Research Highlights | Berezovski Research Group | Maxim Berezovski

Research Highlights

1. Aptamers for biosensing and bioseparation of pathogens

The unique feature of our R&D program is that we thoroughly explore aptamer-related projects, from the selection of new DNA aptamers against cells and viruses to the creation of sensors and bioassays. We have produced several anti-pathogen aptamers and applied them in electrochemical aptamer-based biosensors (aptasensors) to detect viruses (Vaccinia, Vesicular Stomatitis Virus, Norovirus) and bacteria (Salmonella typhimurium, Salmonella enteritidis). The aptamers were tailored to the pathogens by counter selection against other related pathogens. It is important to note that our aptasensors could distinguish between viable and nonviable viruses and bacteria. We tested the sensors with a mixture of live and dead viruses/bacteria and detected as little as 60 virus particles or 6 live bacterial cells in 1 µL of solution. Our aptasensors open a new venue for developing various viability sensors to detect many microorganisms and spores, validate the efficiency of sterilization, and spot bioweapons. This study was featured in C&EN “Biosensor Spots Viable Viruses” and Wiley-VCH ChemVews Magazine “Virus Detection – Dead or Alive”.

In addition to biosensing, we applied aptamers for virus purification. DNA aptamers were selected to bind to an oncolytic virus only in the presence of Ca(II) and Mg(II) ions. These ions stabilize the DNA phosphate backbone through chelation and thereby increase the stability of aptamer folding. Thus, chelation of Ca/Mg ions by EDTA/EGTA decreases aptamer affinity and allows the intact virus release. Magnetic beads coated with the switchable aptamers were used to purify the virus from cell cultures. We patented this application for the positive isolation of viruses and cells. Aptamers with switchable affinity brought much attention from biotech companies specializing in vaccine manufacturing, monoclonal antibody production, cell isolation, and cell therapy.


2. Aptamers for biomarker discovery and molecular therapy

We developed Aptamer-facilitated Biomarker Discovery (AptaBiD) technology and revealed new surface biomarkers for live activated (mature) and non-activated (immature) dendritic cells, cell organelles, and nucleus of cancer cells. AptaBiD transformed the field of biomarker discovery, as it is the only technology that simultaneously discovers biomarkers in their native states and produces affinity probes. Furthermore, our aptamers against salmonella showed a vivid bacteriostatic effect. Aptamers against oncolytic virus and antigen-binding fragments of polyclonal antibodies protected the virus from its neutralization. With this approach, we were able to increase viral infectivity by more than 70% in the presence of neutralizing antibodies. Thus, this method showed its potential to enhance the delivery of oncolytic viruses through the bloodstream without compromising the patient’s immune system. 

In 2016, we selected and identified DNA aptamers as specific affinity probes that bind to lung adenocarcinoma cells derived from postoperative tissues. The unique feature of our selection strategy is that aptamers are produced for lung cancer cell biomarkers in their native state and conformation without previous knowledge of the biomarkers. We applied these aptamers to detect circulating tumor cells in clinical samples of peripheral blood of lung cancer patients. We identified several aptamer-associated protein biomarkers for lung cancer. Tumor-specific aptamers can be produced for individual patients and synthesized many times during anticancer therapy, thereby opening up the possibility of personalized diagnostics.

In 2017, we presented magnetodynamic nanotherapy using DNA aptamer-functionalized 50 nm gold-coated magnetic nanoparticles. The nanoparticles were exposed to a low frequency alternating magnetic field and eliminated tumor cells selectively in vivo. The aptamer-guided nanoparticles and the low frequency alternating magnetic field demonstrated a unique noninvasive nanoscalpel technology for precise cancer surgery at the single-cell level.


