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).