Computational design of sequence-specific DNA-binding proteins

Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove. Universal protein-binding microarray (PBM) experiments were performed for small, recombinant, DNA-binding protein designs 1, 3, 5, 6, 9, 35, & 48. Briefly, universal PBMs involved binding of his-tagged proteins to double-stranded 15K Agilent microarrays containing a DNA library designed to cover all possible 9-bp sequences, with every 7-mer occurring in at least 16 different spots on the array. This design allows comprehensive and unbiased characterization of the binding specificity of transcription factors for all possible 7-bp sequences.