Identification and quantification of intracellular N-acetylglucosamine modified glycoRNAs

GlycoRNAs are recently discovered glycoconjugates, their different components and distributions are urgently-to-study issues. Here, we design a general paradigm to study glycoRNA from multiple perspectives. Using the metabolic labelling technology and the designed fluorescent DNA probes, the N-acetylglucosamine (GlcNAc) modified Y5 RNA is identified to be mainly in nucleus by fluorescence resonance energy transfer imaging. After it is collected by a target-specific capture procedure, the component of GlcNAc conjugated ribonucleotide can be determined by gel electrophoresis and mass spectrometric analysis. The levels of intracellular GlcNAc modified Y5 RNA in different cell lines are in situ quantified with a GlcNAc-specific in situ hybridization-mediated proximity ligation assay with single-cell and single-molecule resolution. These findings innovate the distribution and quantitation information of glycoRNAs, which greatly promotes the functional research of glycoRNAs. Authors develop a general paradigm combining fluorescence resonance energy transfer imaging, gel electrophoresis and mass spectrometric analysis to identify and quantify intracellular N-acetylglucosamine modified glycoRNAs.