Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities

Transcription factors ( TFs) interact with specific DNA regulatory sequences to control gene expression throughout myriad cellular processes. However, the DNA binding specificities of only a small fraction of TFs are sufficiently characterized to predict the sequences that they can and cannot bind. We present a maximally compact, synthetic DNA sequence design for protein binding microarray ( PBM) experiments(1) that represents all possible DNA sequence variants of a given length k ( that is, all 'k- mers') on a single, universal microarray. We constructed such all k- mer microarrays covering all 10 - base pair ( bp) binding sites by converting high- density single- stranded oligonucleotide arrays to double- stranded ( ds) DNA arrays. Using these microarrays we comprehensively determined the binding specificities over a full range of affinities for five TFs of different structural classes from yeast, worm, mouse and human. The unbiased coverage of all k-mers permits high- throughput interrogation of binding site preferences, including nucleotide interdependencies, at unprecedented resolution.