The elegant structure of DNA has inspired chemists for decades. The simple pairings of the four native nucleobases (adenine, guanine, cytosine, and thymine) produces all of the genetic variations in every known living organism. Based on that basic principle, scientists have made modifications to DNA for a variety of purposes, from creating photonic wires for nanomachines to making completely unnatural DNA for engineered genetics. One aspect of DNA's natural behavior, however, has eluded scientists: DNA replication.
DNA polymerases, which produce copies of DNA molecules, are enzymes that have evolved for millennia, and there has been little success in getting them to work on artificial DNA in living cells. For the development of engineered genetics, it would be advantageous to find ways to hijack a cell’s enzymes for the replication of unnatural DNA. In working towards that goal, Eric Kool and his colleagues from Stanford University and the Massachusetts Institute of Technology managed to insert and replicate artificial DNA in E. coli cells. This first successful example of bypassing the natural cellular replication machinery was published as an advanced article in the Angewandte Chemie International Edition.
The researchers supersized native nucleobases to create unnatural ones that are 2.4 angstroms bigger, which is slightly less than the diameter of a water molecule (2.75 angstroms). While the artificial nucleobases are noticeably bigger, they still maintain the ability to hydrogen bond with their natural counterparts.
The significant size difference will probably hinder most DNA polymerases, but every organism produces a number of specialized polymerases; the normal hydrogen bonding of these extended bases might allow a few of these to work efficiently. Some of these polymerases have evolved to work around damaged DNA, which makes them good candidates to operate with nucleobases that are larger than normal.
To determine if any of the polymerases in a living cell would work on completely unnatural DNA, the authors placed strands of artificial DNA into a viral genome and inserted it into E. coli cells. They then measured the efficiency and accuracy of DNA replication in both healthy and UV-damaged bacteria cells. They hypothesized that the UV damage would activate more specialized polymerases that can handle greater variation in DNA structure, making replication easier for unnatural DNA.
In terms of replication efficiency, UV exposure was advantageous for artificial DNA. Replication for xAdenine and xThymine were just as efficient as natural adenine and thymine—that’s a 20 to 27 percent improvement from their replication in undamaged cells. The efficiency for xCytosine was 53 percent and xGuanine was 45 percent. While those values are low, they are significantly better than what was obtained in undamaged cells, where the efficiencies were 29 percent for xCytosine and 11 percent for xGuanine.
As for replication accuracy, xAdenine did the best, as 99 percent of the daughter phage had the correct placement of adenine. Next best was xCytosine with 88 percent fidelity. The other two nucleobases had negligible replication accuracy.
Even though the replication accuracy and efficiency were uneven for the four unnatural nucleobases, the authors have successfully shown that it is possible for DNA polymerase to process the genetic information stored in artificial DNA. This paves the way for further engineering to improve replication, which would give scientists better control of biological systems.
Angewandte Chemie Int. Ed., 2009. DOI: 10.1002/anie.200805683