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.