Saponin is Essential for the Isolation of Proteins and RNA from Biological Nanoparticles.

Autor: Brezgin S; Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia.; Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia., Frolova A; Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia.; Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia., Bayurova E; Chumakov Federal Scientific Center for Research and Development of Immunobiological Products, Russian Academy of Sciences (Polio Institute), Moscow 142782, Russia., Slatinskaya O; Faculty of Biology, Lomonosov Moscow State University, Moscow 119234, Russia., Ponomareva N; Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia.; Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia., Parshina E; Faculty of Biology, Lomonosov Moscow State University, Moscow 119234, Russia., Bochkova Z; Faculty of Biology, Lomonosov Moscow State University, Moscow 119234, Russia., Kachanov A; Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia., Tikhonov A; Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia., Kostyusheva A; Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia., Karandashov I; Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia., Demina P; Moscow Pedagogical State University, Moscow 119435, Russia., Latyshev O; N. F. Gamaleya National Research Center for Epidemiology and Microbiology of the Ministry of Health of the Russian Federation, Moscow 123098, Russia., Eliseeva O; N. F. Gamaleya National Research Center for Epidemiology and Microbiology of the Ministry of Health of the Russian Federation, Moscow 123098, Russia., Belikova M; Chumakov Federal Scientific Center for Research and Development of Immunobiological Products, Russian Academy of Sciences (Polio Institute), Moscow 142782, Russia., Pokrovsky VS; Blokhin National Medical Research Center of Oncology, Moscow 115478, Russia.; People's Friendship University, Moscow 117198, Russia., Gegechkory V; Department of Pharmaceutical and Toxicological Chemistry, Sechenov First Moscow State Medical University, Moscow 119146, Russia., Khaydukov E; Moscow Pedagogical State University, Moscow 119435, Russia., Silachev D; V.I. Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology, Moscow 117997, Russia.; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia., Zamyatnin AA Jr; Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia.; Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia.; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, Russia.; Department of Biological Chemistry, Sechenov First Moscow State Medical University, Trubetskaya Str. 8-2, Moscow 119991, Russia., Maksimov G; Faculty of Biology, Lomonosov Moscow State University, Moscow 119234, Russia., Lukashev A; Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia.; Research Institute for Systems Biology and Medicine, Moscow 117246, Russia., Gordeychuk I; Chumakov Federal Scientific Center for Research and Development of Immunobiological Products, Russian Academy of Sciences (Polio Institute), Moscow 142782, Russia., Chulanov V; Department of Infectious Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia., Kostyushev D; Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119991, Russia.; Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia.; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, Russia.
Jazyk: angličtina
Zdroj: Analytical chemistry [Anal Chem] 2024 Oct 29; Vol. 96 (43), pp. 17432-17443. Date of Electronic Publication: 2024 Oct 14.
DOI: 10.1021/acs.analchem.4c04607
Abstrakt: Extracellular vesicles (EVs), biomimetics, and other biological nanoparticles (BNs) produced from human cells are gaining increasing attention in the fields of molecular diagnostics and nanomedicine for the delivery of therapeutic cargo. In particular, BNs are considered prospective delivery vehicles for different biologics, including protein and RNA therapeutics. Moreover, EVs are widely used in molecular diagnostics for early detection of disease-associated proteins and RNA. Technical approaches for measuring biologics mostly originated from the field of EVs and were later adopted for other BNs, such as extracellular vesicle-mimetic nanovesicles, membrane nanoparticles (nanoghosts), and hybrid nanoparticles, with minimal modifications. Here, we demonstrate that BNs are highly resistant to protocols that severely underestimate the protein and RNA content of BNs, and provide the relevance of these data both for general BNs characterization and practical applications of CRISPR/Cas-based therapies. We demonstrate that the addition of saponin leads to an ∼2- to 7-fold enhancement in protein isolation and an ∼2- to 242-fold improvement in RNA recovery rates and detection efficiency. Differences in the proteolipid contents of BNs, measured by Raman and surface-enhanced Raman spectroscopy, correlate with their susceptibility to saponin treatment for cargo extraction. Finally, we develop a unified protocol using saponin to efficiently isolate proteins and RNA from the BNs. These data demonstrate that previously utilized protocols underestimate BN cargo contents and offer gold standard protocols that can be broadly adopted into the field of nanobiologics, molecular diagnostics, and analytical chemistry.
Databáze: MEDLINE
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