Blocker-SELEX: A Structure-Guided Strategy for Developing Inhibitory Aptamers Disrupting Transcription Factor Interactions

Despite the well-established significance of transcription factors (TFs) in pathogenesis, their utilization as pharmacological targets has been limited by the inherent challenges mainly associated with modulating their protein-protein and protein-DNA interactions. The lack of defined small-molecule binding pockets and the nuclear localization of TFs makes neither small molecule inhibitors nor neutral antibodies suitable in blocking TF interactions. Aptamers are short oligonucleotides exhibiting high affinity and specificity for a diverse range of targets. The large molecular weights, expansive blocking surfaces and efficient cellular internalization make aptamers as a compelling molecular tool for traditional TF interaction modulators. Here, we report a structure-guided design strategy called Blocker-SELEX for developing inhibitory aptamers (iAptamer) that selectively block TF interactions. Our approach led to the discovery of an iAptamer that cooperatively disrupts SCAF4/SCAF8-RNA Polymerase II (RNAP2) interactions, thus dysregulates RNAP2 dependent gene expression and splicing, leading to the impairing of cell proliferation. This approach was further applied to develop iAptamers efficiently block WDR5-MYC interaction. Together, our study highlights the potential of Blocker-SELEX in developing iAptamers that effectively disrupt TF interactions, and the generated iAptamers hold promising implications as chemical tools in studying biological functions of TF interactions and the potential for nucleic acids drug development. Overall design: To validate the targeted effect of SRiApt-1 on gene transcription and mRNA splicing by inhibiting the interaction between RNAP2 and SCAF4/SCAF8 proteins at the cellular level, we first optimized the stability of SRiApt-1 by introducing two 2'-O-Me modifications, resulting in SRiApt-1_m (5'-G(2'-O-Me)GGGTGGGGA(2'-O-Me)G-3') . Subsequently, mRNA sequencing was conducted on HCT116 cells cell line treated with SRiApt-1_m or control aptamers for 24 hours. Analysis of the RNA-seq data revealed significant alterations in the transcriptome of HCT116 cells upon SRiApt-1_m aptamer treatment.