Paramjit S. Arora

RNA, unlike DNA, folds into a multitude of secondary and tertiary structures. This structural diversity has impeded the development of ligands that can sequence‐specifically target this biomolecule. We sought to develop ligands for double‐stranded RNA (dsRNA) segments, which are ubiquitous in RNA tertiary structure. The major groove of double‐stranded DNA is sequence‐specifically recognized by a range of dimeric helical transcription factors, including the basic leucine zippers (bZIP) and basic helix‐loop‐helix (bHLH) proteins; however, such simple structural motifs are not prevalent in RNA‐binding proteins. We interrogated the high‐resolution structures of DNA and RNA to identify requirements for a helix fork motif to occupy dsRNA major grooves akin to dsDNA. Our analysis suggested that the rigidity and angle of approach of dimeric helices in bZIP/bHLH motifs are not ideal for the binding of dsRNA major grooves. This investigation revealed that the replacement of the leucine zipper motifs in bHLH proteins with synthetic crosslinkers would allow recognition of dsRNA. We show that a model bHLH DNA‐binding motif does not bind dsRNA but can be reengineered as an RNA ligand. Based on this hypothesis, we rationally designed a miniature synthetic crosslinked helix fork (CHF) as a generalizable proteomimetic scaffold for targeting dsRNA. We evaluated several CHF constructs against a set of RNA and DNA hairpins to probe the specificity of the designed construct. Our studies reveal a new class of proteomimetics as an encodable platform for sequence‐specific recognition of dsRNA.