Single‐Cell Imaging of Site‐Specific RNA‐Protein Interactions

ABSTRACT

Detecting and quantifying specific RNA‐protein interaction pairs under diverse physiological conditions is crucial for elucidating disease mechanisms and exploring potential therapeutic targets. However, it still remains a major challenge to visualize specific intracellular RNA‐protein interactions at near‐single‐molecule resolution. In this work, we introduce an RNA‐protein interaction visualization method by antibody‐guided proximal in situ reverse‐transcription detection (RAPID), to enable spatial localization and quantification of specific RNA‐protein interaction pairs within single cells at near‐single‐molecule resolution. We demonstrate the high specificity and robustness of RAPID by validating the interaction between the heterogeneous nuclear ribonucleoprotein hnRNPC and the long noncoding RNA (lncRNA) MALAT1 in HeLa cells. Using RAPID, we found that SFPQ‐NEAT1 interactions exhibited distinct spatial organization and dynamic changes upon cellular stimulation, indicating that the SFPQ‐NEAT1 axis is closely associated with nuclear reorganization and stimulus‐responsive gene regulation. RAPID, providing a new perspective on RNA‐protein interactions, represents a novel analytical tool that facilitates further investigation of their roles in biology and disease.