R2D ligase: Unveiling a novel DNA ligase with surprising DNA-to-RNA ligation activity

DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5' phosphates and 3' hydroxyl groups on one strand of the duplex. While DNA ligases preferentially ligate single breaks in double-stranded DNA (dsDNA), they are capable of ligating a multitude of other nucleic acid substrates like blunt-ended dsDNA, TA overhangs, short overhangs and various DNA-RNA hybrids. Here we report a novel DNA ligase from Cronobacter phage CR 9 (R2D Ligase) with an unexpected DNA-to-RNA ligation activity. The R2D ligase shows excellent efficiency when ligating DNA to either end of RNA molecules using a DNA template. Furthermore, we show that DNA can be ligated simultaneously to both the 5' and 3' ends of microRNA-like molecules in a single reaction mixture. Abortive adenylated side product formation is suppressed at lower ATP concentrations and the ligase reaction reaches near completion when ligating RNA-to-DNA or DNA-to-RNA. The ligation of a DNA strand to the 5'-PO₄ ^2- end of RNA is unique among the commercially available ligases and may facilitate novel workflows in microRNA analysis, RNA sequencing and the preparation of chimeric guide DNA-RNA for gene editing applications.