Non - canonical amino acids
Human cells have been taught to embed non-canonical amino acids into proteins
Polina Loseva, N+1
American molecular biologists forced human hematopoietic cells to encode non-classical amino acids and embed them into proteins. It turned out that at the same time, the cells retain the ability to differentiate and can survive in the body of mice. This can be useful to obtain human proteins with altered properties – and, for example, immediately check their pharmacological properties in vivo. The work was published in the journal Proceedings of the National Academy of Sciences (Shao et al., Expanding the genetic code of the human hematopoietic system).
Writing a new amino acid into the genetic code is not as difficult as it may seem. The fact is that in our genetic code there are already free "words" of three nucleotides that do not encode anything by themselves. They serve as stop codons. As soon as the ribosome that builds the protein reaches such a "meaningless word", it stops, and the protein is considered ready. And such stop codons can be artificially assigned a new value.
In order for a triplet of nucleotides to have a meaning, it is necessary that the cell be able to decipher it. This usually happens like this: a transport RNA sticks to the information RNA, which carries the necessary amino acid. For stop codons, there is no such transport RNA in the cell, but it can be added from the outside. And we'll have to add another enzyme that connects the transport RNA with a non-canonical amino acid. Then the cell will embed a new amino acid in the place of the stop codon.
Similar experiments have already been performed on various organisms, from bacteria to mice. And so a group of scientists from the Scripps Institute, led by Peter Schultz, got to human cells. In their work, the researchers forced one of the stop codons (UAG) to encode the amino acid pyrrolysine - just as some bacteria and archaea do.
To achieve their goal, the authors of the work infected human cells with a viral vector based on the Epstein-Barr virus. The genes of the virus were necessary for this section of DNA to continue to exist and multiply inside the nucleus. In addition, the vector contained a gene for transport RNA for pyrrolysin and a transferase enzyme that connects pyrrolysin to RNA. Finally, inside the vector there was a gene of a green fluorescent protein with a mutation: one of its codons was artificially replaced with a stop codon of UAG. This is necessary to test the effectiveness of rewriting: if the cell has learned to embed pyrrolysin instead of a stop codon, then the protein will turn out to be full-sized and will glow. If you have not learned, then the protein will break in the middle, and the glow is not visible.
First, scientists tested their method on human embryonic kidney cells. As expected, they began to glow only when pyrrolysin was added. The researchers then took hematopoietic stem cells from human umbilical cord blood. They achieved successful embedding in about half of the cells, and almost a third of them acquired the ability to glow.
Then these cells were planted in immunodeficient mice to find out how much they retain their viability and new properties in a living organism. It turned out that they survive three times worse than cells that have not been injected with a viral vector (9.5 percent of cells in the bloodstream and 27.8 percent, respectively). Nevertheless, when scientists increased the number of transplanted cells, they achieved that a quarter of the hematopoietic cells in the mouse body turned out to be human. About 20 percent of them continued to glow. Then they traced that these human cells produce different types of shaped elements, including different types of leukocytes, and some of them retain the ability to glow.
Thus, the authors of the work managed to force human stem cells to encode non-canonical amino acids. This method may be useful to obtain human proteins with altered properties – structure or functions – for research or drug development. The authors of the work, for example, suggested that immune cells that originate from such blood cells can produce modified antibodies, so it would be interesting to test the reaction of chimeric mice from their experiment to some allergens.
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