NHAPL analysis of glycoRNA reveals sialic acid-containing glycosylated mRNA 3UTRs and enables sensitive SLE diagnostics

GlycoRNA, newly identified RNA molecules bearing glycan modifications on cell membranes, is implicated in cell communication and immune regulation. However, current technological 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 glycoRNA analysis from 160pg total cell RNA and 1{micro}l 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 glycoRNA. Using NHAPL, we uncover for the first time that protein-coding mRNAs, specifically 3' untranslated region (3'UTR) fragments of FNDC3B and CTSS, undergo sialic acid-containing N-glycosylation on the cell surface. These glycoRNAs functionally promote monocyte adhesion to endothelial cells and hepatoma cell migration, revealing a direct role in cell-cell interactions and cancer-related phenotypes. Applying multiplexed NHAPL to human serum, we identify glycoRNA signatures highly specific to systemic lupus erythematosus (SLE). In particular, glycoY5 and glycoU1 achieve near-complete discrimination between patients and healthy controls (area under the curve (AUC) = 1.00 and 0.9977), whereas conventional total RNA analysis fails to capture these differences, highlighting RNA glycosylation modification as a distinct regulatory layer. Its simplicity and flexibility make it well suited for clinical glycoRNA profiling and biomarker discovery. Overall, NHAPL represents a robust and versatile platform for advancing glycoRNA research and diagnostic development.