A customizable purification assay designed for small-RNA biomarker identification and functional evaluation of circulating exosomes: Exosome Capture by AnTibody of CHoice and Enzymatic Release (exo-CATCHER)

ABSTRACT: Circulating nucleic acids, encapsulated within exosomes, can provide a remote cellular snapshot of biomarkers associated with disease, however selective exosome isolation is critical. Current laboratory-based purification techniques rely on the physical properties of exosomes for selection, rather than their inherited cellular characteristics. Therefore, we established a highly selective purification assay, termed exosome Capture by AnTibody of CHoice and Enzymatic Release (exo-CATCHER), designed for high-throughput analysis of low-abundance small-RNA cargos by next-generation sequencing. We demonstrated its sensitivity by specifically isolating and sequencing small-RNA from mouse exosomes spiked into human plasma. Furthermore, we used Western blotting, nanoparticle tracking, and transmission electron microscopy to validate, quantify, and demonstrate capture and release of intact exosomes. As proof of principle, we utilized exo-CATCHER to purify serum exosomes from mildly and severely ill Covid-19 patients, identified, and validated hsa-miR-146a and hsa-miR-126-3p to be significantly downregulated in a small sample set of severely ill patients. We used exo-CATCHER to purify intact exosomes from high IgG titer convalescent sera and confirmed unexpected neutralizing properties, against SARS-CoV-2 in vitro, identified using ultracentrifuged exosomes, not observable with exosomes from sera with IgG titers below quantification level. Exo-CATCHER represents a versatile molecular assay for highly specific purification of circulating exosomes.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE158948 | GEO | 2021/05/13

REPOSITORIES: GEO