T4 RNA Ligase 2 is derived from T4 bacteriophage and recombinantly expressed in Escherichia coli. It is an ATP-dependent double-stranded RNA ligase used for intramolecular circular ligation and intermolecular linear ligation of double-stranded RNA.

Unlike T4 RNA Ligase 1, T4 RNA Ligase 2 shows much higher ligation activity toward nicks in double-stranded RNA than toward single-stranded RNA termini. It can also ligate the 3'-hydroxyl of an RNA strand to the 5'-phosphate of a DNA strand in intramolecular or intermolecular duplex nucleic acids (double-stranded RNA, RNA/DNA hybrids, and/or double-stranded DNA).Ligation by T4 RNA Ligase 2 requires the presence of a 5'-phosphate and a 3'-hydroxyl, and can occur between the 5'-phosphate of an RNA or DNA strand and the 3'-hydroxyl of an RNA strand.

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