Pip-HoGu: An Artificial Assembly with Cooperative DNA Recognition Capable of Mimicking Transcription Factor Pairs

Cooperation between pairs of transcription factors (TFs) has been widely demonstrated to play a pivotal role in the spatiotemporal regulation of gene expression, but blocking cooperative TF pair-DNA interactions synergistically has been challenging. To achieve this, we designed programmable DNA binder pyrrole-imidazole polyamides conjugated to host-guest assemblies (Pip-HoGu) to mimic the cooperation between natural TF pairs. By incorporating cyclodextrin (Cyd)-adamantane (Ada), we synthesized Ada1 (PIP1-Ada) and Cyd1 (PIP2-Cyd), which were evaluated using T_m, EMSA, competitive, and SPR assays and molecular dynamics studies. The results consistently demonstrated that Pip-HoGu system formed stable noncovalent cooperative complexes, thereby meeting key criteria for mimicking a TF pair. The system also had a longer recognition sequence (two-PIP binding length plus gap distance), favorable sequence selectivity, higher binding affinity, and in particular, a flexible gap distance (0-5 bp). For example, Ada1-Cyd1 showed thermal stability of 7.2 °C and a minimum free energy of interaction of -2.32 kcal·mol^-1 with a targeting length of 14 bp. Furthermore, cell-based evaluation validated the capability of Pip-HoGu to exhibit potent cooperative inhibitory effects on gene expression under physiological conditions by disrupting TF pair-DNA function. In conclusion, the modular design of Pip-HoGu defines a general framework for mimicking naturally occurring cooperative TF pair-DNA interactions that offers a promising strategy for applications in the precise manipulation of cell fate.