Helix-forming properties of size-expanded DNA, an alternative four-base genetic form

We describe the chemical and biophysical characterization of a new four-base genetic system, in which all base pairs are larger than the natural pairs. A recent preliminary study showed that three sequences containing size-expanded DNA (xDNA) bases could form stable cooperative complexes. However, many of the standard and essential properties that natural DNA possesses were unexplored for this new class of helical assembly. We therefore undertook a study of several properties of this new genetic complex: strand stoichiometry, preferred strand polarity (i.e., parallel vs antiparallel), mismatch selectivity, base size selectivity, ionic strength dependence, fluorescence behavior, CD spectra, and sequence generality. Results showed that several sequences formed double-stranded helical complexes, and interestingly, a pyrimidine-rich strand of xDNA bases was shown to form a triple helical complex as well. A test of strand polarity showed a preference for antiparallel orientation, as does natural DNA. Mismatch and size selectivity were generally moderate to strong, with one exception. Ionic strength dependence varied by relatively small degrees from that of natural DNA, although a triple helical complex of xDNA showed more marked dependence. Spectral characteristics (fluorescence, CD) were found to be quite different than those of natural DNA, apparently because of large differences in the electronic character of the expanded pi-systems. Finally, several sequence contexts were found to form helices in a sequence-predictable manner. Two exceptions were noted and may be explained by competition from alternative folding structures and/or strong, single-stranded stacking. The viability of xDNA as an alternative genetic system and its possible biotechnological applications are discussed.