The possibility of increasing the number of replicable nucleotides in nucleic acids above the standard four found in terran DNA and RNA was noted a quarter-century ago. However, only recently has the broader scientific community come to recognize that expanded genetic "alphabets" also expand the functional potential of nucleic acids. Key events contributing to this recognition include the observation by Hirao that adding a fifth nucleotide to a DNA aptamer increased its affinity for its target, the demonstration by Perrin of improved performance of DNA that appends functional groups to the standard four nucleobases, and the engineering by Romesberg of a strain of <i>E. coli</i> that maintained a non-standard base pair in a plasmid for nine hours. How far might synthetic biology rewrite the biopolymers that lie at the center of terran biology? If the added nucleotide pairs deviate too greatly from the canonical Watson-Crick geometry, standard polymerases will be unable to explore the vastly larger genetic “sequence space” opened up by synthetic biology. Here, we add two new nucleotides (Z and P) designed to pair with a strict Watson-Crick geometry. We then report laboratory <i>in vitro</i> evolution (LIVE) experiments where the GACTZP system has evidently explored the entire expanded sequence space, evolving and adapting to deliver GACTZP products containing multiple, nearby, and adjacent Z’s and P’s. These bind specifically to cancer cells better than GACT species.

Exploring the sequence space from expanded genetic alphabets

249th National Meeting (2015)

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