Introducing GlycoRNAs, a new frontier in small RNA biology.

Figure 1. Scheme of a glycoRNA on the surface of a cell. Modified from Disney (2021)³.

Given the extraordinary nature of this discovery, the initial reports sparked debate. Were these glycoRNAs real biological entities, or artifacts of preparation? A follow up study by Flynn's lab addressed this by using advanced chemical labelling tools, northern blots, and mass spectrometry, further supporting glycoRNAs as authentic biological molecules⁴.

Growing evidence suggests that glycoRNA are detectable both in vitro and in vivo, particularly on the cell surface. Among small RNAs, Y RNAs often represent the largest fraction of glycoRNA species, followed by tRNAs, snRNAs, snoRNAs, and others¹.

Advanced mass spectrometry-based workflows have profiled N-glycans on RNA quantitatively across tissues. These analyses revealed that glycoRNA glycans differ significantly in composition from protein-bound glycans and exhibit tissue-specific abundance patterns, suggesting that glycoRNA levels may vary based on cellular context and physiological state⁵.

Finally, in a transcriptome-wide draft of human glycoRNA glycan profiles, the number of distinct glycan species was limited, yet they were present at relatively high abundance levels. This suggests that although the glycan variety is narrow, their representation on RNA in the glycoRNA pool is significant⁶.

Mechanism of RNA glycosylation

A pivotal study by Xie et al identified the modified nucleotide acp³U (3-(3-amino-3-carboxypropyl)uridine) as the key site for N-glycan attachment on tRNAs and related RNA. It is worth noting that in tRNA this modification is commonly present in eukaryotes and bacteria⁵.

The authors proposed a 3-step model pathway revealing how classical protein-focused glycosylation machinery also extends its reach to RNA substrates. According to this model first acp³U is introduced during tRNA maturation in the nucleus/cytosol. Then the tRNA enters the secretory pathway, allowing the N-glycosylation machinery to attach syalyated glycans. Finally, the resulting glycoRNAs are displayed on the cell surface⁴.

Functional implications

GlycoRNAs appear to extend RNA's functional repertoire into extracellular and immunological realms, acting as novel mediators in cell–cell communication and immune regulation

• Immune modulation via Siglecs: GlycoRNAs, decorated with sialylated N‑glycans, are recognized by Siglec receptors, immune regulatory proteins that bind sialic acid–terminated glycans⁸. This interaction may tune immune activation, tolerance, and inflammatory responses, positioning glycoRNAs as RNA-based immune modulators.
• Surface signaling and cellular entry: GlycoRNAs have been found to assemble into specialized membrane domains, interacting with RNA-binding proteins and facilitating entry of cell-penetrating peptides, a potential new route for molecular signaling or therapeutic delivery⁹.
• Extracellular vesicle-associated biomarkers: GlycoRNAs carried in small extracellular vesicles can be profiled using drFRET, yielding remarkable diagnostic performance: 100% accuracy distinguishing cancer versus control, and ~90% accuracy in subclassifying cancer types within patient cohorts¹⁰.

These insights support the idea that glycoRNAs constitute a relevant, regulated layer of extracellular RNA biology, with intriguing links to immune signaling, disease states, and diagnostic potential.

Detecting and characterizing GlycoRNAs

Small RNA sequencing

Because glycoRNAs are derived from small RNAs, sequencing these fractions has been essential to identify their RNA backbones. When combined with glycan-specific enrichment (lectins, click chemistry, or rPAL), small RNA sequencing reveals exactly which RNAs are glycosylated. In fact Flynn et al. confirmed in their landmark paper that glycoRNAs correspond to intact small RNAs, not degradation products, using sequencing. Pairing NGS with chemical labeling and/or mass spectrometry seems to be key to achieving comprehensive glycoRNA maps in the near future.

Metabolic labeling + northwestern blotting

Cells can be fed unnatural sugars (e.g., Ac₄ManNAz), which become incorporated into glycans. These modified glycans can then be “clicked” to detection reagents. Coupled with northwestern blots, this method first revealed cell-surface glycoRNAs. A detailed step-by-step protocol has been recently published¹¹.

rPAL (RNA-optimized periodate oxidation and aldehyde ligation)

The rPAL technique exploits periodate to oxidize vicinal diols within RNA, creating aldehydes that can be ligated to tagging molecules. Compared to metabolic labeling, rPAL provides a ~25-fold increase in sensitivity and better signal recovery¹².

Lectin-based proximity labeling

Lectins are proteins that bind specifically and reversibly to carbohydrate structures. Lectins such as Wheat Germ Agglutining (binding sialic acid/GlcNAc) enrich glycoRNAs for downstream analysis¹².

drFRET for extracellular vesicle GlycoRNA profiling

The drFRET assay uses dual probes recognizing both RNA and glycan moieties, enabling ultrasensitive detection of glycoRNAs in biofluids from as little as 10 µL of biofluid. In clinical studies, drFRET was combined with Small RNA sequencing to correlate glycoRNA presence with specific small RNA species, supporting its potential for diagnostic stratification¹⁰.

Conclusions and outlook

GlycoRNAs are small RNAs covalently coated with glycans, prominently sialylated N-glycans. Other glycans may be present. In fact, a recent study suggests that O-glycosylation also contributes to glycoRNA biogenesis in some systems¹².

Their discovery reshapes RNA biology and introduces new paradigms for cell communication and immune regulation. For scientists and innovators in RNA research, glycoRNAs represent a frontier comparable to the discovery of microRNAs or RNA modifications two decades ago. As detection tools evolve, we may uncover glycoRNAs as key regulators in health, disease, and therapeutic development.