Aptamers for cancer diagnosis
They will find and neutralize
Natalia Bulgakova, "Search" No. 21-2015
It all started with the fact that Tatiana Zamai, a professor at Siberian Federal University (SFU), went to Canada for an internship, where she met Professor Maxim Berezovsky, by the way, a graduate of Novosibirsk State University, now working at the University of Ottawa. One of the topics of the research group headed by him – the development of biosensors based on aptamers – interested her very much. In Russia at that time, no one was doing anything like that. Why not start developing this direction in Krasnoyarsk?
Cooperation has developed. Tatiana Zamai, returning to her native Siberian city, enthusiastically began to develop a new scientific field. The topic of Aptamers and her daughter, Anna Zamai, a graduate of the physics department of Krasnoyarsk State University with a degree in Biophysics, seemed attractive. It was 2009. At the expense of an individual youth grant from the Krasnoyarsk Regional Fund for the Support of Scientific and Scientific-Technical Activities, the first reagents and everything necessary to start research were purchased. And the work went.
Today Anna Zamai is a Doctor of Biological Sciences, head of the Laboratory of Biomolecular and Medical Technologies of the Krasnoyarsk State Medical University (KrasSMU) named after V.F. Voino-Yasenetsky. In addition, she is a leading technologist of Laboratory 2.1 of the Institute of Chemistry and Chemical Technology of the Siberian Branch of the Russian Academy of Sciences (IHXT SB RAS) and a project manager supported by the Ministry of Education and Science of the Russian Federation under the Federal Target Program "Research and Development in priority areas of development of the scientific and Technological complex of Russia for 2014-2020". The theme of this project is as follows: "Development of multiplex electrochemical sensor systems based on precious metal nanoparticles and DNA aptamers for the diagnosis of lung cancer", cipher for lot 2014-14-576-0114.
Aptamers are synthetic single–stranded RNA or DNA molecules capable of binding to desired targets. In other words, a single-stranded DNA molecule forms a three-dimensional structure resembling a ring with "tails" of different lengths extending from it. It is with them that it "binds" to the sections of the molecule of a particular protein. The conformation (geometric shapes that molecules can take) of the aptamer depends on the sequence of the oligonucleotides forming the DNA molecule, and hence its affinity to the target (that is, the binding strength to the molecule of the protein chosen by the researcher). By selecting the right sequence from a large library of aptamers, scientists get the ones they need, for example, those that bind to malignant tumor cells. Selection is, frankly, not an easy task: after all, such a library includes 10 to the 12th degree of different sequences (types of aptamers), and only a few are suitable for solving the task. Aptamers are called "digital medicines": diagnostic and therapeutic drugs can be made on their basis. Abroad, research in this area began in the late 1990s, and they are considered very promising.
– Everyone knows about protein antibodies, they are used in diagnostics in laboratories, – explains Anna Zamay. – DNA aptamers are analogs of protein antibodies. In terms of functional characteristics, they are absolutely the same. Even the features of the work are very similar. However, aptamers have several advantages: protein is demanding to storage conditions, expensive to produce. And aptamers with a known sequence of nucleotides can be easily and fairly cheaply synthesized in any quantities. You can modify them with fluorescent or other labels – then, by contacting the tumor cell, the aptamer will give out its presence by glow. You can attach medicines to it and ensure the targeting of their delivery. For example, drugs for the treatment of brain diseases have very undesirable side effects, and the use of aptamers allows you to deliver the drug precisely to the affected tissues and reduce the negative impact on healthy cells by reducing the concentration of the drug.
– How do you select the desired sequence?
– If we select aptamers for tumor tissue, first we take blood cells of a healthy person and healthy tissue. We don't want the aptamers to mess with them. We incubate a library of aptamers with these cells. We don't need those aptamers that have bound to healthy cells, and we separate them. The remaining aptamers are incubated with cancer cells. We remove the unrelated aptamers, and leave those that are connected. We increase the number of their amplified copies. During the amplification of oligonucleotides, random changes in the sequence appear, which are necessary for the implementation of directed "evolution in vitro".
We do several such rounds, we go 10-15 times in this circle. And then we check in which round we got aptamers that bind best to the desired targets – cancer cells – and do not bind to other cells. It is possible to similarly test the obtained aptamers for cells of other types of tumor. The stricter the negative selection, the better the aptamers are obtained.
Then we carry out sequencing, we find out the sequences of nucleotides in all the aptamers present in this pool. Previously, a simpler cloning method was used, the picture was not quite complete. Now we are doing genome-wide sequencing and we get absolutely all the sequences. We use bioinformatics methods to select whole families of aptamers, and of them those that are more common: this means that they bind better to target cells.
In the course of projects supported by grants from the Federal Target Program "Research and Development" in the past years, aptamers to human lung cancer tissue were obtained in the laboratory, which make it possible to identify cells and tumor decay products in clinical samples. If the tumor tissue is stained with aptamers, clusters of cancer cells and healthy areas are clearly discernible under the microscope.
