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. Author manuscript; available in PMC: 2020 Feb 15.
Published in final edited form as: Methods. 2018 Nov 10;155:3–9. doi: 10.1016/j.ymeth.2018.10.023

Figure 4. Detection of diphosphorylated 5′ ends by PACO.

Figure 4.

PACO detects diphosphorylated RNA 5′ ends on the basis of their reactivity with Schizosaccharomyces pombe Pce1, a guanylyltransferase that preferentially caps RNA 5′ ends that bear two phosphates rather than three or one. The cap added by Pce1 is detected by its ability to retard the electrophoretic migration of RNA on a boronate gel after site-specific cleavage with a DNAzyme to generate a 5′-terminal fragment bearing a 2′,3′ cyclic phosphate at its 3′ end. TriP, triphosphorylated RNA; DiP, diphosphorylated RNA; MonoP, monophosphorylated RNA. p, phosphate; Gppp, unmethylated cap; OH, hydroxyl.