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Abstract 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. 

Characterizing the relative onset time, strength, and duration of molecular signals is critical for understanding the operation of signal transduction and genetic regulatory networks. However, detecting multiple such molecules as they are produced and then quickly consumed is challenging. A MER can encode information about transient molecular events as stable DNA sequences and are amenable to downstream sequencing or other analysis. Here, we report the development of a de novo molecular event recorder that processes information using a strand displacement reaction network and encodes the information using the primer exchange reaction, which can be decoded and quantified by DNA sequencing. The event recorder was able to classify the order at which different molecular signals appeared in time with 88% accuracy, the concentrations with 100% accuracy, and the duration with 75% accuracy. This simultaneous and highly programmable multiparameter recording could enable the large-scale deciphering of molecular events such as within dynamic reaction environments, living cells, or tissues.