Determining the sequence-recognition properties of DNA-binding proteins and small molecules remains a major challenge. To address this need, we have developed a high-throughput approach that provides a comprehensive profile of the binding properties of DNA-binding molecules. The approach is based on displaying every permutation of a duplex DNA sequence (up to 10 positional variants) on a microfabricated array. The entire sequence space is interrogated simultaneously, and the affinity of a DNA-binding molecule for every sequence is obtained in a rapid, unbiased, and unsupervised manner. Using this platform, we have determined the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor. The approach also yielded unique insights into the altered sequence-recognition landscapes as a result of cooperative assembly of DNA-binding molecules in a ternary complex. Solution studies strongly corroborated the sequence preferences identified by the array analysis.chemical genomics ͉ ligand-DNA recognition

The cooperative assembly of multiprotein complexes results from allosteric modulations of DNA structure as well as direct intermolecular contacts between proteins. Such cooperative binding plays a critical role in imparting exquisite sequence specificity on the homeobox transcription factor (Hox) family of developmental transcription factors. A well-characterized example includes the interaction of Hox proteins with extradenticle (Exd), a highly conserved DNA binding transcription factor. Although direct interactions are important, the contribution of indirect interactions toward cooperative assembly of Hox and Exd remains unresolved. Here we use minor groove binding polyamides as structural wedges to induce perturbations at specific base steps within the Exd binding site. We find that allosteric modulation of DNA structure contributes nearly 1.5 kcal/mol to the binding of Exd to DNA, even in the absence of direct Hox contacts. In contrast to previous studies, the sequence-targeted chemical wedges reveal the role of DNA geometry in cooperative assembly of Hox-Exd complexes. Programmable polyamides may well serve as general probes to investigate the role of DNA modulation in the cooperative and highly specific assembly of other protein-DNA complexes.The physical basis of DNA sequence recognition by its ligands (proteins or small molecules) has been investigated in great detail over the years, and several underlying principles have been defined (1-3). The main contributors to the specificity of DNA sequence recognition are interactions between protein side chains and the edges of the base pairs in the cognate DNA site. Efforts to rationally redesign these direct interactions have yielded some exciting successes (4-8). A major limitation of focusing on re-engineering direct interactions, however, is that recognition of sequence-dependent structural properties of DNA and other modes of indirect readout are also critical specificity determinants (9-15). Often a combination of direct and indirect interactions governs exquisite DNA sequence specificity displayed by its ligands (9,11,(14)(15)(16)(17)(18)(19)(20)(21)(22). Specificity in molecular recognition is also achieved by cooperative binding of multiple proteins to closely appositioned DNA sequences (23). Binding of one protein to its DNA cognate site in the enhancer can alter DNA structure and conformational flexibility of a juxtaposed site, thus allosterically improving the energetics

A protein-DNA dimerizer constructed from a DNA-binding polyamide and the peptide FYPWMKG facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. The Exd binding domain can be reduced to a dipeptide WM attached to the polyamide through an ε-aminohexanoic acid linker with retention of protein-DNA dimerizer activity. Screening a library of analogues indicated that the tryptophan indole moiety is more important than methionine's side chain or the N-terminal acetamide. Remarkably, switching the stereochemistry of the tryptophan residue (L to D) stabilizes the dimerizer•Exd•DNA ternary complex at 37 °C. These observations provide design principles for artificial transcription factors that may function in concert with the cellular regulatory circuitry.