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The large-scale integration project involving a number of academic institutes is being carried out in Novosibirsk. This project became the subject of the report "Technological Platform for Synthetic Biology", delivered at the meeting of the RAS SB Presidium in March 2012 by Alexander Sinyakov, Cand. Sc. (Chem.), Head of the Medical Chemistry Laboratory under the RAS SB Institute of Chemical Biology and Fundamental Medicine--he is responsible for an overall coordination and progress of the project. He spoke about new horizons opened up by gene synthesis, biomedical research, development of biotechnologies and design of a microchip DNA synthesizer. He told Yulia Alexandrova, journalist of the Science in Siberia newspaper, about the essence of these works.

Today, a new interdisciplinary science--synthetic biology aimed to create artificial living systems based on the same genes--is being formed now. This science is developing on the basis of a revolutionary breakthrough in the sphere of gene synthesis. For this purpose microchip reactors are designed and constructed: they will simultaneously produce up to several hundred thousand oligonucleotides--relatively short (some dozens of DNA bases) DNA fragments. From them are collected fragments of genomes. What does the process of such synthesis represent?

There are some computer-based DNA synthesizers operating at the Laboratory of the abovementioned institute. These instruments are controlled by a computer and "collect" oligonucleotides--specific "bricks" for gene engineering and molecular biology research. In particular, they are used to detect pathologies, mutations, pathogens. To fulfill all these tasks, manufacturing of oligonucleotide probe diagnostic chips was launched there.

Artificial DNAs are synthesized on three said devices capable of producing up to 200-300 oligonucleotides of a different composition daily. But the science is rapidly developing and the need for them is also increasing (together with potentialities of studies). In other words, if a modern device produces hundreds of thousands of oligonucleotides and researchers have a lot of cheap and available DNA fragments, there will appear an opportunity to artificially synthesize genomes of various organisms. This very task is to be solved by the scientific community today.

The simplest genomes have already been synthesized. The first one--poliovirus of about 7,500 nucleotides--was constructed in 2002 by US Professor Wimmer and his colleagues. Then, a bacteriophage parasitizing on E.coli was constructed. In 2003, the American biologist Craig Venter was the first who decoded a human genome and synthesized over a million of pairs of nucleotides to construct an artificial bacterium.

According to Alexander Sinyakov, he and his colleagues are trying to design special computer-based devices that would help scientists to construct artificial genomes. However, Novosibirsk specialists are not planning to synthesize highly-complex constructions from the start; they think it fit to move forward gradually, for example, to set up a plant of useful genes. Thus, it is possible to make a series of important proteins, for example, interferon--a non-specific antiviral agent--produce special bacteria with an artificial gene in the genome. This is simpler than isolation of interferon from donor's blood. As for target genes, there is a great variety of them: pathogen antigens used in diagnostics, and genes responsible for coding of mediators of cell immunity, and appropriate constructions necessary to produce live vaccines.

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As an example, the scientist presented a silicon chip of the DNA microchip synthesizer. In theory, it is capable of producing 20,000 oligonucleotides of a different composition on its surface, but today there are less than two hundred cells. First, scientists should learn how to precipitate silicon oxide in them, then to "bind" the first link of a future oligonucleotide, and finally to synthesize DNA fragments of a different composition in each cell. To reach a higher stage--construction of a genome of a living organism, it is necessary to correctly "sew" these oligonucleotides into a target sequence (the process of synthesis is accompanied by errors to be corrected by special enzymes).

One group of scientists cannot solve such complex tasks; only joint efforts within integration projects can bring positive results. Proceeding from high importance of synthetic biology, a consortium was organized at the RAS SB to design a microchip oligonucleotide synthesizer--in addition to the RAS SB Institute of Chemical Biology and Fundamental Medicine, the consortium incorporated the Institute of Automatics and Electrometry, Institute of Semi-Conductor Physics named after A. Rzhanov, Novosibirsk Institute of Organic Chemistry named after N. Vorozhtsov, Institute of Theoretical and Applied Mechanics named after S. Khristianovich. It is an interdisciplinary project intended for the development of chemical reagents, photochemical methods to control the oligonucleotide chain expansion reaction, manufacture of a microchip-reactor, and search for gene synthesis methods.

