dsRADAR: Imaging and Detecting Cellular dsRNA by Repurposing RNA-Binding Proteins

Double-stranded RNA (dsRNA) is recognized by cellular receptors as a sign of viral infection, triggering the innate immune response. Cellular dysregulation can also lead to accumulation of endogenously produced dsRNA that stimulates a viral-like immune response. Additionally, dsRNA contamination in RNA therapeutics can lead to harmful side effects via a similar pathway. Despite the clinical relevance of dsRNA, reliable tools for its detection remain limited. Currently, dsRNA detection relies almost exclusively on the monoclonal antibodies J2 and K1, which suffer from sequence bias and low sensitivity. To address this challenge, we repurposed naturally occurring dsRNA-binding domains (dsRBDs) to produce reliable, pan-specific affinity reagents for dsRNA. We systematically screened the dsRBDs of the three human adenosine deaminases acting on RNA (ADARs) and identified ADAR3 dsRBDs as the most promising candidates. We then engineered ADAR3-derived dsRBD constructs having varying linker lengths and domain combinations to create dsRNA accumulation detected by ADAR3 RBDs (dsRADAR) affinity reagents. Finally, we demonstrate the superior performance of dsRADAR over currently available dsRNA antibodies in a cell model of viral infection and a tissue model of gastric inflammation. Together, dsRADAR provides a sensitive and reliable approach for imaging and detecting diverse dsRNA structures in biological contexts.