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.