DNA recognition by quinoxaline antibiotics: use of base-modified DNA molecules to investigate determinants of sequence-specific binding of triostin A and TANDEM

Abstract

The methodology of DNAase I footprinting has been adapted to investigate the sequence-specific binding of two quinoxaline drugs to DNA fragments containing natural and modified bases. In order to help comprehend the molecular origin of selectivity in the bis-intercalation of triostin A and TANDEM at CpG and TpA sites respectively, we have specifically examined the effect of the 2-amino group of guanine on their sequence specificity by using DNA in which that group has been either removed from guanine, added to adenine or both. Previous studies suggested that the recognition of particular nucleotide sequences by these drugs might be dependent upon the placement of the purine 2-amino group, serving as a positive or a negative effector for triostin A and TANDEM respectively. However, the footprinting data reported here indicate that this is not entirely correct, since they show that the 2-amino group of guanine is important for the binding of triostin A to DNA but has absolutely no influence on the interaction of TANDEM with TpA steps. Apparently the binding of triostin A to CpG sites is primarily due to hydrogen bonding interaction between the cyclic peptide of the antibiotic and the 2-amino group of guanine residues, whereas the selective binding of TANDEM to TpA sites is not hydrogen-bond driven and probably originates mainly from steric and/or hydrophobic interactions, perhaps involving indirect recognition of a suitable minor groove structure.

Role of stacking interactions in the binding sequence preferences of DNA bis-intercalators: insight from thermodynamic integration free energy simulations

“…As expected, due to the reported ascendancy of the exocyclic 2-amino group in the binding of these compounds, it was found that replacement of all Gs by Is resulted in triostin A (25), echinomycin (26) and 2QN (10) completely failing to bind to these modified nucleotides. Strikingly, however, replacing all As with Ds led to an unexpected redistribution of binding sites for echinomycin (23) and 2QN (10) to other pyrimidine–purine combinations, such as TpD and CpD, in preference to the usual CpG step despite the fact that, from a minor-groove viewpoint, CpG and TpD steps share the same hydrogen bonding capabilities (Figure 2).…”

“…TANDEM does not bind to CpI steps) (25) and, similarly, that binding of CysMeTANDEM to CpI is much weaker than binding to the high-affinity TpA sites (16) despite the fact that the minor grooves of CpI and TpA steps provide the same hydrogen bonding possibilities (Figure 2). Furthermore, the affinity of CysMeTANDEM (21,22) and TANDEM (8,18) for binding TpA-containing sequences is clearly affected by the flanking bases, with the tetranucleotides ATAT and TTAA being the best and poorest binding sites, respectively.…”

“…Since the bis-intercalative interaction of echinomycin has been shown to be entropically driven (28), suggesting that the process is predominantly stabilized by hydrophobic interactions, it has been hypothesized that the binding selectivity of these compounds might originate from differences in steric and/or hydrophobic interactions with a minor groove of suitable dimensions rather than with specific hydrogen bond formation (25). Nonetheless, two independent lines of evidence appear to suggest that aromatic stacking interactions can be particularly relevant in determining the sequence specificity for this group of bis-intercalating ligands.…”

Preferred Binding Sites for [N-MeCys3,N-MeCys7]TANDEM Determined Using a Universal Footprinting Substrate

“…However, the binding is strongly influenced by the identity of the surrounding bases and all TpA steps do not constitute good binding sites. This is not unexpected since other studies have shown that TANDEM does not bind to IpC, for which the hydrogen bonding face exposed to the minor groove is identical to TpA (18). It has therefore been suggested that the selectivity is not determined by hydrogen bond formation but may originate from steric and/or hydrophobic interactions with a minor groove of suitable dimensions (18).…”

“…Subsequent footprinting studies showed that TANDEM and its derivative [N-MeCys 3 ,N-MeCys 7 ]TANDEM bind to the dinucleotide TpA (8 -11), and this has been confirmed by NMR studies which have suggested mechanisms by which the ligand targets this sequence (12)(13)(14)(15)(16). However, several studies have suggested that TpA alone does not constitute the best binding sequence, and that the preferred sites are flanked by other AT residues (8,9,17,18), consistent with the observation that the ligand binds cooperatively to poly(dA-dT) (7). We have therefore analyzed the binding of this ligand to TpA when flanked by different base pairs, by quantitative DNase I footprinting using a novel DNA substrate which contains all possible 136 tetranucleotide sequences.…”