Developing a Novel Cell Surface RNA Detecting and Profiling Method via RNA Metabolic Labeling

Cell surface RNA (csRNA) is a recent discovery in the field of RNA biology and has been implicated in playing important roles in many biological processes due to its extracellular properties. To understand the biogenesis, regulation, and function of csRNA, it is critical to develop methods to detect, isolate, and confidently characterize membrane-bound csRNA. Previously, csRNA has been profiled using methods based on cell membrane isolation that are expensive, laborious, and with unsatisfactory specificity and sensitivity . In this study, we use metabolic labeling and chemical cross-linking techniques to specifically label csRNA with biotin handles. We intended to use this technique for separating biotin-labeled csRNA from total RNA samples for characterization purposes. The primary materials that were used to label such csRNAs are 4-Thiouridine (4sU), an unnatural nucleotide analogue, and S-(2-aminoethyl)-ester-methanesulfonothioic-acid-biotin (MTSEA-biotin), a crosslinker designed specifically to label 4sU. By deploying these tools to cell lines such as HEK293T and HeLa, csRNA is detectable by Enhanced Chemiluminescent detection via Dot Blot. Furthermore, to separate biotin-labeled csRNA from total RNA, streptavidin-coated magnetic bead separation procedures could be used as a promising method for purifying csRNA from total RNA, for RNAseq characterization. This study highlights the processes of establishing the csRNA detection protocol and describes the current status and issues with developing the streptavidin-coated magnetic beads separation method.