Bacteria build unnatural proteins using unnatural DNA | Investigation



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The ability of life to use alternative biochemists has been demonstrated by US researchers. UU., Which have demonstrated for the first time that the battle-horse bacterium Escherichia coli can be designed to use unnatural base pairs in its DNA to produce proteins that contain unnatural amino acids. 1

While DNA replication containing unnatural base pairs has been previously reported, this is the first time that the largest available information is used. in living cells.

Raises the possibility that life in other worlds may not use the same biochemical basis as ours.

  Semisynthetic organism (E. coli) that produces green fluorescent protein through the decoding of an unnatural codon [19659005] It has been argued for a long time that the molecules of life are somehow special and different from other types of molecules, "says Floyd Romesberg of the Scripps Research Institute in La Jolla in the United States. "We have shown that this may not be true: our artificial part, the unnatural pair of bases, can function in what could be said to be the most central of all biological processes: the storage and retrieval of information." </p>
<p>  It is also a step towards creating totally new drugs and organisms functions. "The immediate goal that really drives us is to use the semi-synthetic organism to create new clbades of protein drugs," says Romesberg. "Protein drugs have revolutionized medicine, but the properties they have, and therefore the diseases that can be developed to treat, must somehow be limited by what they are made of. "He hopes that his approach can broaden that repertoire. </p>
<h2 id= Translation task

A job Previous Romesberg and other groups showed that unnatural base pairs can be inserted into the DNA of living organisms, but use them to write Going and reading genetic information is another matter. Genes are translated into proteins through messenger RNA (mRNA) and transfer RNA (tRNA), which bind in the multi-protein enzyme called ribosome to bind the amino acids that the tRNA has in proteins.

Each amino acid is transported by a different tRNA, the code is read by coincidence of a codon of three nucleotides in the mRNA with a complementary anticodon in the tRNA. Then, to read the information in a pair of unnatural nucleotides, the complementary pairs must be incorporated into both the mRNA and the tRNA and function in the ribosome. This is what Romesberg and his colleagues have achieved.

  Schematic representation of the incorporation of a non-canonical amino acid (ncAA) into a protein by ribosomal decoding of a non-natural codon in a semi-synthetic organism

What is particularly remarkable about its alternative nucleic acid chemistry is that its novel base pair is not linked together, like normal DNA bases, by hydrogen bonds. Instead, it incorporates bulky organic heterocyclic groups that stick to each other in the water by mutual attraction of hydrophobic units. The Scripps group has shown that a bacterial tRNA designed to have an anticodon with an unnatural pair component can recognize and adhere to the complementary codon in the mRNA transcribed from a corresponding gene within a plasmid of DNA added to the cells.

A crucial trick is to use tRNA-loading enzymes that are not sensitive to their unnatural anticodons, such as E. coli tRNA synthetase that charges tRNA with the amino acid serine. The researchers first used this system to insert serine into a position in a standard protein – green fluorescent protein (GFP) – encoded by the non-natural base pair.

  CW2411 nature24659 Romesberg 2 Figure a and b

Then they got more ambitious, using the same approach to insert unnatural amino acids into the protein. They first replaced the serine-tRNA synthetase gene with one that produces an enzyme used in a different microorganism, Methanosarcina barkeri that uses a rare amino acid, pyrrolysin. The researchers used this tRNA synthetase to add an unnatural pyrrolysin derivative to GFP. As an additional illustration of the expanded amino acid alphabet, they also used a tRNA synthetase from the Methanococcus jannaschii to make GFP contain the markedly heterodox amino acid p -azido-phenylalanine. [19659015] The immediate objective that really impels us is to use the semisynthetic organism to create new clbades of protein drugs

Floyd Romesberg, Scripps Research Institute

This system provides a great boost to the amount of information that can be genetically encoded The pair of unnatural bases makes 152 new codons available, says Romesberg's Scripps colleague, Yorke Zhang. "As we continue to develop it, it could be used to simultaneously and efficiently direct the incorporation of as many unnatural amino acids as you wish," he says. The team is now working to demonstrate the simultaneous insertion of four different non-natural amino acids into a protein.

"This is an important step in the development of technology towards the expression of proteins with unnatural amino acids," says Eric Kool of Stanford University in the United States, who has also worked on the expansion of the genetic code. "If the technology finally allows the expression of high efficiency of proteins with multiple non-natural amino acids, it could be very important for future protein and biotechnology therapies."

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