To investigate the effect of inserting non-proteinogenic amino acids, we decided to focus on transcription factor (TF) proteins, which represent the largest family in the human proteome. Specifically, we selected the bHLH-LZ TF network Myc-Max-Mad. This network controls the expression of approximately 15% of the human genome by forming dimeric complexes that selectively bind to promoter regions containing the enhancer box DNA sequence (5′-CACGTG-3′, E-box) to either activate or suppress gene expression (Figure ). The Myc-Max-Mad network is involved in key cellular processes such as cell proliferation, differentiation, and apoptosis. Its deregulation is implicated in many human disease pathogeneses, including various types of cancer (~50% of human tumors). 16 Gene expression is primarily initiated and regulated by TFs interacting with specific DNA sequences through their DNA-binding domains. 17 DNA-binding activity and sequence specificity are mainly governed by the unique topology of the DNA-binding motif. We sought to build our abiotic TFs primarily based on the DNA-binding domain of the TF Max, which governs the dimerization and DNA-binding activity of the Myc-Max-Mad network. Crystal structure and sequence analysis revealed that six key residues make specific base pair contacts or interact with the phosphate backbone to bind to the canonical E-box DNA sequence. We envisioned the insertion of novel orthogonal transformations into these key residues and/or other crucial sites that can expand the functional space to discover new protein analogs with refined activity. In this study, we combined structure-based design and total synthesis to develop novel abiotic TF analogs derived from natural bHLH-LZ TFs, e.g., Max. We demonstrated the power of combining chemical protein synthesis and late-stage transformations to produce a library of abiotic TF analogs for systematic DNA binding analysis. Importantly, we employed our approach to prepare 27 abiotic TF analogs, with the insertion of up to six novel transformations at strategic positions. Comprehensive DNA binding analysis revealed that the mutation site plays a critical role in the DNA binding of TFs. Furthermore, we discovered novel abiotic TF analogs, e.g., μMax20, bearing two mutations (Lys31hArg and Lys57hArg mutations), which exhibited potent DNA binding activity at the core E-box site and exceptional intrinsic cell permeability at submicromolar concentrations. Taken together, our findings demonstrate the power of combining structure-based design and total synthesis to develop novel proteins with unique properties.