Cells use only two base pairs in their DNA—adenine and thymine, and cytosine and guanine. Now, chemists have expanded that number to three.
In a feat of synthetic biology, Floyd E. Romesberg of Scripps Research Institute, La Jolla, Calif., and coworkers put DNA with three types of base pairs into living bacterial cells. Natural bacterial DNA polymerase recognizes and copies the expanded DNA, and natural DNA-repair enzymes do not break it down, they report (Nature 2014, DOI: 10.1038/nature13314).
“An entire new life-form is emerging through the combination of chemistry and biology,” comments Steven A. Benner of the Foundation for Applied Molecular Evolution, in Gainesville, Fla. “This result is a step toward future development of living cells that use synthetic alien DNA that encodes additional genetic information to support biotechnology.”
DNAs with expanded genetic codes have been created, copied with DNA polymerases, transcribed into RNA, and translated into nonnatural amino acids, but only in vitro. The Scripps study now moves such efforts into living cells. The work could one day ease the development of modified RNAs, proteins, and cells with customized traits and functions. La Jolla-based Synthorx, cofounded by Romesberg, plans to use the technology to also develop new vaccines, aptamers, and nanomaterials.
In the new study, Romesberg and coworkers incorporate nonnatural bases called d5SICS and dNaM into DNA. These bases pair with one another by hydrophobic interactions, whereas natural DNA bases pair by hydrogen bonding.
Getting cells to biosynthesize modified nucleoside triphosphates, the substrates needed to replicate expanded DNA, has proven difficult. So the Scripps team instead engineered bacteria to express transporter enzymes that import nucleoside triphosphates through the cell membrane from the outside. And the researchers satiated the bacteria with inorganic phosphate, making them too full to eat the imported nucleoside triphosphates as food, as they would normally do.
When the supply of substrate is cut off, bacterial replication of the nonnatural bases shuts down. This reversibility means that modifying microorganisms this way is safer than engineering them with recombinant DNA technology, which is a permanent modification, says Ichiro Hirao of the RIKEN Center for Life Science Technologies, Yokohama, Japan. Therefore, “if the organism escapes Floyd’s laboratory and makes it to the San Diego Zoo, it’s not going to eat the penguins—it isn’t a Frankenstein monster,” Benner says.
If expanded DNA can eventually be transcribed into messenger RNA in vivo, the mRNA transcripts would have 216 amino acid-coding units (codons) instead of the current 64. Ali Tavassoli of the University of Southampton, in England, notes that such expanded mRNAs could make it possible to insert several types of nonnatural amino acids—instead of the current single type—into individual modified proteins.
Benner predicts the technology could eventually spawn far more than just novel proteins for medical and biotechnology use: “If you set the grand challenge of creating an artificial life-form using an alien genetic alphabet, it will drag scientists across uncharted territory, forcing them to ask and answer yet unscripted questions.”