Mirror-image polymerase makes mirror gene and more

Life can look very different in a mirror. DNA’s famous double helix normally has a right-handed twist, for example, but its reflection corkscrews the other way, forming a left-handed helix called L-DNA. Researchers at Tsinghua University have now created a set of molecular tools that could help to make mirror-image biology a reality. As well as assembling an entire mirror gene from L-DNA, the team has used mirror DNA to store a passage of text and conceal a hidden message within it (Nat. Biotechnol. 2021, DOI: 10.1038/s41587-021-00969-6).

Ting F. Zhu, who led the work, says it is a step on the way to creating a mirror version of the so-called central dogma of molecular biology, which describes how organisms use the information encoded in DNA. This information can be copied to another DNA molecule by replication, or into RNA by transcription. RNA then acts as a template to build a protein, a process called translation that relies on a molecular machine called the ribosome. “Our long term goal is to build a mirror-image ribosome, to realize the mirror-image central dogma,” Zhu says.

Zhu has previously been able to create and copy mirror DNA using mirror versions of enzymes called polymerases, which contain D-amino acids rather than the L-amino acids typically found in normal proteins. But the small mirror polymerases he built for these tasks suffer from poor activity, and make too many errors when copying DNA.

Now his team has built a mirror version of the 775-amino acid Pfu DNA polymerase, which Zhu says is the largest functional protein to be chemically synthesized to date. In its natural form, this enzyme is widely used to amplify DNA in the polymerase chain reaction (PCR), because it is stable, efficient, and very accurate.

Synthesizing a protein of this size was a huge challenge. Although solid phase peptide synthesis can reliably string together a few dozen amino acids, the process gradually becomes less efficient as the chain grows longer. So the researchers made some judicious alterations to the enzyme’s usual amino acid sequence, which enabled them to build up smaller mirror peptides into two larger fragments, before fitting them together to make the mirror polymerase.

They used this mirror polymerase to accurately construct an entire mirror gene, uniting chemically-synthesized sections of nucleotides into a complete sequence of double-stranded L-DNA. The gene they chose is 1500 bases long and codes for a piece of mirror RNA called 16S rRNA, which will be a core component of the mirror ribosome. Zhu has already made other parts of the mirror ribosome, and once the whole machine is assembled it should be able to take over the task of building mirror proteins, rather than using these laborious chemical methods.

“I think it’s a milestone to be celebrated,” says Jeffrey W. Bode at ETH Zurich, who works on protein synthesis and was not involved in the research. “It validates the idea that synthesis is actually going to get you into this mirror image world.”

Meanwhile, Zhu is also trying to develop practical applications for his mirror molecules, including information storage. As a proof of principle, the researchers used mirror DNA to store a paragraph of text written by Louis Pasteur in 1860, in which the scientist speculated on the possibility of a mirror-image world of biology. With each letter of the text represented by a trio of DNA bases, the whole paragraph was spread over 11 double-stranded mirror DNA sequences, crafted by the mirror polymerase. The researchers retrieved the text by sequencing the mirror DNA with one of their smaller mirror polymerases, demonstrating that it’s possible to reliably write and read mirror DNA sequences.

Being unnatural, mirror DNA might offer an advantage over other forms of DNA data storage, because it resists degradation in the environment. The team found that a short section of double-stranded mirror DNA could be amplified and sequenced after spending more than a year in some local pond water, whereas the equivalent sequence of natural DNA could not be amplified after 1 day in the same water. “I think information storage is an example of how mirror DNA could become useful in the future,” Zhu says.