FIG. 1.

Binding of PPRH to dsDNA. This binding occurs by triplex formation with the polypyrimidine strand, causing the displacement of the polypurine strand, allowing the binding of a specific oligonucleotide (RO13 or pPy13). Lane 1, labeled ³²P-polypurine/³²P-polypyrimidine duplex (Dup13) (1.2 nM); lane 2, ³²P-polypurine/polypyrimidine duplex (1.33 nM); lane 3, ³²P-Dup13 plus hairpin-13 (Hp13); lane 4, ³²P-Dup13 plus Hp13 and RO13; lane 5, ³²P-RO13 (13.14 nM) plus Hp13; lane 6, ³²P-Dup13 plus RO13; lane 7, ³²P-polypurine ssDNA (pPu13) (2.28 nM); lane 8, ³²P-polypurine/polypyrimidine duplex plus Hp13; lane 9, ³²P-polypurine/polypyrimidine duplex plus Hp13 and RO13; lane 10, polypurine/³²P-polypyrimidine duplex (12.6 nM) plus Hp13; lane 11, polypurine/³²P-polypyrimidine duplex; lane 12, ³²P-RO13 plus Hp13 and Dup13; lane 13, ³²P-RO13. All unlabeled species were used at 10 nM. Incubations were performed at 37°C for 30 min. As unspecific DNA, 1 μg of Salmon Sperm DNA was added. ssDNA targets, duplexes and triplexes are indicated by arrows. Labeled oligonucleotides are indicated as •. (+) indicate unlabelled oligonucleotides present in the reaction. (A) Triplex structure between the PPRH and the polypyrimidine strand (lanes 3, 4, 5, and 12). (B) Binding between the displaced polypurine strand and a specific polypyrimidine oligonucleotide (lanes 1, 2, 3, 4, 6, 9, 10, 11, and 12). (C) Displaced polypurine strand (lanes 3, 7, and 8). PPRH, polypurine reverse Hoogsteen hairpin. Color images available online at www.liebertpub.com/hgtb