1. Analysis of Proteins and Nucleic Acids Using Capillary Electrophoresis and Mass Spectrometry (CE-MS)
Capillary electrophoresis (CE), the focus of Berezovski’s Ph.D. dissertation, remains a pivotal research tool in the Berezovski lab. Due to Berezovski's contributions, CE coupled with mass spectrometry (CE-MS) has become a platform for studying the conformational dynamics and affinity interactions of DNA and proteins, as well as screening new enzymes and substrates. In 2016, Berezovski and his research team demonstrated, for the first time, the simultaneous analysis of protein conformational isomers with their enzymatic activity and inhibition in a single experiment, with results published in Nature Chemical Biology [Nature Chemical Biology, 2016, 12, 918].
In addition to proteins, Berezovski is also interested in analyzing microRNAs using CE. His team developed a novel protein-facilitated affinity CE assay with fluorescent detection for the rapid quantification of ultralow microRNA levels in blood serum, utilizing DNA/RNA binding proteins (SSB and p19) as separation enhancers. This technique enabled the detection of endogenous microRNAs and their posttranscriptional modifications by CE-MS [Analytical Chemistry, 2018, 90, 6618].
2. Aptamers for Biosensing and Molecular Therapy
The Berezovski lab’s R&D program uniquely explores aptamer-related projects, ranging from the selection of new DNA and RNA aptamers [Molecular Therapy-Nucleic Acid, 2020, 20, 176] and the identification of aptamers’ 3D structures and binding epitopes [Analytical and Bioanalytical Chemistry, 2019, 1, 1; ChemMedChem, 2020, 15, 363, COVER PAGE ARTICLE], to the creation of novel aptamer-based sensors and bioassays. His team has developed aptamers for detecting viruses (Vaccinia, Vesicular Stomatitis Virus, Norovirus, and SARS-CoV-2) [Molecular Therapy - Nucleic Acids 2023, 31, 731; Chemistry–A European Journal, 2022, 28, e202104481, COVER PAGE ARTICLE], bacteria (Salmonella typhimurium, Salmonella enteritidis), and cancers [Molecular Therapy-Nucleic Acids, 2017, 6, 150; Cancers, 2019, 11, 351; Biomedicines, 2020, 8, 14].
Berezovski pioneered the selection of DNA aptamers for primary tumor cells and the follow-up detection of circulating tumor cells (CTCs) in blood and cerebrospinal fluid [Molecular Therapy - Nucleic Acids 2023, 32, 267]. His strategy selects aptamers for cell biomarkers in their native state and conformation without prior knowledge of the biomarkers. Tumor-specific aptamers can be produced for individual patients and synthesized repeatedly during anticancer therapy, opening the possibility for personalized diagnostics. His team was the first to use DNA aptamers in mass cytometry to detect cancer cells [Analytical and Bioanalytical Chemistry, 2018, 410(13): 3047-3051], a growing research area with several commercial applications.
In 2021, his team labeled an aptamer with the isotope 11C for imaging tumors and metastases in vivo using positron emission tomography-computed tomography (PET/CT) [Molecular Therapy-Nucleic Acids, 2021, 26, 1159, COVER PAGE ARTICLE; Molecular Therapy - Nucleic Acids 2023, 32, 267]. The radiolabeled aptamer provided higher specificity and contrast imaging of tumors compared to the standard radiotracer, 18F-FDG.
Additionally, the team applied aptamers for virus purification. The DNA aptamers were selected to bind viruses only in the presence of Ca(II) and Mg(II) ions. Chelation of these ions by EDTA/EGTA reduces aptamer affinity, allowing the release of free and intact viruses. They patented switchable aptamers for the positive isolation of viruses and cells (US patent issued in 2019, US10233442), attracting significant attention from biotech companies specializing in vaccine manufacturing, antibody production, and cell isolation.
In 2017, Berezovski presented magnetodynamic nanotherapy using DNA aptamer-functionalized 50 nm gold-coated magnetic nanoparticles [Theranostics, 2017, 7, 3326; Cancers, 2020, 12, 216] and magnetic microdisks [Nucleic acid therapeutics, 2017, 27, 105]. Exposed to a low-frequency alternating magnetic field, these nanoparticles and microdisks selectively eliminated tumor cells in vivo, demonstrating a unique noninvasive nanoscalpel technology for precise cancer surgery at the single-cell level.
3. Proteomics and Metabolomics of Extracellular Vesicles (EVs)
Berezovski's research also focuses on developing new technologies for the affinity isolation, detection, and comprehensive analysis of EV biomarkers. EVs, which include small (exosomes, 30-150 nm) and large (microvesicles, 100 – 1000 nm; apoptotic bodies, 50 – 5000 nm) vesicles, are small membrane-enclosed structures secreted by many cell types and released into various body fluids. EVs contain a lipid bilayer that protects the cargo of nucleic acids, proteins, lipids, and metabolites from degradation, constituting a new system of cell-cell communication.
The Berezovski lab developed “EVqCE” to measure the average RNA mass in EVs, determine EV concentrations, and assess the degree of EV degradation after sample handling [Separations, 2021, 8, 110]. They found that aggressive cancer cells have less RNA in EVs (200 ag per particle) compared to non-aggressive cancer cells (350 ag per particle).
In 2020, the lab published a comprehensive study on the proteome of exosomes from the highly aggressive breast cancer cell line MDA-MB-231 and the noncancerous MCF10A cell line [Scientific Reports, 2020, 10, 1]. This article was selected as “Top 100 in Cancer” due to its high interest from the research community. They identified three exosomal membrane/surface proteins, glucose transporter 1 (GLUT-1), glypican 1 (GPC-1), and disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), as potential breast cancer biomarkers and validated them using Western blotting and flow cytometry.
In a follow-up study, the lab compared the proteome of microvesicles (MVs) derived from the same cell lines [Biomedicines, 2021, 9, 107]. Among the 89 proteins unique to MDA-MB-231 MVs, three enzymes—ornithine aminotransferase (OAT), transaldolase (TALDO1), and bleomycin hydrolase (BLMH)—were proposed as cancer therapy targets and enzymatically validated in cells and MVs. This study demonstrates that MVs carry functional metabolic enzymes and provides a framework for future studies on their biological role in breast cancer.
The lab also performed a phosphoproteomic analysis [Biomedicines, 2022, 10, 408] and lysine acetylation analysis [Biomedicines, 2023, 11, 1076] of breast cancer-derived exosomes to provide insights into the molecular and cellular regulatory mechanisms crucial for breast cancer progression and metastasis. They mapped 2450 phosphorylation sites to 855 distinct proteins, covering a wide range of functions. Among the identified proteins, they validated four cancer-associated enzymes: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), sirtuin-1 (SIRT1), and sirtuin-6 (SIRT6).
Additionally, they validated five acetylated enzymes from the glycolytic pathway, present only in cancer-derived sEVs: aldolase (ALDOA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK1), enolase (ENO), and pyruvate kinase M1/2 (PKM). The specific enzymatic activity of ALDOA, PGK1, and ENO was significantly higher in EVs from highly metastatic MDA-MB-231 cells compared to non-metastatic MCF10A cells.
Their study demonstrated that exosomes contain functional metabolic enzymes, which could be further explored for potential early cancer diagnostic and therapeutic applications.