4 research outputs found
Induced topological changes in DNA complexes: influence of DNA sequences and small molecule structures
Heterocyclic diamidines are compounds with antiparasitic properties that target the minor groove of kinetoplast DNA. The mechanism of action of these compounds is unknown, but topological changes to DNA structures are likely to be involved. In this study, we have developed a polyacrylamide gel electrophoresis-based screening method to determine topological effects of heterocyclic diamidines on four minor groove target sequences: AAAAA, TTTAA, AAATT and ATATA. The AAAAA and AAATT sequences have the largest intrinsic bend, whereas the TTTAA and ATATA sequences are relatively straight. The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AAATT are similar as are the effects on TTTAA and ATATA. A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA. All compounds decrease the mobility of the ATATA sequence that is consistent with decreased minor groove width and bending of the relatively straight DNA into the minor groove. Similar, but generally smaller, effects are seen with TTTAA. The intrinsically bent AAAAA and AAATT sequences, which have more narrow minor grooves, have smaller mobility changes on binding that are consistent with increased or decreased bending depending on compound structure
Microscopic Rearrangement of Bound Minor Groove Binders Detected by NMR
Thermodynamic and structural studies are commonly utilized
to optimize
small molecules for specific DNA interactions, and, thus, a significant
amount of binding data is available. However, the dynamic processes
that are involved in minor groove complex formation and maintenance
are not fully understood. To help define the processes involved, we
have conducted 1D and 2D NMR in conjunction with biosensor-SPR experiments
with a variety of compounds and symmetric, as well as asymmetric,
AT tract DNA sequences. Surprisingly, the NMR data clearly show exchange
between equivalent binding sites for strongly binding compounds like
netropsin and DB921 (<i>K</i><sub>a</sub> > 10<sup>8</sup> M<sup>–1</sup>) that does not involve dissociation off the
DNA. A quantitative analysis of the data revealed that these bound
exchange rates are indeed much faster than the macroscopic dissociation
rates which were independently determined by biosensor-SPR. Additionally,
we could show the existence of at least two 1:1 compound DNA complexes
at the same site for the interaction of these compounds with an asymmetric
DNA sequence. To explain this behavior we introduced a model in which
the ligand is rapidly flipping between two orientations while in close
association with the DNA. The ligand reorientation will contribute
favorably to the binding entropy. As the potential of minor groove
binders to form more than a single complex with asymmetric, as well
as symmetric, duplexes is widely unknown, the consequences for binding
thermodynamics and compound design are discussed