HPLC analysis of Cv-cGLR1, Sp-cGLR1, and Sp-cGLR3 reactions, compared with 2'3'-cUA and 3'3'-cUA synthetic standards. Data are representative of n = 3 independent experiments. Multiple products can be identified from the Sp-cGLR1 reaction; the ratio of HPLC-integrated concentration of 3'3'-cUA, 3'3'-cGAMP, and 3'3'-cAA is 3.3:1:2.1 (corrected with extinction coefficient 22,500, 25,050, and 27,000 M-1cm-1, respectively), confirming that 3'3'-cUA is the major product in vitro. See also Figure S3B for HPLC trace of Sp-cGLR1 reaction supplemented with only ATP and UTP, where the ratio of HPLC-integrated concentration of 3'3'-cUA and 3'3'-cAA is 2.5:1. The peak eluted around 3 min in Sp-cGLR3 reaction is likely an intermediate derived from ATP, as the same peak was also observed when the reaction was supplemented with only ATP (see Figure S3B).
