Inhibition of sequence-specific protein-DNA interaction and restriction endonuclease cleavage via triplex stabilization by poly(L-lysine)-graft-dextran copolymer.
Triplex stabilization by poly(L-lysine)-graft-dextran copolymer within a mammalian gene promoter inhibits the DNA binding activity of nuclear proteins from HeLa cells as well as restriction endonuclease cleavage at physiological pH and ionic conditions in vitro. Electrophoretic mobility shift assays using a 30-mer homopurine-homopyrimidine stretch (located between -170 and -141 bp) of rat alpha 1 (I) collagen gene promoter reveal that the copolymer, at its wide range of charge ratio with DNA, stabilizes triplex DNA and enhances triplex-specific inhibition of the protein-DNA interaction. When the triplex-forming region (located between -165 and -146 bp) of the promoter is engineered at the Bam H1 and Pst 1 sites of a plasmid DNA, copolymer-mediated triplex stabilization also remarkably competes endonuclease activity of BamH1. Finally, the triplex-stabilizing efficiency of the copolymer is remarkably higher than that of spermine and benzo[e]pyridoindole. Our results indicate that the copolymer, regardless of the length of the target duplex, stabilizes triplexes for significant inhibition of protein-DNA interaction and endonuclease activity. Since stable triplex formation within a short region out of a long native duplex is a prerequisite to confer the therapeutic potential of antigene strategy, triplex stabilization on a long target duplex and inhibition of nuclear protein-DNA interaction may open the possible in vivo applicability of the copolymer.
Ferdous
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Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Akaike
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T
Maruyama
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D000818:Animals; D002417:Cattle; D004247:DNA / Q000737:chemistry*; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D003911:Dextrans / Q000737:chemistry*; D004586:Electrophoresis; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry*; D011108:Polymers; D011188:Potassium / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D051381:Rats; D013950:Thymus Gland / Q000737:chemistry
D003911:Dextrans; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011108:Polymers; D011743:Pyrimidines; D011107:Polylysine; D004247:DNA; D004262:DNA Restriction Enzymes; D011188:Potassium