Towards a new genetic system with expanded size

This thesis describes studies towards a new family of DNA analogues with altered sizes. In studies towards making compressed duplexes with a shorter diameter by ca. 2.4 Å, the size of a benzene ring, several shortened pyrimidine analogues were chemically synthesized and incorporated into oligonucleotides. Although these shortened bases were designed to fully maintain the Watson-Crick hydrogen bonding potentials, they failed to pair strongly or selectively with any natural or extended bases in the context of normal B-DNA. Attempts to form compressed duplex were unfruitful as well. In studies towards expanded duplexes with a larger diameter by ca. 2.4 Å, expanded base analogues for A, G, T, and C were synthesized: called xA and xG (made by the author), and xT and xC (made by Gao). These expanded base analogues generally prefer pairing with their hydrogen bonding partners in normal DNA (i.e. xA selectively pairs with T) although such expanded base pairs decrease the overall stability of DNA due to their larger size. Because of their expanded size, all these analogues are fluorescent with emission maxima around 400 nm. The added size significantly increases stacking ability of these expanded analogues and they can successfully form fully expanded duplexes (called xDNA) with greatly enhanced stability as compared to their DNA counterparts. Studies have also revealed that xDNAs maintain most features of normal DNA such as pairing selectivity and pairing direction. Structure elucidation of one xDNA duplex by solution phase NMR showed that xDNA resembles DNA in many structural characteristics such as right-handedness, 2′-endo sugar conformation and Watson-Crick type hydrogen bonding. Furthermore, all expanded nucleoside triphosphate except dxGTP can be incorporated into DNA by Klenow polymerase with certain preference for their natural H-bonding partner or an abasic site.