The occurrence of cancer can be judged by the appearance of cancer markers in the blood – proteins characteristic of a particular cancer. However, the presence of a tumor cannot be determined only by a single protein. And the concentration of many well-known biomarkers in the blood of different patients, even with the same diagnosis, varies greatly. One cancer sufferer has a very high concentration, the other has a low concentration, but this does not mean that he does not have cancer! And the disease can be missed. Therefore, now scientists have come to the conclusion that a complex of different biomarkers – a signature - is needed for a more accurate diagnosis. However, not all cancer markers that are used now have sufficient specificity, that is, they lack strict selectivity with respect to the targets with which they are associated. A multiplex system using six aptamers obtained for tumor tissue will make it possible to diagnose cancer more accurately.
Now, at the second stage of the project, biologists are selecting aptamers for six standard cancer marker proteins, choosing the best pool. Chemists conduct their research in parallel. And there are already results. The idea was suggested by a portable electrochemical station – a device slightly larger than a cigarette box, brought from Spain. The principle of its operation is as follows: when binding proteins-cancer markers of the patient's blood plasma with aptamers "sitting" on the golden electrode, the resistance of the biosensor surface changes. The information is displayed on the computer monitor, and it can be used to judge the presence of the disease. But the Spanish electrochemical station, A.Zamay notes, is based on only one biosensor. In addition, the error with which it gives the result is large enough for accurate studies that are conducted in the laboratory. Taking as a basis the principle of operation of this device, Krasnoyarsk residents want to make its improved analogue – for multiplex detection of the signature of six standard cancer marker proteins and six new candidates for biomarkers of lung cancer. This is import substitution.
– Cancer is an individual disease; in fact, everyone has their own tumor with the same name, – explains Anna Zamai. – Some of the patients have more biomarker proteins than others. Therefore, in order to make an accurate diagnosis, a comprehensive study is necessary. And, accordingly, a multiplex biosensor.
The principle of operation of an electrochemical biosensor for the diagnosis of oncological diseases based on DNA aptamers
Since the work is carried out in close cooperation with oncologists of the Krasnoyarsk Regional Oncological Dispensary named after A.I.Kryzhanovsky, scientists know exactly what result is needed for use in clinical practice.
By the third stage of the project, which will involve sequencing and synthesis of aptamers, the multiplex sensor is expected to be ready, and it will be possible to test it.
To solve the complex task of diagnosing lung cancer in Krasnoyarsk, not only scientists and specialists from many branches of science and areas of practical activity – biologists, doctors, chemists, surgeons, engineers, there is even a mathematician, but also various organizations have united. At the beginning of April this year, an agreement was signed on joint activities for the development and production of biosensor test systems for the diagnosis of oncological diseases based on DNA aptamers between V.F.Voino-Yasenetsky KrasSMU, A.I.Kryzhanovsky Regional Oncological Dispensary (hereinafter referred to as the Oncological Dispensary), Krasnoyarsk Scientific Center SB RAS, Institute Chemistry and Chemical Technologies SB RAS, Institute of Physics SB RAS and KB "Iskra".
– What is the peculiarity of our research? We choose aptamers specifically for tissues," says A. Zamai. – Samples of tumor tissues are received from the oncological dispensary to the laboratory after operations, and we are looking for aptamers directly to them. The ethics committee passed, everything is fine here. In Russia, few people are still engaged in obtaining new aptamers, their study is mainly from the standpoint of fundamental science and so far without a visible practical way out. Abroad, proteins or cell cultures are used in such studies: after receiving a colony of cells from a patient, it is repeatedly cultivated. Of course, the cell line obtained after long-term cultivation is already very different from the cells of the original tumor. I repeat, we are working on fresh postoperative material. The aptamers we receive are very common – we checked! – they do not bind to cultures, but they bind very well to diseased cells – both circulating in the blood and isolated from the tumor. Conversely, aptamers obtained for cultures do not work well on clinical samples.
As soon as the device is ready and certified, reagent kits are created, the oncological dispensary is ready to start implementation immediately. They are looking forward to it! The device will be manufactured by Iskra Design Bureau, which specializes in the development of satellite communication systems. The "clean rooms" necessary for production are already being prepared.
– Modern science is not done in isolation. The results are obtained only when people cooperate, – says Tatiana Zamai, a leading researcher at the Laboratory of Biomolecular and Medical Technologies of KrasSMU. – If a person sits and cooks in his own juice, there will be no practical results. When we received the first aptamers, we had no way to create biosensors based on them. No equipment, no people who understand this. In Russia, the Krasnoyarsk Medical University became the cradle of aptamers, where we were accepted with all our, as it seemed to many, adventurous ideas. The state tasks of the Ministry of Health of Russia supported the development of methods for the medical diagnosis of lung cancer and salmonellosis. A high-resolution mass spectrometer was used to search for cancer marker proteins and targets for therapy. But it was only when we cooperated with experts in the field of chemical technologies for the production of hybrid nanoparticles, physics of nanostructures and magnetic phenomena, mathematics and engineering, that things really moved. And now we are working as a big friendly team, we are supported by the leadership of the Medical University, the leadership of the Krasnoyarsk Scientific Center, and our Canadian colleagues, who are very helpful with equipment and reagents. The support of our projects within the framework of the Federal Target Program "Research and Development" has become a great help. The obtained results attracted the attention of industrial partners: engineers and production workers of the Iskra Design Bureau joined the work on the creation of hardware and software for multiplex electrochemical sensor systems. Now we are preparing to move – the rector has allocated a spacious special room for the laboratory.
Portal "Eternal youth" http://vechnayamolodost.ru05.06.2015