Transcription Factor Decoy Molecules Based on a Peptide Nucleic Acid (PNA)-DNA Chimera Mimicking Sp1 Binding Sites
2003; Elsevier BV; Volume: 278; Issue: 9 Linguagem: Inglês
10.1074/jbc.m206780200
ISSN1083-351X
AutoresMonica Borgatti, Ilaria Lampronti, Alessandra Romanelli, Carlo Pedone, Michele Saviano, Nicoletta Bianchi, Carlo Mischiati, Roberto Gambari,
Tópico(s)RNA and protein synthesis mechanisms
ResumoPeptide nucleic acids (PNAs) are DNA-mimicking molecules in which the sugar-phosphate backbone is replaced by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. We determined whether double-stranded molecules based on PNAs and PNA-DNA-PNA (PDP) chimeras could be capable of stable interactions with nuclear proteins belonging to the Sp1 transcription factor family and, therefore, could act as decoy reagents able to inhibit molecular interactions between Sp1 and DNA. Since the structure of PNA/PNA hybrids is very different from that of the DNA/DNA double helix, they could theoretically alter the molecular structure of the double-stranded PNA-DNA-PNA chimeras, perturbing interactions with specific transcription factors. We found that PNA-based hybrids do not inhibit Sp1/DNA interactions. In contrast, hybrid molecules based on PNA-DNA-PNA chimeras are very effective decoy molecules, encouraging further experiments focused on the possible use of these molecules for the development of potential agents for a decoy approach in gene therapy. In this respect, the finding that PDP-based decoy molecules are more resistant than DNA/DNA hybrids to enzymatic degradation appears to be of great interest. Furthermore, their resistance can even be improved after complexation with cationic liposomes to which PDP/PDP chimeras are able to bind by virtue of their internal DNA structure. Peptide nucleic acids (PNAs) are DNA-mimicking molecules in which the sugar-phosphate backbone is replaced by a pseudopeptide backbone composed of N-(2-aminoethyl)glycine units. We determined whether double-stranded molecules based on PNAs and PNA-DNA-PNA (PDP) chimeras could be capable of stable interactions with nuclear proteins belonging to the Sp1 transcription factor family and, therefore, could act as decoy reagents able to inhibit molecular interactions between Sp1 and DNA. Since the structure of PNA/PNA hybrids is very different from that of the DNA/DNA double helix, they could theoretically alter the molecular structure of the double-stranded PNA-DNA-PNA chimeras, perturbing interactions with specific transcription factors. We found that PNA-based hybrids do not inhibit Sp1/DNA interactions. In contrast, hybrid molecules based on PNA-DNA-PNA chimeras are very effective decoy molecules, encouraging further experiments focused on the possible use of these molecules for the development of potential agents for a decoy approach in gene therapy. In this respect, the finding that PDP-based decoy molecules are more resistant than DNA/DNA hybrids to enzymatic degradation appears to be of great interest. Furthermore, their resistance can even be improved after complexation with cationic liposomes to which PDP/PDP chimeras are able to bind by virtue of their internal DNA structure. nuclear factor κB promoter-specific transcription factor Sp1 human immunodeficiency virus type 1 long terminal repeat oligodeoxyribonucleotide peptide nucleic acid PNA-DNA-PNA chimera urokinase-type plasminogen activator urokinase-type plasminogen activator receptor cytosine arabinoside high pressure liquid chromatography tetralysine-cholesterol tetralysine-palmitate liposomal formulation of Lys4-Chol liposomal formulation of Lys4-Palm N-(9-fluorenyl)methoxycarbonyl globin In vitro transfection of cis elements that decoy against nuclear factors leads to alteration of gene expression and was recently proposed in molecular medicine as a novel tool for the possible therapy of several disorders (1–12). 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In addition, it was found that the in vitroinvasiveness, synthesis of mRNA for uPA, and cell proliferation were also inhibited by the transfection of Sp1 decoy ODNs, suggesting that Sp1 decoy strategy could be effective for regulating tumor growth by reducing in cancer cell (a) angiogenic growth factor expression, (b) proliferation, and (c) invasiveness. In another report, Verrecchia et al. (21Verrecchia F. Rossert J. Mauviel A. J. Invest. Dermatol. 