Figure 1: Initial substrate specificity panel of R2D ligase along with other commercially available ligases.

Effect of Magnesium, KCl and ATP on ligation of RNA to DNA

To investigate the effect of buffer components on ligation performance using different substrates, we tested the R2D ligase with three substrates (S1, S7 and S8, Figure 2A) with increasing amounts of MgCl2, and MnCl2 and ATP in the ligation mixture (figure 2B-D). In a typical ligation buffer such as for the T4 DNA Ligases these components are used at concentrations of 5 mM MgCl2/MnCl2 and 1 mM ATP (the arrows in the figure shows standard concentrations to which the results have been normalized).
The effect of magnesium is similar when using both DNA-DNA substrate (S1) and the DNA-RNA hybrids (S8) with an increase in activity followed by a plateau at 2 mM (S1) and 5 mM (S8) (Figure 2B and supporting information S5). With RNA-DNA the increase in activity with metal ion concentration continued until 10 mM which was the highest concentration tested. Interestingly, when substituting Mg2+ with Mn2+ as the divalent metal ligand, a shift in optimum metal concentration from more than 5 mM to 3 mM or less is observed when ligating DNA-DNA (S1) and RNA-DNA (S7) (Figure 2C). A similar shift was previously reported in archaea DNA ligases [13], as well as in human DNA Ligase I [14]. The Mn2+ concentration shows less impact on the activity of R2D when ligating DNA-RNA (S8), as the activity at all Mn2+ concentrations is within 70 % of maximum activity. Additionally, while a higher R2D ligase concentration was necessary to detect ligation of S1 and S7 with Mn2+, ligation of S8 showed a 23-fold increase in efficiency compared with using Mg2+ (figure 2D). In the case of ATP, an increase above 0.1 mM enhanced ligation efficiency when ligating DNA-DNA (S1) and RNA-DNA (S7) (Figure 2E). When ligating DNA-RNA however, a sharp decrease was observed at ATP concentrations above 0.1 mM, indicating a clear advantage in reducing ATP with this substrate. A higher amount of side product corresponding to approximately 1 nucleotide larger than the donor oligo was also observed at higher ATP concentrations (supporting information figure S4). This observation suggests that R2D is defective in completing step 3 of strand joining with DNA-RNA (S8) relative to other substrates meaning that an increased rate of Step 1 (enzyme adenylation) with high ATP causes rapid re-adenylation of the enzyme, resulting in premature release of the ‘dead-end’ adenylated RNA product. This may be either due to decreased affinity for the 5’adenylated form of this duplex relative to the DNA-DNA or RNA-DNA forms, or due to decreased rate of catalysis with a ribonucleotide at the 5’ end. Similar effects have been observed for other ligases acting on mixed DNA/RNA hybrids which required tuning of ATP concentration for optimal performance [12]. Reducing the ATP concentration to 0.1 mM as well as increasing MgCl2concentration to 10 mM gave a 2-fold increase in ligated product when ligating S8, as well as minimizing abortive adenylation (Figure 2F and supporting figure S3).