Visualizing the Interaction of lncRNA and Proteins with Intrinsically Disordered Regions (IDRs) at the Single‑Molecule Level Using High‑Speed Atomic Force Microscopy (HS‑AFM) to Study Liquid–Liquid Phase Separation (LLPS)

Long non‑coding RNAs (lncRNAs), such as XIST, NEAT1_2, and MALAT1, play a central role in nuclear organization, partly by recruiting RNA‑binding proteins (RBPs) that contain intrinsically disordered regions (IDRs). These IDRs often promote liquid–liquid phase separation (LLPS), yet the molecular mechanisms by which individual lncRNAs engage IDR‑rich RBPs to initiate condensate formation remain unclear. The transient nature of these interactions has also made them difficult to study using traditional structural approaches.In this project, we will use high‑speed atomic force microscopy (HS‑AFM), which enables real‑time imaging at nanometer resolution, to observe lncRNA–RBP interactions at the single‑molecule level.Our work will (1) map how key domains of XIST and NEAT1_2 interact with their RNA‑binding proteins (RBPs); (2) track binding dynamics and early events leading to liquid–liquid phase separation (LLPS); and (3) examine how RNA motifs and features within intrinsically disordered regions (IDRs) affect the efficiency and stability of condensate formation. HS‑AFM data will be combined with biochemical and biophysical analyses to clarify how RNA structure and IDR properties contribute to phase behavior. By elucidating how XIST and NEAT1_2 form structures such as the Barr body and paraspeckles, this study will provide a foundation for future efforts to target pathological condensates associated with cancer, neurodegeneration, and developmental disorders.