All together, mutations in residues that directly interact with the E-box nucleotides led to a reduction in DNA binding activity, whereas mutations in other strategic residues, e.g., Ala17/21 or amino acids that can interact with the DNA backbone, e.g., Lys31/57 can enhance DNA binding (Figure ). The mutation site in Max TF is critical for DNA sequence specificity. Next, we focused on combining productive mutations to produce advanced Max analogs bearing 2-5 mutations (Figure ). To this end, we synthesized three µMax analogs with combined mutations to probe their DNA binding activity: