Results
R2D ligase shows excellent activity on nicked DNA/RNA
hybrids
Initially, R2D ligase was tested in a substrate specificity assay
adapted from Bullard and Bowater [10], along with other well-known
DNA and RNA ligases. The substrate specificity assay evaluates each
ligase’s ability to ligate nicked
duplex substrates composed of three individual oligos, where each oligo
may be DNA or RNA. In the present study we focus on the DNA-splinted
substrates only. For ligation performance on the full panel, see the
supporting information (Figure S1-S2). Figure 1 shows that R2D was the
only ligase tested that was able to ligate all four substrates S1, S6,
S7 and S8 to completion. Specifically, complete attachment of DNA- to
the 5’ends of RNA was only observed with R2D ligase and while both
PBCV-1 DNA ligase and T3 DNA ligase carried out some ligation on this
substrate, this was at a much lower level. The DNA-splinted RNA
substrate was also ligated to near-completion by R2D ligase, as well as
T4 DNA Ligase and T4 RNA Ligase 2. All DNA ligases were able to ligate
the nicked DNA substrate, and all ligases were able to ligate RNA- to
5’DNA with a DNA splint. To our knowledge, this is the first report of a
ligase that exhibits the functionality of ligating DNA to both sides of
RNA efficiently [10]. Crystal structures of a range of ligases show
that upon DNA binding prior to step 2, ligase enzymes enforce an
RNA-like A-structure on the 3’OH terminal nucleotide of acceptor strand,
while the 5’P terminal nucleotide is retained in the DNA-B conformation,
providing a rationale for the high rates of joining of S7 by all ligases
tested here [11], [12]. The broader tolerance of an RNA acceptor
strand by R2D ligase may lie either in an ability to tolerate an A-form
of the nucleotide in this position, or conversely an enhanced ability to
impose a B-conformation on the 5’ terminal ribonucleotide.