Longitudinal and large-scale monitoring of transcriptome and RBP-RNA interactome in living cells by engineered protein nanocages

Nondestructive sequencing of RNA from live cells is essential for monitoring and understanding dynamic biological processes. However, most existing RNA sequencing methods rely on cell lysis or fixation, limiting their applicability for longitudinal studies. Here, we introduce POND-seq ( P rotein nanocage-emp O wered N on- D estructive seq uencing), a novel approach that employs secretory protein nanocages fused with RNA-binding proteins (RBPs) to capture the RBP-RNA interactome and transcriptome in live cells. POND-seq reliably identifies RNA targets of canonical RBPs across multiple cell types. By fusing poly(A)-binding protein (PABPC1) to the nanocage, we demonstrate that POND-seq can monitor transcriptomic changes in response to signaling stimuli and selectively capture cell-type-specific transcriptomes from mixed populations. Additionally, POND-seq facilitates the dissection of RNA-binding domains and key amino acid residues critical for RBP-RNA interactions. We further highlight its utility in large-scale screening, offering compelling evidence for the pathogenicity of FMR1 variants. POND-seq represents a transformative advancement in RNA biology, cell biology and precision medicine, enabling unprecedented insights into cellular dynamics and disease mechanisms.