DNA-sequence specific recognition by a thiazole analogue of netropsin: a comparative footprinting study

Abstract

Four different footprinting techniques have been used to probe the DNA sequence selectivity of Thia-Net, a bis-cationic analogue of the minor groove binder netropsin in which the N-methylpyrrole moieties are replaced by thiazole groups. In Thia-Net the ring nitrogen atoms are directed into the minor groove where they could accept hydrogen bonds from the exocyclic 2-amino group of guanine. Three nucleases (DNAase 1, DNAase 11, and micrococcal nuclease) were employed to detect binding sites on the 160bp tyr T fragment obtained from plasmid pKMA-98, and further experiments were performed with 1 1 7mer and 253mer fragments cut out of the plasmid pBS. MPE-Fe(ll) was used to footprint binding sites on an EcoRI/Hindlll fragment from pBR322. Thia-Net binds to sites in the minor groove containing 4 or 5 base pairs which are predominantly composed of alternating A and T residues, but with significant acceptance of intrusive GC base pairs. Unlike the parent antibiotic netropsin, Thia-Net discriminates against homooligomeric runs of A and T. The evident preference of Thia-Net for AT-rich sites, despite its containing thiazole nitrogens capable of accepting GC sites by hydrogen bonding, supports the view that the biscationic nature of the ligand imposes a bias due to the electrostatic potential differences in the receptor which favour the ligand reading alternating AT sequences. INTRODUCTIONSequence-specific molecular recognition of DNA by proteins is central to the regulation of many cellular processes (1,2). Examination of the interaction between oligopeptides and DNA can be useful as a means of elucidating the structural basis for such selectivity. Moreover, studies of the interaction of oligopeptides with DNA have become increasingly important for

Small ligands that neither bind to nor alter the structure of d(GA·TC)n sequences in DNA

“…They contain various isolated GA sites (Figs. 1 and 3A) in agreement with previous studies [21]. In any case, Thia‐Net had no effect on the pattern of DNAse I cleavage in the d(GA·CT) 5 sequence.…”

“…Nevertheless, the preference for cutting ApG bonds over GpA in the large d(GA·CT) sequences was always sustained. The footprints in regions other than the (GA) 5 inserts were largely corroborative of the binding selectivity of the three ligands to DNA, previously analyzed [12, 18–21]. A summary map showing the difference in susceptibility of this DNA fragment to DNAse I in the presence of the higher concentrations of the three ligands is displayed in Fig.…”

Binding properties and DNA sequence-specific recognition of two bithiazole-linked netropsin hybrid molecules

“…These digestions also yielded fragments suitable for 3'-end labeling by the reverse transcriptase. The detailed procedures for isolation, purification, and labeling of these duplex DNA fragments have been described recently (Bailly et al, 1990b(Bailly et al, , 1992bPlouvier et al, 1991).…”

Single Strand Targeted Triplex Formation: Strand Displacement of Duplex DNA by Foldback Triplex-Forming Oligonucleotides

“…The reaction was stopped by adding 0.5 M EDTA solution (18,19). The samples were then mixed with formamide dye solution, heated at 95°C for 5 min, snap-cooled on ice, and analyzed on a 10% denaturing polyacrylamide gel containing 7 M urea (20).…”