Sequence-directed single strand cleavage of DNA by a netropsin-flavin hybrid molecule

In an attempt to obtain sequence specific DNA-cleaving molecules, we have synthesized a series of hybrid minor groove binders composed of a photoactiveable isoalloxazine (flavin) chromophore linked through a polymethylenic chain to a bis-pyrrolecarboxamide moiety related to netropsin. Like netropsin, the hybrid derivatives preferentially bind to A+T-rich sequences. Activation of the flavin chromophore by visible light results in the appearance of single strand breaks in the vicinity of the DNA binding site. We have further investigated the cleavage affinity properties of one of these compounds referred to as netropsin-flavin (Net-Fla) and considered as representative of the series. Net-Fla cleaves only one strand at a specific locus downstream of 5'-AAAT-3', upstream of 5'-TAAA-3' and on either side of a 5'-AAAA-3' sequence. Net-Fla cleaves both strands downstream to 5'-AATT-3'. This makes the properties of Net-Fla similar to that of a restriction endonuclease and provides additional insight into establishing the rules for the readout of B-DNA helix by non-nucleotidic compounds. Using molecular modeling, we show that Net-Fla binds to an asymmetric site in one orientation. The values of the energetic minima lie in the same order as expected from the cleavage patterns, which suggests that the oriented cleavage is a consequence of a sequence-oriented binding of Net-Fla in the DNA minor groove.