Triple Helix-Mediated Inhibition of Gene Expression Is Increased by PUVA Robert Besch, Christoph Marschall, Theda Schuh, Carine Giovannangeli, Claudia.

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Triple Helix-Mediated Inhibition of Gene Expression Is Increased by PUVA Robert Besch, Christoph Marschall, Theda Schuh, Carine Giovannangeli, Claudia Kammerbauer, Klaus Degitz Journal of Investigative Dermatology Volume 122, Issue 5, Pages 1114-1120 (May 2004) DOI: 10.1111/j.0022-202X.2004.22521.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Triple helix target sequence and oligonucleotides. (A) The ICAM-1 gene structure is indicated with numbered exons. A 16 bp target site is localized in the seventh exon and corresponds to positions #2465–#2450 of Genbank accession number NM_000201. (B) A 32 bp gene segment is depicted, which includes the 16 bp oligopyrimidine·oligopurine target sequence (underlined) and a 5′-TpA site (boxed) suitable for psoralen/UVA photoadduct formation. Sequences of the triplex-forming oligonucleotide (TFO) and the scrambled control oligonucleotide (CO) are juxtaposed to the target sequence. Oligonucleotides are psoralen and triethyleneglycol (TEG) conjugated as indicated. Journal of Investigative Dermatology 2004 122, 1114-1120DOI: (10.1111/j.0022-202X.2004.22521.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Triplex formation at the target sequence. (A) A digoxigenin-labeled (*) 32 bp double-stranded target fragment (5 nM) containing the ICAM-1-target sequence (as shown in Figure 1) was incubated with various concentrations of triplex-forming oligonucleotide (TFO) or control oligonucleotide (CO), as indicated (see Materials and Methods for details). Triple helices (a) were retarded under non-denaturing conditions relative to unbound double-stranded target sequences (b). (B) The digoxigenin-labeled 32 bp double-stranded target fragment (5 nM) was incubated with oligonucleotides (5 μM) and irradiated with various UVA dosages, as indicated. Photoadducts (c, cross-links; b, monoadducts) were retarded under denaturing conditions relative to unbound single-stranded target sequence (a). Journal of Investigative Dermatology 2004 122, 1114-1120DOI: (10.1111/j.0022-202X.2004.22521.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Expression plasmids containing different target sequences. The expression plasmid pCM51 was constructed as described under Materials and Methods and contains a multiple cloning site between the SV40 promoter and the chloramphenicol acetyltransferase (CAT) gene (A, ‘no target sequence’). Different sequences were cloned into the multiple cloning site: plasmid pCM52 (B) contains the 16 bp triple helix target sequence (underlined); pCM52m (C) contains the target sequence in which one central base position is mutated (boxed); and pCM53 (D) contains a 16 bp scrambled polypurine sequence (underlined). Journal of Investigative Dermatology 2004 122, 1114-1120DOI: (10.1111/j.0022-202X.2004.22521.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Triple helix-mediated inhibition of chloramphenicol acetyltransferase (CAT) expression is enhanced by psoralen/UVA. The plasmid pCM52 containing the 16 bp triple helix target sequence was incubated with psoralen-modified triplex-forming (TFO) or control (CO) oligonucleotides and either UVA irradiated (5 J per cm2) or not. The reactions were then used to transfect A431 cells, which were lyzed 6 h after transfection. The amount of CAT protein was determined in lysates by ELISA. Bars represent the percentage of CAT expression (mean±SD) relative to CAT expression of control cells transfected with reactions containing the plasmid, but no oligonucleotides. Statistical analysis was performed with ANOVA (Fisher's protected least significant difference test). Journal of Investigative Dermatology 2004 122, 1114-1120DOI: (10.1111/j.0022-202X.2004.22521.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Triple helix formation at the target site is sequence specific. Plasmids containing the triple helix target sequence (pCM52), no target sequence (pCM51), a scrambled control sequence (pCM53), or a mismatched target sequence (pCM52m) were incubated with triplex-forming (TFO) or control (CO) oligonucleotides, and irradiated with UVA (2 J per cm2). The reactions were then used to transfect A431 cells. Cells were lyzed 6 h after transfection, and chloramphenicol acetyltransferase (CAT) protein determined in lysates by ELISA. Bars (mean±SD of three independent experiments) represent the percentage of CAT expression of cells transfected with TFO-treated plasmids relative to cells transfected with CO-treated plasmids. Journal of Investigative Dermatology 2004 122, 1114-1120DOI: (10.1111/j.0022-202X.2004.22521.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions