DNA sequence preferences of several AT-selective minor groove binding ligands

Abstract

We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand.

Sequence and length dependent thermodynamic differences in heterocyclic diamidine interactions at AT base pairs in the DNA minor groove

“…The largest variations in affinity were found in comparison of sites such as AATT, which binds all three ligands quite strongly, and sites with a TA step such as TATA and TTAA, which bind the ligands significantly more weakly. With Hoechst 33258, for example, there is a fifty fold decrease in binding from the AATT to the TATA site [4]. Footprinting results with the AAAA and ATAT sequences, which are the same as in the synthetic polymers, poly(dA) ·poly(dT) and poly(dA-dT)·poly(dA-dT), vary significantly among the compounds.…”

“…The number of binding sites, equilibrium constants, binding enthalpy and entropy were obtained by fitting the corrected data to an appropriate binding model. 4 ] with DB75 were determined using the "model-free ITC" protocol to obtain multiple estimates of ΔH° and to avoid any possible fitting bias [35,36]. This protocol, which uses a high DNA concentration, ensures that all titrated drug is effectively bound after each addition.…”

“…The importance of the G-NH 2 group for binding specificity was verified by Bailly and Waring [13] who showed that replacement of G by inosine and substitution of 2,6-diaminopurine (DAP) for adenosine completely reversed the AT/GC binding specificity of minor groove binding agents [4]. Fox conducted a detailed footprinting study with netropsin, Hoechst 33258 and berenil with a designed DNA sequence that contained all possible sequences of four AT base pairs.…”

“…As shown by DNA footprinting methods, well-characterized and structurally-diverse compounds, such as netropsin, Hoechst 33258 and berenil, which bind at AT sites in the DNA minor groove, can interact with a large variety of AT sequences [1,2]. For compounds of this size, a site of at least four base pairs in length is required but the affinities of the compounds for different AT sites can vary by a surprisingly large amount [3][4][5]. Early studies of netropsin binding to synthetic polydeoxyribonucleotides by Zimmer and coworkers [6,7] revealed sequence dependent differences in affinities and complex structures.…”

“…Hoechst 33258 binds approximately ten fold better to the A 4 sequence, netropsin binds approximately three times better to that sequence but berenil shows no significant difference in affinity between the alternating and nonalternating sites. Berenil does bind at least ten fold more weakly to TTAA than to AATT, however, and it is clear that all of the minor groove binding compounds have different abilities to distinguish among equivalent length AT binding sequences [4].…”