Part I: Fluorescence of size-expanded DNA bases: Reporting on DNA sequence and structure with an unnatural genetic set. Part II: Toward replication of xDNA, a size-expanded, unnatural genetic system

This thesis describes two separate studies involving an unnatural genetic set containing size-expanded DNA base pairs which are 2.4 A larger than natural DNA pairs (xDNA). This designed genetic helix is under study with the goals of mimicking the functions of the natural DNA-based genetic system, and of developing useful research tools as well as biotechnological applications. Unlike the natural genetic system, xDNA bases are fluorescent in monomeric and oligomeric form; they are also capable of binding DNA with high affinity. The first described study involved exploring the photophysical properties of xDNA bases in a variety of contexts, such as conjugated to natural DNA and in oligomers composed of all xDNAs. These fluorescent nucleobase analogues revealed several unique fluorescence responses (excimers, enhancements, quenching) in both single stranded oligomers, and also on hybridization with natural DNAs. Overall, it was found that xDNAs can self-report selectively on specific structures and sequences of natural DNA, which may prove useful in the arena of nucleic acid detection, and reporting/probing of biological structures and events. The second study involved evaluating the potential of this unnatural genetic set to further function as an alternative genetic system beyond selective duplex assembly by templating and encoding genetic information in the context of natural replication machinery. In addition to the summarized in vitro studies, the described in vivo research involved transforming various xDNA-containing gene inserts encoding for green fluorescent protein into E. coli via a modified plasmid. Results showed formation of green colonies in cases involving single as well as consecutive xDNA base pairs suggesting faithful reading of xDNAs by the E. coli polymerases, which was confirmed by plasmid sequencing. These results suggest that it is possible to redesign the chemical information of a cell while retaining biological function.