NHAPL enables homogeneous detection of RNA-associated glycan signals.

GlycoRNAs, newly identified RNA molecules bearing glycan modifications on cell membranes, are implicated in cell communication and immune regulation. However, current methodological limitations impede a thorough elucidation of their biological roles and clinical significance. Here, we developed Nucleotides Hybridization and Aptamer-based Proximity Ligation (NHAPL), a homogeneous assay enabling sensitive and quantitative RNA-associated glycan analysis from total cell RNA and serum. NHAPL integrates dual recognition by a sialic acid aptamer and RNA binding probe, followed by ligation and qPCR amplification. We further established multiplexed NHAPL for simultaneous detection of multiple RNA-associated glycan signals. Using this platform, we discovered that a subset of FNDC3B- and CTSS-derived 3'UTR fragments generate RNA-associated glycan signals and promote cell adhesion and migration. Importantly, serum RNA-associated glycan signals detected by NHAPL showed relatively low inter-individual variability among healthy individuals, whereas signals associated with Y5 and U1 RNAs were markedly elevated in patients with systemic lupus erythematosus in our cohort. The AUROC (area under the receiver operating characteristic curve) reached 1.000 for Y5-associated signals and 0.9977 for U1-associated signals in our cohort, supporting their potential as candidate biomarkers for systemic lupus erythematosus. The NHAPL platform requires no specialized instrumentation and enables rapid multiplexed detection of RNA-associated glycan signals. Owing to its simplicity, sensitivity, and flexibility, NHAPL provides a practical platform for profiling these signals and biomarker discovery. Overall, this work establishes NHAPL as a versatile analytical strategy for investigating RNA-associated glycan signals in biological samples.