Preferred binding sites for the bifunctional intercalator TANDEM determined using DNA fragments that contain every symmetrical hexanucleotide sequence

We have prepared novel DNA footprinting substrates that contain all 64 symmetrical hexanucleotide sequences. These were contained in two restriction fragments that were cloned into the pUC19 polylinker site; each fragment was also obtained in both orientations. These fragments were used to assess the sequence binding preferences of the synthetic quinoxaline antibiotic TANDEM. We found that, although the ligand binds to most TpA steps, the affinity is affected by the flanking sequences. The best binding sites contain the tetranucleotide sequence ATAT, although YATATR is a better site than RATATY. TTAA always is a poor binding site, especially TTTAAA. The binding to GTAC is strongly dependent on the flanking bases, with good binding to GGTACC but none at all to CGTACG.