Playing God
The 2018 Nobel Prize in Chemistry was awarded for evolution in vitro
Alexander Dubov, N+1
The Nobel Prize in Chemistry in 2018 was awarded for the development of methods for the directed evolution of enzymes, peptide sequences and antibodies. The award will be shared by Frances H. Arnold, George P. Smith and Sir Gregory P. Winter. Frances Arnold received the prize for the development of a method of directed evolution of enzymes, and two other scientists – for the creation of a method of phage display of peptides and antibodies.
The methods, for the development of which the Nobel Prize in Chemistry was awarded in 2018, are used for the synthesis in laboratory conditions of various types of proteins with the necessary properties – catalytic activity, the ability to bind to certain substances and cells, or a range of conditions in which these proteins work. Both developed approaches are based on the fact that the structure of proteins that are synthesized in living organisms is encoded by a sequence of nucleotides in genes. With the help of transcription and translation mechanisms, the sequence of nucleotides at a certain site of the DNA molecule is converted into a sequence of amino acids, as a result of which a protein with a certain primary structure is synthesized, which takes the desired shape and performs certain functions in the body.
Accordingly, if you slightly disrupt the sequence of nucleotides in DNA by changing a pair of nitrogenous bases, then you can also influence the structure of the proteins that they encode, while changing their physical and chemical properties. In natural conditions, such changes in the nucleotide sequence occur due to random mutations – changes in the genome under the influence of external conditions, for example, due to errors during DNA replication. It is these errors that lead to the evolution of living organisms: small changes accumulate in the structure of the gene, after which the most advantageous variants from the point of view of further development of the species are naturally selected.
In the process of natural evolution, the selection of the most effective proteins and their corresponding genes can take a very long time, covering the life of many generations. But in order for a similar approach to be used for the directed synthesis of proteins with specified properties in the laboratory, it would be desirable to carry it out much faster. Initially, for the directed synthesis of proteins based on DNA, scientists tried to make changes to the structure of the molecule in known places based on theoretical predictions. However, it is almost impossible to predict in advance exactly how point substitutions of nitrogenous bases will affect protein functions, so this approach turned out to be not very effective.
Directed evolution
In 1993, Frances Arnold proposed an alternative approach. She showed that changing the structure of the gene responsible for the synthesis of a certain protein can be carried out not point–by-point, but on the contrary - getting a lot of gene variants with small changes in random places. Nowadays, polymerase chain reaction with an increased probability of mutations is most often used for this – the process of multiple successive doubling of the original DNA molecule with the help of DNA polymerase. As a result of this process, many copies of the original gene are formed, and the structure of many of them, due to random mutations, differs very slightly from the structure of the original molecule. All these genes encode almost the same protein as DNA with the original sequence of nucleotides, but its properties are still slightly different. The set of all these numerous modifications – the gene library– contains molecules that encode both more effective enzymes and less effective ones.
The main difficulty after creating such a library is to select from it exactly those genes that correspond to the most effective – from the point of view of further use – proteins. To do this, it was proposed to use relatively small libraries, which were gradually enriched with "useful" options due to selection after each round of mutagenesis. This process essentially repeats evolution, and at each stage the gene that encodes the more efficient protein is selected.
"You are playing God and evolution to some extent," Konstantin Severinov, a professor at Skoltech and Rutgers University, comments on this approach. – Evolution according to Darwin also happens completely undirected, all changes happen without regard to the result, by chance, and then selection finds in the available diversity what we need in this situation. In the experiment, you just dramatically increase the rate of accumulation of mutations, and then select as you want."
The theoretical possibility of such a method was predicted in 1984 by Manfred Eigen (also a Nobel Laureate), but it was Francis Arnold who managed to implement it in an experiment for the first time in 1993. In her work (Engineering proteins for nonnatural environments), she synthesized subtilisin E, an enzyme from the hydrolase class. As a result of four stages of mutagenesis, she managed to increase its activity by 250 times compared to the original modification.
The proposed methodology included the following main stages. First, those enzymes that are best suited for possible evolution are determined. Then gene libraries are compiled for them, after which criteria are developed by which the desired dynamics of the enzyme characteristics is determined (for example, Arnold looked at how fast the hydrolysis of casein under the action of the enzyme proceeds). Those DNA that produce "good" proteins are preserved, and those that lead to the synthesis of enzymes with degraded properties are discarded. Then the selected DNA undergoes the next stage of mutagenesis, and a new library is created. By repeating this procedure several times, it is possible to obtain an enzyme with the required properties.
Schematic diagram of the method of directed evolution of enzymes (Nobel Prize)
"In fact, Arnold proposed a method that allows for dramatically accelerated evolution of DNA molecules in the laboratory. Previously, when people cloned DNA, multiplied molecules in a test tube, they tried to make these modifications as accurately as possible in order to increase the amount of the original gene and God forbid to change it," Severinov says. – In fact, she came up with the idea that this should not be done very accurately, but, on the contrary, a little inaccurately, and then you will have material for evolution. If you have a certain protein with a certain set of physical or biological properties that you would like to improve, then you can not wait for the mercy of nature, but try to do it yourself."
The enzymes, the artificial evolution of which Arnold studied in her works, are proteins that regulate the rate of synthesis or decomposition of certain compounds in the cell, responsible for the transfer of chemical groups from one molecule to another. At the same time, it is not necessary to accelerate the reaction that the enzyme catalyzes – it is possible, for example, to increase the stability of the protein at different temperatures or selectivity in the synthesis of optical isomers. Thus, the approach developed by Arnold helped not only to improve the method of synthesis of biocatalysts with specified properties, but also to investigate the process of protein modification in the process of evolution in general – the dependence of protein properties on external conditions, mutagenesis rate, population size or selection rigidity.
