The comparison of pre-adenylated RNA or DNA donors in step 3 of the ligation reaction with the K97A MthRnl mutant or truncated T4Rnl2. Enzymes were assayed using 2’-O-methylated at 3’-end RNA (RNA-OMe) acceptor in reactions with 5’ pre-adenylated RNA (AppRNA21-NH2) or DNA (AppDNA21-NH2) with identical sequence. (A) Reactions without (input) or with truncated T4Rnl2. (B) Reactions without (input) or with K97A MthRnl mutant. Reactions were carried out as described in the Methods at 25°C for T4Rnl2tr and 65°C for K97A mutant. The products were analysed on 15% urea-PAGE and stained with SYBR Gold stain. Positions of substrates and ligated products are indicated on the right. Donor substrates used in the reactions are indicated on the top. Mr indicates molecular weight RNA markers.