The God games continue

Semi-synthetic bacteria have been taught to decipher artificial genetic code

Daria Spasskaya, N+1

With the help of an artificial third base pair in DNA, scientists were able to encode an amino acid, thereby expanding the natural genetic code. Earlier, the same research group embedded a third pair of X-Y bases in the DNA of bacteria and thus created the first semi-synthetic living organism. In a new paper published in Nature, bacteria were taught not just to reproduce artificial DNA, but also to implement the information encoded in it in the form of a protein.

Experiments with E. coli with an expanded genetic alphabet are conducted by a group led by Floyd Romesberg from the Scripps Research Institute in California. Earlier, scientists from Romesberg's laboratory created semi-synthetic bacteria capable of stably reproducing (replicating) DNA containing, in addition to the "natural" pairs of nucleotides A-T and G-C, an extra pair of X-Y. The synthetic deoxynucleoside DNAm is hidden under X here, and dTPT3 is hidden under Y. Since bacteria are unable to synthesize additional bases on their own, X and Y in the form of triphosphates have to be added to the growth medium. In order for the substances to enter the cell, scientists embedded a transporter gene from the genome of the algae Phaeodactylum tricornutum into the bacterial genome.

In order for semi-synthetic DNA to be copied and transmitted from generation to generation, DNA polymerase must recognize the extra bases. But in order to use such DNA to encode a protein (actually, this is the main function of DNA), it is necessary to adapt a much more cumbersome translation apparatus to synthetic molecules. In the new work, the scientists managed to do this.

The realization of hereditary information occurs in the cell in several stages. First, a matrix RNA (mRNA) molecule is read from DNA, which serves as an "instruction" for the ribosome in the process of protein synthesis. The sequence of amino acids is encoded in mRNA as a sequence of three-letter codons. The codon and amino acid correspondence is provided by transport RNA (tRNA), which contains an anticodon complementary to the codon in mRNA. The enzyme aminoacyl-tRNA synthetase is responsible for the correct attachment of an amino acid to tRNA. The tRNA molecule, "charged" with the right amino acid, delivers the latter to the ribosome, where the amino acid is included in the protein chain in the right place due to the codon-anticodon correspondence.

Protein Synthesis (Wikimedia Commons)

To demonstrate the ability of semi-synthetic DNA to encode information, scientists introduced an artificial base into the green fluorescent protein (GFP) gene. It was decided to replace tyrosine (TAC) at position 151 with serine, but instead of the AGC codon, the AXC codon was embedded in the gene. Serine was chosen for the reason that its aminoacyl-tRNA synthetase has a wide specificity - six codons correspond to this amino acid in the table of the genetic code. To ensure codon-anticodon correspondence, the sequence GYT was introduced into the gene for serine tRNA. The GFP gene mRNA and serine tRNA were to be synthesized by bacteriophage T7 RNA polymerase.

The artificial base pair DNAm-dTPT3 (dX-dY) is shown above, complementarity in which is ensured by hydrophobic interactions, and not by hydrogen bonds, as in the canonical pair dA-dT. Below is a diagram of a genetic construct containing artificial bases in the composition of the green fluorescent protein gene (sfGFP) and serine tRNA (serT). Zhang et al / Nature 2017.

In order for semisynthetic RNA to be synthesized in the cell, ribonucleoside phosphates NaM and TPT3 also had to be added to the growth medium. It turned out that the algae transporter can pump them into the cell, and the viral RNA polymerase effectively embeds new "letters" into the RNA. Serine minoacyl-tRNA synthetase successfully attached serine to the modified tRNA, and the amino acid was embedded in the protein, which became clear from the green glow of the cells. If there was no mutant gene for tRNA, there was no glow either, since synthesis stopped at 151 positions.

At the second stage, scientists assigned an artificial codon a separate, non-canonical (that is, not included in the twenty most common) amino acid. To do this, tRNA genes and the corresponding aminoacyl-tRNA synthetase for pyrrolysin from the microorganism Methanosarcina barkeri were additionally introduced into the "experimental" E. coli. The inclusion of pyrrolysin in the protein was detected primarily by the glow of bacteria, which reached almost 70 percent of the glow of cells with "natural" protein. The inclusion of an amino acid was also detected by the specific addition of a fluorescent dye in the purified protein to the pyrrolysin residue.

In addition to the AXC codon, the researchers tested the GXC codon and the corresponding GYC anticodon, which also worked successfully for pyrrolysin. Thus, the natural genetic code was expanded into two positions at once.

The efficiency of embedding pyrrolysin (PrK) in the protein at position 151 encoded by the AXC or GXC codon, compared with tyrosine (Y), which stands in this position in the wild-type protein. It can be seen that with a small probability (1-2 percent), other amino acids, for example isoleucine (I) or valine (V), can be embedded instead of the artificial base, which is due to incorrect embedding of the base opposite X during replication (Zhang et al / Nature 2017).

There is no practical application for this work yet – scientists are simply "playing gods" and experimenting with the possibilities of the genetic apparatus of living organisms. Unlike Craig Venter, who claimed to create a synthetic organism, meaning mycoplasma with a "truncated" genome, Romesberg's group is actually much closer to creating "synthetic life". However, in addition to synthetic bacteria, his laboratory is also engaged in more applied things – for example, we wrote about a gel made of modified DNA that can be used for long-term storage of proteins. Also, the laboratory staff is engaged in the development of new antibiotics. To put the research results into practice, Romesberg created the biotech company Synthorx.

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