An international team of scientists from the United States, France and China has created a semi-synthetic life form. Although attempts to obtain bacteria with modified DNA have already been made, microorganisms multiplied poorly, required special growing conditions, and eventually got rid of the modifications introduced into them. "Lenta.ru" talks about a new work in which the researchers managed to solve these problems, having obtained a creature that is radically different from all natural life on Earth.
More recently, the DNA of all living organisms on our planet consisted of four types of nucleotides containing adenine (A), or thymine (T), or guanine (G), or cytosine ©. Strings of tens or hundreds of millions of nucleotides form separate chromosomes. The genes found on chromosomes are essentially long nucleotide sequences in which the amino acid sequences of proteins are encoded. The combination of three consecutive nucleotides (codon or triplet) corresponds to one of 20 amino acids. Thus, life uses a three-letter genetic code (ATG, CGC, and so on) based on a four-letter alphabet (A, C, T, G).
When a cell of an organism needs a protein (polypeptide), the gene encoding it is turned on. The latter is attached to a special enzyme called RNA polymerase, which, during the process of transcription, begins to follow the sequence of nucleotides and create a copy of it in the form of a molecule called messenger RNA (mRNA). RNA is very similar to DNA, but instead of thymine, it contains uracil (U). After that, the mRNA leaves the cell nucleus and goes to the ribosomes, where it serves as a recipe for creating the amino acid chain of the protein during translation.
The researchers decided to change the genetic code of Escherichia coli by adding two additional "letters" to it. The fact is that DNA in living organisms is double, that is, it is formed by two chains that are paired with each other by complementary bonds. Such bonds are formed between the base of the A-nucleotide from one strand and the base of the T-nucleotide from the other (similarly, between C and G). That is why the two new synthetic nucleotides must also be able to complementarily pair. The choice fell on dNaM and d5SICS.
E. coli Escherichia coli
Photo: Rocky Mountain Laboratories / NIAID / NIH
One pair of synthetic nucleotides was inserted into a plasmid - a double-stranded circular DNA molecule capable of multiplying separately from the rest of the bacterial genome. They replaced a pair of complementary nucleotides A and T, which were part of the lactose operon - a set of genes that metabolize lactose sugar, and the non-coding DNA sequences associated with them. Synthetic nucleotides were not included in the region that the polymerase copies in mRNA.
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Why did scientists decide not to insert synthetic nucleotides directly into the gene, but next to it? The fact is that it is very difficult to change a gene in this way so that it remains functional. After all, for this you need to bind the resulting new codons to any amino acid. For this, in turn, it is necessary to teach the cell to produce various types of transport RNA (tRNA) that can recognize these codons.
The tRNA molecules perform the following function. They, like trucks, carry a certain amino acid at one end, approach the mRNA in the ribosomes and, in turn, begin to match the triplet of nucleotides at the other end with the codon. If they match, the amino acid is stripped off and incorporated into the protein. However, if there is no suitable tRNA, the protein will not be synthesized, which can negatively affect the cell viability. Therefore, by inserting synthetic nucleotides into genes, scientists would have to create genes that encode new tRNAs that can recognize artificial codons and attach the correct amino acid to the polypeptide. However, the researchers' task was simpler. They needed to make sure that the plasmid with synthetic nucleotides would successfully multiply and be passed on to daughter organisms.
Plasmids used to transform Escherichia coli
Image: Denis A. Malyshev / Kirandeep Dhami / Thomas Lavergne / Tingjian Chen / Nan Dai / Jeremy M. Foster / Ivan R. Correa / Floyd E. Romesberg / Nature / Department of Chemistry / The Scripps Research Institute
This plasmid, designated pINF, was introduced into E. coli. However, to copy it, it is necessary that many nucleotides are present inside the bacterial cell. For this purpose, another plasmid, pCDF-1b, was inserted into E. coli. It contained the gene for the diatom Phaeodactylum tricornutum PtNTT2, which encodes the NTT protein, which transports nucleotides from the nutrient medium into the cell.
However, scientists faced a number of difficulties. First, the proteins of Phaeodactylum tricornutum have a toxic effect on the E. coli cell. All because of the presence in them of a fragment of the amino acid sequence, which carries a signaling function. Thanks to her, the protein takes the correct position in the alga cell, after which the sequence is removed. E. coli is unable to remove this fragment, so the researchers helped her. They were able to remove the first 65 amino acids from NTT. This significantly reduced toxicity, although it also reduced the rate of nucleotide transport.
Another problem was that synthetic nucleotides were retained in plasmids for a long time, and not replaced when DNA was copied. As it turned out, their safety depended on what nucleotides surrounded them. To find out, the scientists analyzed various combinations embedded in 16 plasmids. To understand if a synthetic nucleotide had dropped out of the sequence, the researchers used CRISPR / Cas9 technology.
CRISPR / Cas9
Image: Steve Dixon / Feng Zhang / MIT
CRISPR / Cas9 is a molecular mechanism that exists inside bacteria and allows them to fight bacteriophages. In other words, this technology represents immunity against viral infections. CRISPR are special sections of DNA. They contain short fragments of DNA viruses that once infected the ancestors of today's bacteria, but were defeated by their internal defenses.
When the bacteriophage enters the bacteria, these fragments are used as a template for the synthesis of molecules called crRNA. Many different RNA chains are formed, they bind to the Cas9 protein, whose task is to cut the virus DNA. He can do this only after crRNA finds a complementary fragment of viral DNA.
If, instead of crRNA, an RNA sequence complementary to a particular fragment of the plasmid is used, Cas9 will cut the plasmid as well. But if there are synthetic nucleotides in that fragment, then the protein will not work. Thus, using CRISPR, it is possible to isolate those plasmids that are resistant to unwanted mutations. It turned out that in 13 out of 16 plasmids, the loss of synthetic nucleotides was insignificant.
Thus, the researchers managed to create an organism with fundamental changes in DNA, capable of retaining them in itself indefinitely.
Although a semi-synthetic life form has only two unnatural nucleotides in its genome, which are not found in codons and are not involved in the coding of amino acids, it is the first resistant organism whose DNA alphabet consists of six letters. In the future, scientists will most likely be able to use this innovation to synthesize proteins, thereby creating a full-fledged artificial genetic code.
Alexander Enikeev
