Lys57hArg and Lys31hArg (µMax20); Lys57hArg, Lys31hArg, Ala21Aib, and His19Tha (µMax21); Lys57hArg, Lys31hArg, Glu23Aad, Ala21Aib, and His19Tha (µMax22). All µMax20-22 analogs were prepared by employing the one-pot NCL-desulfurization approach described in Figure (Figure ; SI section 5). Subsequently, we incubated each of the µMax20-22 analogs separately with the E-box probe and analyzed their DNA binding activity via EMSA (Figure ). The assays revealed that µMax20 and µMax21 exhibited potent DNA-binding activity, whereas µMax22 showed a slightly reduced binding activity. We further confirmed that inserting sequence mutations in our lead analogs (µMax20 and µMax21) did not interfere with the secondary structure of Max TF. We characterized the secondary structure of µMax20, µMax21, and WT Max using circular dichroism (CD) spectroscopy. In these experiments, all analogs exhibited a similar α-helical pattern, with deep double minima at 208 and 222 nm, as depicted in their CD spectra (Figure ). Next, we investigated the DNA sequence specificity of our lead analogs to the core E-box sequence. More specifically, we examined the binding specificity of µMax20 and µMax21 across a diverse range of DNA sequences using the protein binding microarray experiments and compared their preferences to those of the wild-type analog (WT Max). The binding of the WT Max and the µMax20-21 analogs was measured on a universal DNA microarray chip, encompassing all possible 10-mer sequences (SI section 8). Interestingly, µMax20 retained binding preferences similar to those of the wild-type protein WT Max (Figure , showing binding profiles). In contrast, µMax21 showed a marked loss of selectivity for the canonical palindromic E-box sequence, instead displaying generally reduced specificity and moderate preferences for shorter, non-palindromic sequences (Figure , illustrating altered binding patterns). These experiments revealed that despite sequence mutations at strategic sites of Max, our synthetic Max analog, µMax20, retains potent and specific DNA-binding activity with the canonical E-box binding motif. Finally, we analyzed the DNA binding affinity of the µMax20 analog by determining the dissociation constant to the E-box DNA probe via the biolayer interferometry (BLI) assay (SI section 9). We determined a KD value of 4.38 ± 0.50 nM for µMax20 and 8.10 ± 2.24 nM for the WT Max (Figure ). All together, these assays demonstrate the preservation of folding and the DNA-binding activity of the abiotic Max analog µMax20 with target E-box sequence.