A folding motif formed with an expanded genetic alphabet

Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5 °C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ−:dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ−:dZ pairs join parallel strands in a four-stranded compact down–up–down–up fold. These have two possible structures: one with intercalated dZ−:dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC+-reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis. Standard DNA is limited by low information density and functional diversity. Now it has been shown that an expanded genetic alphabet—incorporating a synthetic nucleotide, dZ—allows for the creation of stable three-dimensional DNA structures under mild alkaline conditions. Such stable structures enrich our understanding of DNA’s structural diversity and its potential in synthetic biology applications.