Figure 2.

Reaction of carbodiimides with pseudouridine and uridine. Top: acylation of pseudouridine (Ψ) with the carbodiimide CMCT (full name in text). The carbodiimide group reacts with both N1 and N3, but after alkaline treatment the N1-CMCT is cleaved. The remaining CMC at N3 enables detection by mass spectrometry, reverse transcription, sequencing or even biotin pulldown in case of the CMC-azide (encircled, blue). Bottom panel: Like pseudouridine, uridine gets labeled at the N3 position, which is removed after alkaline treatment. The same was found to be true for guanosine and inosine. Insert: Structure of CMCT.