Introduction

Nucleic acid ligases catalyze the formation of a phosphodiester bond between the 3’ OH group and an adjacent 5’ phosphorylated end of nucleic acids using energy from an adenylate-donating nucleotide cofactor) [1], [2]. The reaction mechanism involves three steps that are highly conserved among all family members. In the first step, the ligase self-adenylates through bond formation between an AMP group, donated by either ATP or NAD, and an active site lysine residue. In the second step, the AMP group is transferred to the 5’ phosphate of the nucleic acid, and finally the 3’OH end of the adjacent strand performs a nucleophilic attack on this activated 5’ end resulting in the formation of a diester bond in the nucleic acid backbone [3].
DNA ligases are key enzymes in molecular biology workflows including molecular cloning and adapter ligation prior to sequencing [4]–[6]. These applications have traditionally been dominated by viral DNA ligases such as T3 and T7 DNA Ligases, with the T4 DNA Ligase (T4 DnL) being by far the most widely used [7]. T4 DNA ligase shows excellent efficiency when ligating nicked duplex DNA, certain chimeric DNA-RNA oligonucleotides and DNA-RNA hybrids [8], [9]. Other double-stranded substrates, however, have not been ligated efficiently by any known ligase. Here we report the novelChronobacter phage CR9 DNA Ligase ( Commercially known as ArcticZymes R2D Ligase™) with hitherto unreported ability to ligate DNA to the 5’-end of RNA. We will also discuss possible application areas where such ligation activity may lead to new innovations.