Optimization of RiPCA for the Live‐Cell Detection of Pre‐MicroRNA‐Protein Interactions

Advancements in methods for identifying RNA‐protein interactions (RPIs) on a large scale has necessitated the development of assays for validation of these interactions, particularly in living cells. We previously reported the development of RiPCA (RNA interaction with protein‐mediated complementation assay) to enable the cellular detection of the well‐characterized interaction between the pre‐microRNA, pre‐let‐7, and its RNA‐binding protein (RBP) partner Lin28. In this study, the applicability of RiPCA for the detection of putative pre‐miRNA‐protein interactions was explored using an improved RiPCA protocol, termed RiPCA 2.0. RiPCA 2.0 was adapted to detect the sequence specificity of the RBPs hnRNP A1, Msi1, and Msi2 for reported pre‐microRNA binding partners. Additionally, the ability of RiPCA 2.0 to detect site‐specific binding was explored. Collectively, this work highlights the versatility of RiPCA 2.0 in detecting cellular RPIs.