Proteins are crucial for the body’s cells since they act as the building material for our organism. Apart from helping cells grow and interact with other substances, proteins, proteins can also cause diseases, including cancer. These days, one particular protein under scrutiny is the recently discovered hnRNPA2B1 (A2B1), which is responsible for regulating RNA processes. Studies that its amount increases in cancer cells. Moreover, hnRNPA2B1 is responsible for the stability of malignant cells, which is why it is a potential target for cancer treatment. Through this protein, it’s also possible to influence the cells directly: destabilize them, hinder their division, and thus stifle cancer development.
However, proteins are challenging to work with. Outside of living cells, they quickly lose their biological functions and become unsuitable for research, while the existing analysis methods require large protein concentrations and special tags to track them – which complicates the process even further.
“Proteins have binding sites – locations through which the protein interacts with molecules of medicine. However, outside a cell, proteins quickly lose shape: their binding sites become deformed and small molecules cannot bind there. Moreover, proteins can be inconveniently located on the sensors used to analyze them – for instance, their binding site could be facing downward and this would make interaction with the molecule impossible,” explains Olga Volkova, one of the paper’s authors and a PhD student at ITMO’s Infochemistry Scientific Center.
Researchers at ITMO have found a way around these limitations that will prolong a protein’s life outside a cell. They turned to the quartz crystal microbalance (QCM) method, which, unlike its counterparts, works with micro- and nano-quantities of protein (the method’s sensitivity is 1 nanogram), doesn’t require tags, and makes it possible to track the interaction between proteins and small molecules in real time.
The electrodes inside the microbalance. Photo by ITMO NEWS / Dmitry Grigoriev
First, the electrodes inside the microbalance are covered with a layer of polyelectrolytes. Like the electrodes, they have a charge and thus easily connect to the charged metal surface. This way, they create an artificial membrane on the sensors by blanketing the protein and preventing it from disintegrating or losing its form and biological functions. After that, excess polyelectrolytes are removed with water and a protein solution is placed on the electrodes. Once the protein “precipitates” on the sensors, a solution of small molecules (cancer treatment or its components) is placed on the electrodes. Next, the microbalance detects real-time mass changes on its electrodes.
“With this method, we track the changes in the electrodes’ oscillation frequency. Next, voltage is applied to the gold electrodes, and the quartz crystal inside them begins to oscillate at a certain frequency; in our case it is 5 MHz. Consequently, when something else “lands” on the electrode, the oscillation frequency changes, and the device immediately records this. By analyzing these changes, we can make our conclusions about how these substances interact. At the same time, we can also determine the amount of treatment molecules that ‘settled’ in the protein. Moreover, thanks to real-time observation, we can exclude the involvement of other factors. Such a qualitative and quantitative evaluation isn’t possible with any other method,” adds Evgeny Smirnov, the head of the research team and a researcher at the Infochemistry Scientific Center.
Apart from demonstrating the method’s efficiency and accuracy, the team described the optimal conditions for its application.
The method’s application in cancer treatment testing will help expand the amount of analyzed materials and in this way facilitate the selection of the most efficient ones, as well as decrease the amount of resources required for testing. In the future, the method can be used to adjust treatments or develop better ones. The technology has already been experimentally tested – the researchers are currently using it to analyze cancer treatments.
This project is supported by the Russian Science Foundation grant No. 22-65-00022.
