Block decoys: Transcription-factor decoys designed for in vitro gene regulation studies

Transcription-factor decoys are short synthetic oligodeoxynucleotides that sequester cognate transcription factors and prevent their binding at target promoters. Current methods of decoy formation have primarily been optimized for potential therapeutic applications. However, they are not ideally suited to in vitro investigations into multi-transcription factor-mediated processes that may require multiple regulatory elements to be inhibited in varying combinations. In this study we describe a novel method for chimeric decoy formation in which blocks containing discrete transcription factor binding sites are combined into circular molecules. Unlike currently available methods, block decoys allow rapid construction of chimeric decoys targeting multiple regulatory elements. Further, they enable fine-tuning of binding-site copy ratios within chimeras, allowing sophisticated control of the cellular transcriptional landscape. We show that block decoys are exonuclease-resistant and specifically inhibit expression from target binding sites. The potential of block decoys to inhibit multiple elements simultaneously was demonstrated using a chimeric decoy containing molar optimized ratios of three regulatory elements, NF-κB-RE, CRE, and E-box. The chimeric decoy inhibited expression from all three elements simultaneously at equivalent levels. The primary intended use of block decoys is in vitro gene regulation studies in which bespoke chimeras can be rapidly constructed and utilized to determine a promoter's functional regulation.