One of the many accomplishments of synthetic biology over the past two decades has been the generation of DNA systems that have additional nucleotide "letters" that form additional nucleobase pairs. These include Artificially Expanded Genetic Information Systems (AEGIS) that use non-standard H-bonding patterns to form up to 12 mutually exclusive base pairs. These base pairs are presumed to have Watson-Crick geometries like standard nucleotides. However, the emergence of non-standard nucleotides that were proposed to pair without hydrogen bonding called this view into question. To address this issue, we synthesized the guanine riboswitch aptamer domain containing one of these unnatural pairs (designated P:Z). We showed that the modified P:Z ribswitch can adopt that native conformation and bind guanine without loss of affinity. We crystallized the unnatural riboswitch and solved the structure at 3.22Å resolution. The results lay to rest the possibility that the P:Z base pair deviates substantially from the Watson-Crick geometry.

Crystallization and structure of a functional riboswitch bearing an unnatural base pair

249th National Meeting (2015)

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