Comprehensive mutational analysis plot of PA1. (Left) Plot of the relative abundance of each sequence motif in each Z score bin. Relative abundance is calculated as the number of sequences in each Z score bin that contain a particular sequence motif divided by the number of total sequences in that Z score bin. These abundances are then scaled to one. (Right) Sequences. S = GorC,W = AorT.

Illustration of a CSI microarray and the experimental approach. Each hairpin probe is composed of a permuted hairpin stem (N¹–N⁸) with a 3-bp flanking sequence (CGC) on either side. N′ represents the exact complement to the permuted (N) forward sequence. A fluorescently tagged ligand is applied to the microarray to obtain a comprehensive ligand-binding profile. In addition to reference grid features, high intensity features are circled, indicating tight binding of the ligand to that specific probe sequence.

CSI profile data for PA2 and PA3 with Exd. (A) (Left) Structures of polyamide-peptide conjugates PA2 and PA3 (ImImPy*Py-γ-ImPyPyPy-β-Dp). The expected DNA-binding sequence is 5′-WGWCCWW-3′ based on the ring-pairing rules for polyamides (2). The peptide sequence, N-FYPWMK-C, is conjugated to Py*. (Right) Schematic of cooperative binding of polyamide and Exd to DNA. (B) Logos for the main motifs found in the CSI profile for PA2-Exd (Left) and PA3-Exd (Center) using motif-finding algorithms (31–33). Logos are based on sequences from the top Z score bin (Z > 5.0). (Right) Representation of expected binding orientation of Exd and polyamide in the motif. Boxes indicate the binding position of Exd and polyamide in the sequence. An underline instead of a box indicates that the polyamide is binding in an inverted orientation. (C) Plot of the relative abundance of each sequence motif in each Z score bin. (Left) PA2 with Exd. (Right) PA3 with Exd.

Solution binding and molecular modeling data. (A) EMSA. (Upper) PA2 (50 nM) incubated with increasing concentrations of Exd (in nM). (Lower) PA3 (50 nM) with an Exd titration. Labels above each pair of EMSAs indicate the binding motif used. The sequences used are shown below each pair of EMSAs. Boxes indicate the Exd- and polyamide-binding sites. An underline instead of a box indicates that the polyamide is binding in an inverted orientation. (B) Molecular models (46, 47) of Exd and polyamide bound in consensus, consensus +1, consensus –1, and inverse orientations. Models are based on aligning the DNA from the Protein Data Bank files 1B8I and 1M18 (47). Distances are calculated from the N-methyl group of the analogous ring to which the linker is connected on PA2 and PA3 to the carboxyl carbon of the methionine of the Hox docking peptide (FYPWM) bound to Exd in the crystal structure. (C) Table listing the K_d calculated from the EMSA and the fluorescence intensity (F.I.) extracted from the CSI profile for each polyamide–Exd complex.

CSI profile of PA1. (A) Structure of polyamide-Cy3 conjugate PA1 (ImPy*PyPy-γ-ImPyPyPy-β-Dp). (B) Histogram of averaged intensities of all replicate features. Intensities are background-subtracted so that the mean intensity is zero. Red numbers indicate Z scores. (C)(Top) Logo (53) based on the sequences from the top Z score bin (Z >25). (Middle) DNA sequence that would be targeted by PA1 based on the ring pairing rules for polyamides; an Im/Py ring pair targets G·C, and a Py/Py pair targets either A·T or T·A (2). Numbers indicate base pair positions. (Bottom) A ball-and-stick schematic of PA1. Im or open circle, N-methylimidazole; Py or filled circle, N-methylpyrrole ring; Py* or open circle with inner dot, N-methylpyrrole ring with a Cy3 dye attached; β or diamond, β-alanine; Dp or a half circle with a positive charge, dimethylaminopripylamide; γ or turn, γ-aminobutyric acid. (D) Intensity profile of all sequence permutations of the core consensus sequence 5′-WGWWCW-3′. The intensities of all probes that contain a specific permutation of the core consensus sequence are averaged together. (E) Plot of the correlation between CSI intensities and equilibrium association constants (K_a) determined from nuclease protection (DNase I footprinting) experiments (Table 1). The intensities of all CSI probe sequences that contain a particular footprinted sequence are averaged together.