A comparison of the K97A MthRnl mutant activity with activities of truncated T4Rnl2 and T4Rnl1 in step 3 of the ligation reaction. Enzymes were assayed using three different acceptors with identical sequence: RNA (RNA30), 2’-O-methylated at 3’-end RNA (RNA-OMe) or ssDNA (DNA30) in reactions with 5’ pre-adenylated DNA donors AppDNA17-NH2 or AppDNA21-NH2 as described in the Methods. (A) Control reactions without enzyme. (B, C and D) Reactions with truncated T4Rnl2, K97A MthRnl mutant and T4Rnl1 respectively. (E) For comparison, T4Rnl1 was assayed in the ligation reactions with 5’-phosphorylated 3’-amino-blocked DNA donor (pDNA17-NH2) and indicated acceptors in presence of ATP. (F) Percentage of ligation of the different acceptors was calculated from experiments shown in Figures 4 (panels B and C) and 5 (panels B, C and D). Mean values and standard deviation were calculated from multiple (2–5) gels analysis described in the Methods are shown.