Abiotic Transcription Factors by Strategic Insertion of Non-Proteinogenic Amino Acids

Nature employs proteins as molecular machines to orchestrate numerous intricate biological processes. Natural proteins are primarily synthesized from 22 proteinogenic amino acids. In principle, developing abiotic proteins with novel non-proteinogenic residues can expand their functional space, enabling the discovery of new reactivities not found in nature. Here, we combined a rational design with total synthesis to develop novel abiotic proteins derived from natural transcription factors (TFs). We synthesized a library of abiotic TFs using solid-phase synthesis and native chemical ligation to afford 27 novel analogs on a milligram scale. Systematic DNA binding analysis revealed that the mutation site plays a key role in the DNA binding activity of TFs. Remarkably, DNA binding and cellular studies enabled the discovery of the μMax20 analog, bearing two non-proteinogenic residues (Lys31/57 to hArg), which displayed potent DNA binding to the core E-box site and exceptional intrinsic cell permeability. Furthermore, the site-specific editing of μMax20 with staples led to an advanced analog, 2s-μMax20, which is amenable to cellular delivery at nanomolar concentrations. Collectively, our findings indicate that inserting non-natural transformations into strategic domains and residues is a powerful strategy for leveraging protein function to engineer novel analogs for basic research and biomedical applications.