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Alkyne modification and CuAAC

The labelling RNA for alkyne modification was transcribed via in vitro transcription. The reaction, modifying the labelling RNA was catalysed by yeast Poly(A) Polymerase (PAP) in the compatible PAP buffer with 3 different alkynes (5-Ethynyl-dUTP, C8-Alkyne-dUTP, C-Alkyne-dCTP) for 2 hours at 37 °C. 200 nM of modified RNA was incubated in 50 mM phosphate buffer (pH 7) with 400 nM Alexa 488 azide (solved in DMSO and stored at 4 °C). 1 mM CuSO4 and 5 mM Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) were mixed and added to the reaction. Eventually, 1 mM sodium ascorbate (NaAsc) was added. The final reaction volume was 50 µL. Incubation time ranges between 12 to 14 hours at 37 °C.