Microscopic Rearrangement
of Bound Minor Groove Binders
Detected by NMR
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Abstract
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