2001; 116: 755-763Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) found that decoy Sp1-binding ODNs inhibited COL1A2promoter activity both in cultured fibroblasts and in vivo, in the skin of transgenic mice, which have integrated a mouseCOL1A2 promoter/luciferase reporter gene construct, indicating that targeting Sp1 efficiently blocks extracellular matrix gene expression, and suggest that such an approach may represent an interesting therapeutic alternative toward the treatment of fibrotic disorders. 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In the present report, we determined whether PNA/PNA and PNA/DNA hybrid could act as decoy molecules for transcription factor Sp1. Furthermore, we investigated binding to Sp1 transcription factor(s) and biological activity of PNA-DNA-PNA chimeras mimicking Sp1 binding sites. The synthetic oligonucleotides used in this study were purchased from Amersham Biosciences. HPLC-purified PNAs were purchased from ISOGEN Biosciences (Maarssen, the Netherlands). Chimera synthesis proceeded by sequential elongation of the PNA fragment, to which DNA first and then PNA were attached. Aminomethyl-polystiren-NH2 resin (loading 37 μmol/g), resin functionalized with an hexamethylene bisacetamide linker bound through an ester bond to a Gly, was used. The DNA part of the chimera was prepared on anAmersham Biosciences Gene Assembler. Chain elongation was performed with 15 eq of methyl DNA phosphoramidites using 5-(o-nitrophenyl)tetrazole (8 eq) as the activator. Standard DNA capping, washing, oxidation, and detritylation cycles were used. Coupling yields were gauged spectrophotometrically (254 nm) by the absorption of the released trityl cation after each deprotection step. In the last DNA elongation step, cyanoethyl 5-amino-5-deoxythymidine phosphoramidite was used (38van der Laan A.C. Havenaar P. Oosting R.S. Kuyl-Yeheskiely E. Uhlmann E. van Boom J.H. Bioorg. Med. Chem. Lett. 1998; 8: 663-668Crossref PubMed Scopus (37) Google Scholar, 39van der Laan A.C. Meeuwenoord N.J. Kuyl-Yeheskiely E. Oosting R.S. Brands R. van Boom J.H. Recl. Trav. Chim. Pays-Bas. 1995; 114: 295-297Crossref Scopus (36) Google Scholar). The PNA part of the chimera was prepared on a full automated PerSeptive Biosystems Expedite 8900 Nucleic Acid synthesizer (PerSeptive Biosystems, Foster City, CA) using standard (designed for 2-μmol scale) PNA coupling cycles and solutions. Fmoc (Bz, benzyl)/(iBu, isobutyl)-protected PNA was used. To improve the coupling efficiency of the first PNA moiety, a double coupling cycle was employed (40Thomson S.A. Josey J.A. Cadilla R. Gaul M.D. Hassman C.F. Luzzio M.J. Pipe A.J. Reed K.L. Ricca D.J. Wiethe R.W. Noble S.A. Tetrahedron. 1995; 51: 6179-6194Crossref Scopus (235) Google Scholar). Upon completion of the last elongation cycle, the terminal Fmoc group was cleaved by piperidine treatment, and the primary amine was acetylated. The methyl groups were removed from the phosphate functions by treatment of the resin with 0.25 ml of thiophenol in 0.5 ml of tetrahydrofuran and 0.5 ml of triethylamine for 45 min. The resin was washed consecutively with tetrahydrofuran, methanol, acetonitrile, and water (5 × 1 ml for each solvent). The oligomers were cleaved from the support with concomitant deprotection of the remaining protective groups by treatment with 0.1 m sodium hydroxide in water/dioxane (1/1, v/v, 1.5 ml) at 55 °C for 16 h. The reaction mixtures were neutralized by the addition of acetic acid, concentrated, and redissolved in 0.15 m ammonium bicarbonate. Desalting was performed using a Sephadex G-25 (superfine, DNA grade) gel filtration column with 0.15 m ammonium bicarbonate buffers. Samples were filtered and then purified by reverse-phase-HPLC on a LiCrosphere 100 RP-18 endcapped column (4 × 250 mm) on a Jasco HPLC system. Gradient elution was performed at 40 °C, building up gradient starting with buffer A (50 mm triethylammonium acetate in water) and applying buffer B (50 mmtriethylammonium acetate in acetonitrile/water 1/1 v/v) with a flow rate of 1 ml/min. