Abstract
An unnatural base-pair architecture with base pairs 2.4 Å larger than the natural DNA-based genetic system (xDNA) is evaluated for its ability to function like DNA, encoding amino acids in the context of living cells. xDNA bases are structurally analogous to natural bases but homologated by the width of a benzene ring, increasing their sizes and resulting in a duplex that is wider than native B-DNA. Plasmids encoding green fluorescent protein were constructed to contain single and multiple xDNA bases (as many as eight) in both strands and were transformed into Escherichia coli. Although they yielded fewer colonies than the natural control plasmid, in all cases in which a modified plasmid (containing one, two, three, or four consecutive size-expanded base pairs) was used, the correct codon bases were substituted, yielding green colonies. All four xDNA bases (xA, xC, xG, and xT) were found to encode the correct partners in the replicated plasmid DNA, both alone and in longer segments of xDNA. Controls with mutant cell lines having repair functions deleted were found to express the gene correctly, ruling out repair of xDNA and confirming polymerase reading of the unnatural bases. Preliminary experiments with polymerase deletion mutants suggested combined roles of replicative and lesion-bypass polymerases in inserting correct bases opposite xDNA bases and in bypassing the xDNA segments. These experiments demonstrate a biologically functioning synthetic genetic set with larger-than-natural architecture.