Figure 2: Biochemical characterization data of R2D Ligase using substrates S1, S7, S8 (A) Substrate panel used in this article. S1, S6, S7 and S8 were used in figure 1, S1, S7 and S8 in figure 2 and S9, S10 and S11 in figure 3. S2-S5 were used in experiments shown in supporting information. (B) MgCl2 dependency. The error bars show the coefficient of variation for each data point (duplicates). (C) MnCl2dependency shows a shift in optima MnCl2 concentration for all three substrates compared to MgCl2. (D) Replacing Mg2+ with Mn2+ in the reaction buffer with substrate S8 increases reaction efficiency with up till 23-fold. (E) ATP dependency on ligase activity. Increase in activity for S1 and S7 at higher ATP concentration, with an opposite effect of ATP on S8. (F) The standard buffer was compared with an optimized buffer (10 mM MgCl2, 0.1 mM ATP, 25 mM KCl) for the S8 substrate. Improvement in overall product yield and minimization of 5’ appRNA intermediate product was observed.

DNA adapters can simultaneously be ligated to both ends of RNA

To investigate the potential of DNA ligation to both ends of a 5’ phosphorylated RNA molecule, we designed a proof of concept using two DNA adaptors and two DNA template strands in a single reaction mixture (Figure 3). Since the 3’RNA to DNA ligation efficiency was predicted to be more efficient than the DNA to 5’RNA ligation, we used the optimized buffer described earlier for the latter substrate. We tested this by separate reactions at either end, as well as for all components in a single mixture. The DNA adapters were marked with different fluorescent labels to differentiate between ligation to the 5’ and 3’ end of the target RNA after Urea/PAGE separation. Successful ligation of both adapters allowed detection of a yellow band composed of both the TAMRA and the FAM fluorophore. For the R2D Ligase, the reaction showed more than 70 % substrate turnover for both adapters, both in isolated and in simultaneous reactions. As expected, T4 DnL showed similar activity when ligating DNA to the 3’ end of RNA but only trace activity (< 1 % turnover) on the 5’ end of the RNA.