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

Abstract

AbstractDNA 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′‐PO42− 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.