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.