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analyses were performed on all chimeras as follows: (a) 5842.6 (M+H)+ and (b) 5602.0 (M+H)+; (a) cag- *TGA GGC GTG GCCA -ggg-Gly and (b) ccc- *TGG CCACGC CTCA -ctg-Gly. The electrophoretic mobility shift assay (41Feriotto G. Mischiati C. Gambari R. Biochem. J. 1994; 299: 451-458Crossref PubMed Scopus (36) Google Scholar, 42Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar) was performed using the double-stranded synthetic oligonucleotides mimicking the Sp1 (nucleotide sequences are presented in Table I and in Fig.1). The DNA stretches of the target molecules were 5′-end-labeled using [γ-32P]ATP and T4 polynucleotide kinase (MBI Fermentas, Milano, Italy) in the case of DNA/DNA, DNA/PNA, and DNA/PDP hybrids. 32P-labeled PDP/PDP molecules were obtained by nick translation, using low concentrations (0.1–0.01 units/reaction) of DNase I and [α-32P]dCTP. Binding reactions were set up as described elsewhere (43Bianchi N. Osti F. Rutigliano C. Ginanni Corradini F. Borsetti E. Tomassetti M. Mischiati C. Feriotto G. Gambari R. Br. J. Haematol. 1999; 104: 258-265Crossref PubMed Scopus (86) Google Scholar) in a total volume of 25 μl of binding buffer plus 5% glycerol, 1 mm dithiothreitol, and 0.25 ng of32P-labeled oligonucleotides. 12 μg of crude nuclear extracts isolated from human cell lines were used, and the binding reaction was carried out in the presence of 1 μg of the nonspecific competitor poly(dIdC)·poly(dIdC) (43Bianchi N. Osti F. Rutigliano C. Ginanni Corradini F. Borsetti E. Tomassetti M. Mischiati C. Feriotto G. Gambari R. Br. J. Haematol. 1999; 104: 258-265Crossref PubMed Scopus (86) Google Scholar). After 20 min of binding at room temperature, the samples were electrophoresed at constant voltage (200 V) under low ionic strength conditions (0.25× TBE buffer = 22 mm Tris borate, 0.4 mm EDTA) on 6% polyacrylamide gels. Gels were dried and subjected to standard autoradiographic procedures (43Bianchi N. Osti F. Rutigliano C. Ginanni Corradini F. Borsetti E. Tomassetti M. Mischiati C. Feriotto G. Gambari R. Br. J. Haematol. 1999; 104: 258-265Crossref PubMed Scopus (86) Google Scholar). In competition experiments, the competitor molecules carrying HIV-1 Sp1 binding sites (DNA/DNA, PNA/PNA, DNA/PNA, PDP/PDP, and DNA/PDP) were preincubated for 20 min with nuclear extracts, before the addition of labeled target DNA. Nuclear extracts were prepared according to Dignam et al.(42Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar). The nucleotide sequences of competitor double-stranded target DNAs used as controls were 5′-TAA TAT GTA AAA ACA TT-3′ (sense strand, NF-IL2A), 5′-CAC TTG ATA ACA GAA AGT GAT AAC TCT-3′ (sense strand, GATA-1), and 5′-CAT GTT ATG CAT ATT CCT GTA AGT G-3′ (sense strand, STAT-1).Table ISynthetic oligonucleotides, PNA, and PNA-DNA chimerasGene/genomeEntrySequenceLengthOligonucleotidesHIV-1HIV-Sp15′-TGAGGCGTGGCCT-3′14-merHIV-Sp1c5′-AGGCCACGCCTCA-3′14-merHIV-Sp1-L5′-CAGTGAGGCGTGGCCAGGG-3′19-merHIV-Sp1-Lc5′-CCCTGGCCACGCCTCACTG-3′19-meruPA receptoruPAR-Sp15′-CCAGCCGGCCGCGCCCCGGGAAGGGA-3′26-meruPAR-Sp1c5′-TCCCTTCCCGGGGCGCGGCCGGCTGG-3′26-merγ-globinγ-glob-Sp15′-CTAAACTCCACCCATGGGTT-3′20-merγ-glob-Sp1c5′-AACCCATGGGTGGAGTTTAG-3′20-merɛ-globinɛ-glob-Sp15′-GGACCTGACTCCACCCCTGAGG-3′22-merɛ-glob-Sp1c5′-CCTCAGGGGTGGAGTCAGGTCC-3′22-merPNAHIV-1HIV-PNA-Sp1NH2-TGA GG CGT GGC CT-Ac14-merHIV-PNA-Sp1cNH2-AGG CCA CGC CTC A-Ac14-merPNA-DNA chimerasHIV-1HIV-PDP-Sp1NH2-CAGTGAGGCGTGGCCAGGG-gly19-merHIV-PDP-Sp1cNH2-CCCTGGCCACGCCTCACTG-gly19-merPNA stretches are underlined. Open table in a new tab PNA stretches are underlined. Human erythroleukemia K562(S) cells (44Bianchi N. Chiarabelli C. Borgatti M. Mischiati C. Fibach E. Gambari R. Br. J. Haematol. 2001; 113: 951-961Crossref PubMed Scopus (74) Google Scholar) were cultured in a humidified atmosphere at 5% CO2 in RPMI 1640 (Flow Laboratories) supplemented with 10% fetal bovine serum (CELBIO), 50 units/ml penicillin, and 50 μg/ml streptomycin (45Gambari R. del Senno L. Barbieri R. Viola L. Tripodi M. Raschellà G. Fantoni A. Cell Differ. 1984; 14: 87-97Crossref PubMed Scopus (70) Google Scholar, 46Bianchi N. Ongaro F. Chiarabelli C. Gualandi L. Mischiati C. Bergamini P. Gambari R. Biochem. Pharmacol. 