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PREDICTOR: A Non‐Enzymatic Catalytic Cascade Tool for in Situ Visualization of Small Extracellular Vesicle Surface glycoRNAs

Year: 2026

Type: article

Abstract: ABSTRACT Glycosylated RNAs (glycoRNAs) are membrane‐displayed RNA‐glycan conjugates, but quantitative in situ analysis of low‐abundance glycoRNAs on small extracellular vesicles (sEVs) remains challenging. Here, we develop PREDICTOR (proximity‐encoded non‐linear hybridization chain reaction circuit), an enzyme‐free catalytic DNA cascade for imaging and quantifying surface glycoRNAs on intact sEVs. PREDICTOR uses a sialic acid aptamer and an RNA‐sequence probe as split recognition modules, whose proximity on a single glycoRNA reconstitutes an initiator that drives dendritic DNA self‐assembly and non‐linear fluorescence amplification on the sEV surface. Across serial sEV inputs (5×10 3 –5×10 9 particles·mL − 1 ), PREDICTOR exhibited a steeper concentration‐response than representative amplification assays, with higher regression slopes in bulk fluorescence (0.372, R 2 = 0.9846) compared with ARPLA (0.301, R 2 = 0.9748) and HieCo 2 (0.230, R 2 = 0.9663), and similarly improved performance in bead‐based imaging (4.105, R 2 = 0.9816 vs. 3.495 and 2.711). Applying PREDICTOR to a breast cancer transformation model (MCF‐10A → MCF‐7 → MDA‐MB‐231) showed that multiple surface glycoRNA candidates decrease with malignancy, accompanied by progressive softening of sEV membranes (Young's modulus reduced by 25.08% and 54.72%). Functionally, enzymatic depletion of surface RNA and disruption of sialylated N‐glycan features attenuated macrophage uptake and inflammatory activation, supporting a contribution of surface glycoRNAs to sEV recognition. Collectively, PREDICTOR provides a rapid, enzyme‐free platform for quantitative and spatial profiling of sEV surface glycoRNAs and links their abundance to vesicle mechanics and immune‐cell interactions.

Source: Journal of Extracellular Vesicles

DOI: 10.1002/jev2.70282