Many uridine modifications have been tested with minimal decrease in RNase 4 and T4 PNK 3′ activities, including pseudo-, N1-methyl-pseudo-, 5-methoxy, and dihydrouridine species (ψ, m1ψ, mo5U, and D). To efficiently digest RNAs containing these modifications, especially m1ψ, we recommend increasing the added volume.
Since RNase 4 endonucleolytic activity requires the 2′-hydroxyl of the uridine nucleotide, modifications such as 2′-O methyluridine (Um) at the cleavage site completely inhibit RNase 4 activity.