2000; 60: 31-40Crossref PubMed Scopus (101) Google Scholar). Cell growth was studied by determining the cell number/ml after different days of in vitro cell culture (46Bianchi N. Ongaro F. Chiarabelli C. Gualandi L. Mischiati C. Bergamini P. Gambari R. Biochem. Pharmacol. 2000; 60: 31-40Crossref PubMed Scopus (101) Google Scholar). Stock solutions of ara-C (100 μm) were stored at −20 °C in the dark and diluted immediately before use. Treatment with the indicated concentrations of DNA- and PNA-based molecules was carried out by adding the appropriate concentrations of the compounds at the beginning of the experiment (cells were usually seeded at 30,000 cells/ml). The medium was not changed during the induction period. K562 cells containing heme or hemoglobin were detected by specific reaction with a benzidine/hydrogen peroxide solution as reported elsewhere (45Gambari R. del Senno L. Barbieri R. Viola L. Tripodi M. Raschellà G. Fantoni A. Cell Differ. 1984; 14: 87-97Crossref PubMed Scopus (70) Google Scholar,46Bianchi N. Ongaro F. Chiarabelli C. Gualandi L. Mischiati C. Bergamini P. Gambari R. Biochem. Pharmacol. 2000; 60: 31-40Crossref PubMed Scopus (101) Google Scholar). The final concentration of benzidine was 0.2% in 5 mglacial acetic acid, 10% H2O2 (45Gambari R. del Senno L. Barbieri R. Viola L. Tripodi M. Raschellà G. Fantoni A. Cell Differ. 1984; 14: 87-97Crossref PubMed Scopus (70) Google Scholar, 46Bianchi N. Ongaro F. Chiarabelli C. Gualandi L. Mischiati C. Bergamini P. Gambari R. Biochem. Pharmacol. 2000; 60: 31-40Crossref PubMed Scopus (101) Google Scholar). The stability of decoy molecules was evaluated after incubation of DNA and PNA-DNA-PNA-based decoys with 3′ → 5′-exonuclease III, 5′ → 3′-λ-exonuclease, and DNase I. ExoIII, λ-exonuclease, and DNase I were purchased from MBI Fermentas (ExoIII and λ-exonucleases) and Promega Corp., Madison, WI (DNase I). In addition, serum (fetal calf serum, Eurobio, 30 g/liter of protein concentration) was also employed. After incubation with increasing amounts of the enzymes (for 10 min in the case of ExoIII, for 30 min in the case of λ-exonuclease and DNase I), the decoy molecules were layered on the top of a 2% agarose gel and detected by ethidium bromide staining. Disappearance of the decoy molecule was considered as an evidence of degradation by the employed enzymes. Results were presented as percentage of recovery with respect to control untreated reaction mixtures. Egg phosphatidyl choline was purchased from Lipid Products (Surrey, England). Tetralysine cationic lipids, tetralysine-cholesterol (Lys4-Chol), and tetralysine-palmitate (Lys4-Palm) were a generous gift of Prof. M. Marastoni (Department of Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy). Positively charged liposomes were produced by a protocol based on reverse phase evaporation followed by extrusion of the liposome suspension through polycarbonate filters with homogeneous pore size. Liposomes were subjected to one extrusion cycle through two stacked 400-nm pore size filters followed by three extrusion cycles through two stacked 200-nm pore size membranes in order to obtain unilamellar liposomes with a homogeneous size distribution. Different cationic detergents were alternatively used for the production of the liposomes, namely Lys4-Chol and Lys4-Palm (47Nastruzzi C. Cortesi R. Esposito E. Gambari R. Borgatti M. Bianchi N. Feriotto G. Mischiati C. J. Controlled Release. 2000; 68: 237-249Crossref PubMed Scopus (52) Google Scholar). The resulting liposomal formulations were named as follows: lipo-Lys4-Chol and lipo-Lys4-Palm. The morphological and dimensional analysis of the produced liposomes was performed by freeze-fracture electron microscopy technique and photo correlation spectroscopy (Zetasizer, Malvern, UK). The freeze-fracture electron micrographs (47Nastruzzi C. Cortesi R. Esposito E. Gambari R. Borgatti M. Bianchi N. Feriotto G. Mischiati C. J. Controlled Release. 2000; 68: 237-249Crossref PubMed Scopus (52) Google Scholar, 48Nastruzzi C. Gambari R. Menegatti E. Walde P. Luisi P.L. J. Pharm. Sci. 1990; 79: 672-677Abstract Full Text PDF PubMed Scopus (11) Google Scholar) confirmed that the extruded liposomal suspension was mainly constituted by unilamellar vesicles. Photon correlation spect
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