Studies towards size-expanded genetic systems

There have been quite a number of examples of DNA base mimics. However, these de novo designed base pairs were studied in the context of natural DNA. Inspired by the "stretched out" adenine analogue lin-benzoadenine, we asked the questions of whether a complete set of such size-expanded bases can form stable duplex structures, and whether such molecular assemblies can function as novel genetic systems. We designed two different sets of DNA base analogues by benzohomologation, namely xDNA and yDNA. Thermal denaturation studies of a single expanded base pair in the context of DNA revealed that yA can selectively pair with T over other DNA bases, although some degree of destabilization occurred compared to an A·T pair. Furthermore, oligonucleotides containing only yA and T bases demonstrated a strong tendency to pair with complementary strands to form higher order structures. Preliminary spectroscopic studies suggested that yDNA may adopt a duplex structure in solution.;To answer whether the size-expanded DNAs can function as novel genetic systems, we explored the replication by DNA polymerases. We measured the enzyme kinetics of xDNA and yDNA by DNA polymerase I (Klenow fragment) and Y family DNA polymerase Dpo4. Our studies showed that while both polymerases had lower incorporation efficiency for these larger base pairs relative to natural ones, they can in most cases retain pairing selectivity for the correct partners. In a single base extension assay, Dpo4 showed higher efficiency in extending the matched xDNA and yDNA pairs. These studies showed that even wild type DNA polymerases replicated xDNA and yDNA with low but significant efficiency. Subsequent studies of full length replication of xDNA by Dpo4 demonstrated that Dpo4 could replicate xDNA with fidelity analogous to natural DNA.;Taken together, the research showed that size-expanded DNA can self-assemble to form duplex structure with higher thermal stability than DNA with analogous sequences. It also suggested that Dpo4 and related enzymes may be candidates for polymerase engineering, leading to more efficient replication of xDNA and yDNA. Preliminary replication results suggested that xDNA could serve as a functional genetic system in vitro.