TY - JOUR
T1 - Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release (EV-CATCHER)
T2 - A customizable purification assay designed for small-RNA biomarker identification and evaluation of circulating small-EVs
AU - Mitchell, Megan I.
AU - Ben-Dov, Iddo Z.
AU - Liu, Christina
AU - Ye, Kenny
AU - Chow, Kar
AU - Kramer, Yael
AU - Gangadharan, Anju
AU - Park, Steven
AU - Fitzgerald, Sean
AU - Ramnauth, Andrew
AU - Perlin, David S.
AU - Donato, Michele
AU - Bhoy, Emily
AU - Manouchehri Doulabi, Ehsan
AU - Poulos, Michael
AU - Kamali-Moghaddam, Masood
AU - Loudig, Olivier
N1 - Publisher Copyright:
© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles
PY - 2021/6
Y1 - 2021/6
N2 - Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory-based purification techniques rely on the physical properties of small-EVs rather than their inherited cellular fingerprints. We established a highly-selective purification assay, termed EV-CATCHER, initially designed for high-throughput analysis of low-abundance small-RNA cargos by next-generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small-RNAs from mouse small-EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small-EVs. As proof-of-principle for sensitive detection of circulating miRNAs, we compared small-RNA sequencing data from a subset of small-EVs serum-purified with EV-CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid-19 patients. We identified and validated, only in small-EVs, hsa-miR-146a and hsa-miR-126-3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid-19 patients with high anti-spike IgG titers, we confirmed the neutralizing properties, against SARS-CoV-2 in vitro, of a subset of small-EVs serum-purified by EV-CATCHER, as initially observed with ultracentrifuged small-EVs. Altogether our data highlight the sensitivity and versatility of EV-CATCHER.
AB - Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory-based purification techniques rely on the physical properties of small-EVs rather than their inherited cellular fingerprints. We established a highly-selective purification assay, termed EV-CATCHER, initially designed for high-throughput analysis of low-abundance small-RNA cargos by next-generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small-RNAs from mouse small-EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small-EVs. As proof-of-principle for sensitive detection of circulating miRNAs, we compared small-RNA sequencing data from a subset of small-EVs serum-purified with EV-CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid-19 patients. We identified and validated, only in small-EVs, hsa-miR-146a and hsa-miR-126-3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid-19 patients with high anti-spike IgG titers, we confirmed the neutralizing properties, against SARS-CoV-2 in vitro, of a subset of small-EVs serum-purified by EV-CATCHER, as initially observed with ultracentrifuged small-EVs. Altogether our data highlight the sensitivity and versatility of EV-CATCHER.
KW - TEM
KW - exosome purification
KW - extracellular vesicles
KW - micro-RNA profiling
KW - sequencing
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U2 - 10.1002/jev2.12110
DO - 10.1002/jev2.12110
M3 - Article
AN - SCOPUS:85111787698
SN - 2001-3078
VL - 10
JO - Journal of Extracellular Vesicles
JF - Journal of Extracellular Vesicles
IS - 8
M1 - e12110
ER -