SEQUENCE-DIRECTED SINGLE-STRAND CLEAVAGE OF DNA BY A NETROPSIN-FLAVINHYBRID MOLECULE

In an attempt to obtain sequence specific DNA-cleaving molecules, we h ave synthesized a series of hybrid minor groove binders composed of a photoactiveable isoalloxazine (flavin) chromophore linked through a po lymethylenic chain to a bis-pyyrolecarboxamide moiety related to netro psin. Like netropsin, the hybrid derivatives preferentially bind to AT-rich sequences. Activation of the flavin chromophore by visible ligh t results in the appearance of single strand breaks in the vicinity of the DNA binding site. We have further investigated the cleavage affin ity properties of one of these compounds referred to as netropsin-flav in (Net-Fla) and considered as representative of the series, Net-Fla c leaves only one strand at a specific locus downstream of 5'-AAAT-3', u pstream 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 pro perties of Net-Fla similar to that of a restriction endonuclease and p rovides 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.