3. Study of biomolecular interactions with kinetic capillary electrophoresis (KCE) and mass spectrometry

KCE coupled with mass spectrometry (KCE-MS) became a platform for studying the conformational dynamics and affinity interactions of proteins and DNA and screening new enzymes and substrates. Different KCE methods were designed for slow affinity interactions and fast interactions depending on the rate of interactions. The range of KCE applications includes:

  1. Selection of DNA aptamers.
  2. Real-time monitoring of protein conformational dynamics in solution.
  3. Study of DNA G-quadruplex folding.

In addition, our article in Nature Chemical Biology showed the simultaneous analysis of protein conformational isomers with their enzymatic activity and inhibition in a single experiment. 

KCE established a new paradigm that separation methods and mass spectrometry could be a comprehensive tool for measuring kinetics and thermodynamics with mass and structure elucidation of interacting molecules. KCE becomes highly attractive for the pharma/biotech industry for screening drug candidates and affinity probes. 

4. Proteomics and metabolomics of extracellular vesicles 

We are interested in developing new technologies for the affinity isolation of extracellular vesicles (EVs), the detection and counting of EVs, and the comprehensive analysis of EV biomarkers. EVs are small membrane-enclosed structures that are secreted into the extracellular milieu by many cell types. These particles include small (exosomes, 30-150 nm) and large (microvesicles, 100 – 1000 nm; apoptotic bodies, 50 – 5000 nm) vesicles. These vesicles are secreted from cells and released in different fluids of the body. EVs contain a lipid bilayer that protects the cargo, consisting of nucleic acids, proteins, lipids, and metabolites, from degradation. EVs are now central to biological sciences because they seem to constitute a new system of cell-cell communication.

Our recently published work investigated the proteome of exosomes from the cancerous breast MDA-MB-231 and the noncancerous MCF10A cell lines. A total of 1,107 exosomal proteins were identified in both cell lines, 726 unique to the MDA-MB-231 breast cancer cell line. Eighty-seven proteins were predicted to be relevant to breast cancer and 16 proteins to cancer metastasis. Three exosomal membrane/surface proteins, glucose transporter 1 (GLUT-1), glypican 1 (GPC-1), and disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), were identified as potential breast cancer biomarkers and validated with Western blotting and high-resolution flow cytometry. This article in Scientific Reports was picked as “Top 100 in Cancer” due to its high interest from the research community. 

In the follow-up article, we compared the proteome of microvesicles (MVs) derived from MDA-MB-231 and MCF10A cell lines. Among the 89 proteins unique to MDA-MB-231 MVs, three enzymes: ornithine aminotransferase (OAT), transaldolase (TALDO1), and bleomycin hydrolase (BLMH), were previously proposed as cancer therapy targets. These proteins were enzymatically validated in cells and MVs. The specific activity of OAT and TALDO1 was significantly higher in MDA-MB-231-derived MVs than in MCF10A MVs. BLMH was highly expressed in MDA-MB-231-derived MVs, compared to MCF10A MVs. This study shows that MVs carry functional metabolic enzymes and provides a framework for future studies of their biological role in breast cancer and potential in therapeutic applications.  

5. Quantitative capillary electrophoresis for counting and quality control of viruses and extracellular vesicles

Viruses as vehicles of antigens and gene therapy agents have progressed rapidly from laboratories to clinics. Furthermore, extraordinary efforts have been taken in oncolytic viral therapy culminating in several ongoing clinical trials. Our lab developed a technology called “Viral qCE” to partition intact DNA/RNA viruses from free nucleic acids and other impurities. It measures an exact virus concentration and host cell DNA contamination. In addition, our method gives the possibility of live monitoring of virus degradation and aggregationscreens virus cryoprotectors, and provides information about the sample stability during storage and handling. 

In additional work, we developed EVqCEto measure an average RNA mass in EVs, determine EV concentrations and the degree of EV degradation after sample handling. The average RNA mass was found to be in the range of 200–400 ag per particle, noting that more aggressive cancer cells have less RNA in EVs (200 ag per particle) than non-aggressive cancer cells (350 ag per particle). 

© Maxim Berezovski 2009-2021