This method has, however, one obvious limitation. "With it, you can't work normally with proteins that you don't know how to grow in bacteria," says Vsevolod Belousov, head of the Laboratory of Molecular Technologies at the Institute of Bioorganic Chemistry of the Russian Academy of Sciences. – If the protein is simple, the bacterium can synthesize it: it will fold properly in it and will work in the bacterium. For example, a fluorescent protein is a small "barrel" of beta layers. Curled up – and that's it. There are no options. Therefore, it evolves easily. For example, we make biosensors for different molecules in a similar way, because in this case it is possible to express this protein inside the bacterium E. Coli. However, if it is, for example, a eukaryotic protein, and even a membrane one, or it has some kind of posttranslational modification that is not present in bacteria, then in this case directed evolution is practically impossible. Only if there is some kind of computer method or after a long and careful study of the structure as a result of attempts at point modifications."
The structure of the enzyme for the cyclopropanation reaction, which was synthesized using the directed evolution method (P. S. Coelho et al./ Nature Chemical Biology, 2013)
Using viruses
In addition to enzymes that help to quickly carry out certain chemical reactions, other proteins can be obtained in a similar way, which are necessary, for example, for the production of medicines. In this context, the attention of scientists is attracted by other compounds that exist in living organisms – antibodies. These are proteins produced by B-lymphocytes when bacteria or viruses enter the body. The antibody binds to epitopes – peptides or saccharides on the surface of bacteria or viruses – and thus suppresses their reproduction or neutralizes the toxic substances released by them. Choosing the right antibody for specific substances is also not an easy task, but it can be solved in a similar way, with the help of directed evolution.
Now the phage display method is most often used for this. For the first time he was offered George Smith, who used bacteriophages – viruses that infect bacteria - to synthesize antibodies that can bind specifically to the desired antigen. Viruses contain a DNA molecule inside, and the shell consists of proteins whose structure is encoded by this DNA. In 1985, the scientist showed that if a sequence encoding another protein in one reading frame is attached to the envelope protein gene, then this protein will appear on the surface of the phage particle. The scientist also showed how the area of appearance of a new peptide sequence depends on the place in the gene in which the new nucleotide sequence is embedded.
Schematic diagram of the phage display method (Nobel Prize)
Five years after Smith's first work, Gregory Winter demonstrated that this method could be used, for example, to produce an antibody on the surface of a bacteriophage capable of selectively binding to the desired antigen. To do this, a gene is embedded in a certain place in the DNA of the virus, which encodes the desired antibody site necessary for antigen recognition. Then the desired peptide site appears in the surface protein, and the virus, thus, actually turns into an antibody itself. After that, it is possible to carry out the procedure of directed evolution: embed random mutations in DNA, create a library of viruses and, as a result of selection, increase the effectiveness of antibodies.
As an example of using this approach Vsevolod Belousov gives the following example: "Let's say you want to get a protein that binds the NADH molecule, but does not bind NADPH. To do this, you take a surface and cover it with a NADH molecule. On it you pour a mixture of phages obtained by the bacterium E. Coli. There are a lot of different modifications in it. Each phage is unique and carries its own version of the protein under study. Everything that is bound, stuck to the surface, and everything that is not bound, you can wash. Then, under tougher conditions, you launder what's bound and sequence the gene–that's what you want to study."
According to Mr. Severinov, the phage display method is the fastest way to obtain antibodies with the best ability to bind to cancer cells. "For example, you already have a certain antibody, and you want to increase its effectiveness a thousand times. To do this, you take the antibody gene, insert it into the virus so that the protein is on the surface, and get a library of viruses. After that, you test it in more and more difficult conditions: you reduce the number of cancer cells that need to be contacted, you add an agent that breaks the binding. You choose those viral particles that turned out to be the "strongest" in the conditions of your selection. Then you can find out how the particles that have passed this functional screening differ from those that have not. As a result, it is possible to obtain a sequence that does not exist in nature, but has the properties we need."
Thanks to the phage display method, it is now actually possible to directly synthesize any proteins with altered properties and use them for the treatment of autoimmune diseases or cancer.
The present of methods of directed evolution
The developed methods of directed evolution and phage display have long become routine. "Now you even stop thinking that they might have an author, like some Pythagorean theorem," Severinov says. "Everyone is working with it, it's really an everyday laboratory practice."
To date, these methods are used to produce biofuels, to develop medicines, to obtain enzymes necessary for the synthesis of compounds that cannot be obtained by classical methods of organic synthesis. "The work of Professor Frances Arnold helped to make qualitative breakthroughs in a large number of syntheses with the help of enzymes, the achievements of Gregory Winter and George Smith made it possible to make significant progress in the synthesis of proteins and new pharmaceuticals, including pro-cancer drugs. We are talking about directed modification in order to obtain new substances, the formation of chemical bonds that could not exist in nature," says Stepan Kalmykov, Acting Dean of the Faculty of Chemistry of Moscow State University. – Such changes can lead to the appearance of new properties of the body – an increase in life expectancy, resistance to diseases, evolutionary changes. This is really a revolution in the field of enzyme research, the consequences of which have enormous applied significance in the medical and biomedical field. This is both targeted drug delivery and a change in the biochemistry of the body. The results of this work can affect every person on Earth."
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