With the growing number and diversity of known genome sequences, there is an increasing opportunity to regulate gene expression through synthetic, cell-permeable small molecules. Enhancing the DNA sequence recognition abilities of minor groove compounds has the potential to broaden their therapeutic applications with significant implications for areas such as modulating transcription factor activity. While various classes of minor groove binding agents can selectively identify pure AT and mixed AT and GC base pair(s) containing sequences, there remains a lack of compounds capable of distinguishing between different AT sequences. In this work, we report on the design compounds that exhibit selective binding to -TTAA- or -TATA- containing DNA minor groove sequences compared with other AT ones. Several studies have shown that the -AATT- and -TTAA- sequences have distinct physical and interaction properties, especially in terms of their different requirements for recognition in the minor groove. Achieving strong, selective minor groove binding at -TTAA- sequences has been challenging, but DB1003, a benzimidazole-furan-furan diamidine, has demonstrated cooperative dimeric binding activity at -TTAA-. It has significantly less binding preference for AATT. To better understand and modify the selectivity, we synthesized a set of rationally designed analogs of DB1003 by altering the position of the five-membered heterocyclic structure. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1992, a benzimidazolefuran-thiophene diamidine, binds strongly to -TTAA- as a positive cooperative dimer with high cooperativity. The high-resolution crystal structure of the TTAA-DNA-DB1992 complex reveals that DB1992 binds as an antiparallel π-stacked dimer with numerous diverse contacts to the DNA minor groove. This distinctive binding arrangement and the properties of diamidines at the -TTAA- minor groove demonstrate that benzimidazole-furan-thiophene is a unique DNA binding pharmacophore. Competition mass spectroscopy and circular dichroism studies confirmed the binding stoichiometry and selectivity preference of the compounds for the -TTAA- sequence.
Read full abstract