Extended Data Figure 6. LC-MS analysis of Sso1393 reactions.
a, Ion chromatograms extracted for m/z 657.1 (cA2-1, A2>P-1, cA4-2, A4>P-2). Individual lanes are labeled. cOAs (mainly cA4), derived from reaction of Csm with ATP, was incubated with Sso1393 for 60 and 150 min or without Ring nuclease for 150 min. A control in which the ATP from the Csm cyclase reaction had been omitted was also analysed as control. Linear oligoadenylates with 2’,3’-cyclic phosphate were derived from hydrolysis of suitable DNA oligonucleotide subtrates with the toxin MazF; whereas cA2 was a commercially available standard. The traces show clearly the difference in retention time between the linear and cyclic isomers.
b, UV traces at 254 nm. Peaks that change in intensity over the course of the enzymatic reaction are indicated by arrows. The three peaks that decreased or increased over the course of the reaction all match the changes in abundance of the m/z 657.1 species. The broad peak at 4.1 – 6 min is an unknown contaminant likely resulting from the phenol-chloroform extraction.
c, Mass spectra for species X and Y. Calculated for C20H23N10O12P2-1 (cA2 / A2>P) m/z 657.0978, found 657.0968 (δm -1.4 ppm); calculated for C40H46N20O24P4-2 (cA4 / A4>P) m/z 657.0978, found 657.0967 (δm -1.6 ppm). The data presented are representative of duplicate experiments.