The binding modes of a rationally designed photoactivated DNA nuclease determined by NMR

Abstract

The complex between the rationally designed synthetic DNA cleaving agent netropsin-diazene and the double-stranded DNA oligomer 5'-CGCAAAGGC-3'.5'-GCCTTTTGCG-3' was characterized by two-dimensional NMR spectroscopy in solution. Photolysis of netropsin-diazene bound to DNA generates a trimethylenemethane diradical intermediate that induces single-strand breaks in the DNA. The pi-diyl trimethylenemethane based compounds are a new class of DNA nucleases. We tested the following design criteria: (i) binding of the diazene and subsequent reactive diyl to the DNA, (ii) sequence selectivity in the ligand binding and (iii) prevention of diyl dimerization. Sixteen NOE derived ligand-DNA distance restraints were used to obtain the energy minimized model of the complex. The ligand is bound to the minor groove of the oligomer with the diazene at the 5' end of the A-tract in the predominant conformation of the complex. This form of the complex exchanges with a minor conformation in which the ligand is in the opposite orientation. The DNA maintains a B-form structure. Netropsin-diazene has fulfilled all of the design criteria, binding to the DNA duplex studied in the minor groove of the central AAAA tract in a 1:1 mode, preventing diyl dimerization and other side reactions from occurring.

Sequence-Specific DNA Minor Groove Binders. Design and Synthesis of Netropsin and Distamycin Analogues

“…In this context, Bregant et al(252) synthesized the netropsin analogue 73 equipped with a trimethylenemethane group (TMM), which can undergo cycloaddition to electron deficient alkenes. Upon photolysis of the diazene moiety, the TMM-netropsin conjugate can transform to a diyl radical and cleaves DNA, predominantly at AT-rich regions(253). Propargylic sulfones are small synthetic molecules that mimic the chemical action of enediynes.…”

Diyl Trapping and Electroreductive Cyclization Reactions

“…On the basis of detailed studies of the cleavage sites, the mode of binding to the double-stranded DNA oligomer 5′-CGCAAAAGGC-3′‚5′-GCCTTTT-GCG-3′, and computer modeling, 64,65 we conclude that the diyl 185 can be positioned so that atom abstraction from the sugar-phosphate backbone is a reasonable possibility. We suggest that there are three pathways which could lead to cleavage: (1) Direct hydrogen atom abstraction.…”

NMR studies of DNA oligomers and their interactions with minor groove binding ligands

“…A critical issue in the design of sequence-specific DNA ligands which contain a reactive group is the effect of the reactive group on the affinity and sequence specificity of the ligand. In the cases of XL-Dst and netropsin-diazene (Spielmann et al, 1995) to 10 -11 (Drake et al, 1969;Saenger, 1983) including the G»T wobble-mispair Hare et al, 1986), A»A and T»T mispairs (Gervais et al, 1995), the C»A mispair (Boulard et al, 1992), and the wellstudied G»A mispair which adopts several different conformations depending on local sequence context and pH (Prive et al, 1987;Gao & Patel, 1988;Nikonowicz et al, 1991;Cheng et al, 1992;Chou et al, 1992;Greene et al, 1994). The surprising variety of base pairing geometries which have been characterized emphasizes the importance of understanding non-standard base pairing interactions and their biological relevance.…”