the hypothesis that a rapid cytoplasmic release profile from nanoparticles would potentiate the anticancer activity of cisplatin. Cisplatin-loaded nanoparticles with pH-responsive poly[2-(N,N-diethylamino)ethyl methacrylate] (PDEA) cores were synthesized from PDEA-block-poly(ethylene glycol) (PDEA-PEG) copolymer by using a solvent-displacement (acetone-water) method. Nanoparticles with pH-nonresponsive poly(epsilon-caprolactone) (PCL) cores made from PCL-block-PEG (PCL-PEG) were used for comparison. Nanoparticle sizes, zeta potentials, drug-loading capacities, and pH responsiveness were characterized. The cellular uptakes and localization in lysosomes were visualized by using confocal fluorescence microscopy. Cytostatic effects of free and encapsulated cis-diammineplatinum(II) dichloride (cisplatin) toward human SKOV-3 epithelial ovarian cancer cells were estimated by using the MTT assay. Intraperitoneal tumor responses to cisplatin and cisplatin/PDEA-PEG were evaluated in athymic mice at 4-6 weeks postinoculation of SKOV-3 cells. PDEA-PEG nanoparticles dissolved at pH < 6 and rapidly internalized and transferred to lysosomes; it therefore was predicted that the PDEA nanoparticles would rapidly release cisplatin into cytoplasm upon integration into acidic lysosomes and thereby overwhelm the chemoresistant properties of SKOV-3 cells. Indeed, relative proportions of viable cells were diminished to a greater extent by exposure in vitro to fast-releasing nanoparticles compared to slow-releasing nanoparticles or an equivalent dose of free cisplatin. Incidences of cellular pyknosis (a morphological indicator of apoptosis) were most evident within intestinal/mesentery tumors of mice treated with cisplatin/PDEA-PEG; tumor burdens were correspondingly reduced. (+info)
Displacement of D1, HP1 and topoisomerase II from satellite heterochromatin by a specific polyamide.
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The functions of DNA satellites of centric heterochromatin are difficult to assess with classical molecular biology tools. Using a chemical approach, we demonstrate that synthetic polyamides that specifically target AT-rich satellite repeats of Drosophila melanogaster can be used to study the function of these sequences. The P9 polyamide, which binds the X-chromosome 1.688 g/cm3 satellite III (SAT III), displaces the D1 protein. This displacement in turn results in a selective loss of HP1 and topoisomerase II from SAT III, while these proteins remain bound to the adjacent rDNA repeats and to other regions not targeted by P9. Conversely, targeting of (AAGAG)n satellite V repeats by the P31 polyamide results in the displacement of HP1 from these sequences, indicating that HP1 interactions with chromatin are sensitive to DNA-binding ligands. P9 fed to larvae suppresses the position-effect variegation phenotype of white-mottled adult flies. We propose that this effect is due to displacement of the heterochromatin proteins D1, HP1 and topoisomerase II from SAT III, hence resulting in stochastic chromatin opening and desilencing of the nearby white gene. (+info)