RNA Dephosphorylation

[Elisa2804]

Good evening everyone,

I'm working with sequencing of tRNA, applying the mim-tRNA Seq protocol (Behrens et al., 2021) but i don't understand the step of dephosphorylation, in which T4 PNK is employed... I know for sure that this step is crucial for adapter ligation on the 3' end of RNA, since when we have tried the ligation without a preliminar dephosphorylation step of total RNA and de-acylation step of tRNA, the ligation was failed. This enzyme has a 3'-phosphatase activity, but tRNA presents a 3'-OH end so, why is this dephosphorylation step important? I supposed that maybe during the de-acylation step (using Tis-HCl) a phosphoryl group remains attached on the 3'-end of tRNA, and so for this reason dephosphorylation is required, but i'm not sure. Can someone give me the answer to my doubt?

Thank you in advance,

Elisa