Discriminating cyclic from linear nucleotides - CRISPR/Cas-related cyclic hexaadenosine monophosphate as a case study

Nucleic acids exist in biological systems as linear and cyclic forms and in most cases the biology of the cyclic form is different from the linear form of exactly the same sequence. Case examples are cyclic nucleotides, second messengers in both prokaryotes and eukaryotes whereby the cyclic forms account for their interesting biological profiles and the actions of the cyclic nucleotides are terminated upon phosphodiesterase hydrolysis into linear forms. For mono and dinucleotides, it has been shown that vast conformational changes that accompany the hydrolysis of the cyclized form allow for discrimination between the cyclized and linear forms. As the ring size increases, it becomes difficult to use conformational or structural differences alone to discriminate between cyclic and linear nucleotides. Here we reveal that for the recently discovered CRISPR/Cas-related cyclic hexaadenosine monophosphate, it is possible to discriminate between the cyclized and linear forms. The structures of c-HexaAMP and linear form are different in acidic media and this structural difference facilitated a simple and practical detection platform for this interesting and new bacterial immunity-related molecule, and we also demonstrate that it is possible to distinguish between linear and cyclized polynucleotides using simple spectroscopic techniques, such as CD and fluorescence-based methods.