To ensure success of the project, physicists should know how to precipitate silicon oxide to reaction cells of a microchip, master photolithography technologies, and how to join quartz glass and silicon. Silicon chips are produced at the Institute of Semi-Conductor Physics by way of photolithography. There is also precipitated silicon oxide from a gaseous phase in the cell, and the channels are treated with plasma to prevent a parallel incidental synthesis of oligonucleotides. To effect reactions on the initial chip, one should master chemistry of oligonucleotide synthesis. This technology is well developed at the RAS SB Institute of Chemical Biology and Fundamental Medicine, and almost all technological processes will be carried out there except for one. When using conventional DNA synthesis plants, special acids are added to reaction columns and a temporary protection of a growing oligonucleotide group is relieved of a blockade. However, as the chip is very small, scientists need other photogenerated acids to effect the same operations. Such acids are produced at Novosibirsk Institute of Organic Chemistry named after N. Vorozhtsov.

The next phase is a target model. An oligonucleotide of a set composition is synthesized in this complex technological machine in each microchip cell; for this purpose a ray of light to relieve protection groups of a blockade is sent to every cell at a definite time. This task is fulfilled by more than 800 thous. of controlled micromirrors. They are very small, about 12 microns, and should light up a strictly determined part of a microchip. This delicate work is effected by the fourth participant of the project--the RAS SB Institute of Automatics and Electrometry. As for enhancement of hydrodynamics of the whole device, this task is assigned to the Institute of Theoretical and Applied Mechanics named after S. Khristianovich.

The microchip synthesizer produces a few target oligonucleotide molecules (100-1,000). It is not enough to build a target gene structure. To solve this problem a well-known polymerase chain reaction is applied. It enables to multiply few target molecules manifold. It also makes it possible to build various structures, obtain hybrid bacteria, viruses, producers, for example, conform bacteria. In other words, after creating a DNA fragment, scientists can "clone" it.

Speaking of the background of these works, Sinyakov pointed out that synthesis of artificial genes was initiated in Novosibirsk 30 years ago; in 2002 biological microchips were first used for diagnostics, and about three

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years ago the first integration project to construct a microchip DNA synthesizer was launched and scientists made first steps in developing a new branch of national science--synthetic biology.

By the way, Western specialists started to use microchip synthesizers not long ago. Since these devices can be used to implement brand-new approaches to develop biological weapons, valuable products for industry, medicine and agriculture, the export restrictions are likely to be expected to our country.

According to the scientist, such export restrictions are already made. Thus, in December 2006, a number of major international companies and American research centers adopted a document "Practical Prospects of the DNA Synthesis and Biological Danger", presenting a plan of a strict control of similar works. Today, only a few companies in the USA, Great Britain and Germany take orders to produce oligonucleotide mixes received on microchip synthesizers, and these orders are analyzed using computers. Besides, a customer must explain what he needs these products for. No doubt, these restrictions make relations between Russia and Western partners more complex.

Back to the project, Sinyakov said: "The task we are facing now is very complicated: we have to master modern photolithography technologies and micromechanics that has already been well developed in the Western countries, to improve chemical processes. We already have a model and are eager to fill it with a meaningful 'content', to 'teach' it how to operate efficiently, to shift from large cell microchips to relatively small ones. This will enable us to increase significantly the number of synthesized oligonucleotides. When we learn to construct thousands, up to 20 thousands, not hundreds of them on a microchip, we'll get a powerful diagnostic device."

Science in Russia, No.6, 2012

Yu. Alexandrova, "Gene Factory is a Useful Thing", "Science in Siberia" newspaper, No. 15, 2012


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