A New Folding Motif Formed with an Expanded Genetic Alphabet

Abstract Adding nucleotide pairs to a DNA alphabet not only increases the linear information density of DNA molecules, but also may increase the diversity of folds available to the molecular system. Reported here is an example of this. A single additional nucleotide, with the 5-nitro-6-aminopyridone ("Z") heterocycle, selected from an artificially expanded genetic information system (AEGIS), was incorporated at twelve sites into a DNA strand 23 nucleotides in length. With a fluor at its 5'-end and a quencher at its 3'-end, this molecule is shown by fluorescence quenching to fold in mildly alkaline (pH 8-9) conditions. This folded structure is quite stable, melting at Tm = 66.5 ℃ at pH 8.5. Spectroscopic, gel, and 2-dimensional NMR (800 MHz) analyses show that the structure is held together by "reverse" Z-:Z base pairs, which have been previously seen in a crystal structure of the free heterocycle, 5-nitro-6-aminopyridone. This, in turn, requires that the strands joined by Z-:Z reverse pairs be oriented in parallel. This, in turn, requires the fold to have a four stranded compact "down-up-down-up" fold. The structure does not form when various Zs are replaced or scrambled. Of two possible structures, we also considered the possibility of "intercalated" Z-:Z base pairs, a fold that resemble the i-motif of standard DNA where an analogous "down-up-down-up" fold is held together by C:C+ pairing at lower pH between parallel strands. An alternative model has a "down-up-down-up" fold without intercalation.