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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE254651 | GEO | 2024/02/01

REPOSITORIES: GEO

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Project description:Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of transcription factor bound sites are actively involved in transcriptional regulation, nor are the key genomic features known that discriminate between active and inactive TF binding events. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a potential marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize systematically the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional assay, we performed cis-regulatory element sequencing (CRE-seq) reporter assays. We tested more than 1,000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB) bound sites in two human cell types, HepG2 and K562. We found that CEBPB bound sites that co-occur with RNAP2 were more likely to exhibit enhancer activity in our reporter assay and these active sequences display significantly stronger RNAP2 read enrichment and local enhancer RNA (eRNA) activity compared to inactive sites. CEBPB-bound sites further maintained substantial cell type specificity, indicating that DNA sequence information at such sites can accurately convey cell type-specific regulatory information, supporting a strong role for local DNA sequence in distinguishing active from inactive TF-bound loci. By comparing our CRE-seq results to a comprehensive set of over 1,000 genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity as well as cell type-specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can discriminate active versus inactive transcription factor bound sites.

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

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