T4 RNA ligase catalyses the ATP-dependent ligation of single-stranded RNA or DNA onto the 5'-phosphoryl termini of single-stranded RNA or DNA. The enzyme, purified from recombinant E. coli CA4 (RNase I-deficient), has an apparent molecular weight of 43,5 kDa. It also catalyses the addition of [5'-³²P] nucleoside 3',5'-bis (phosphate) onto single-stranded RNA.

This enzyme is used in ligation of single-stranded nucleic acids, 3' end-labeling of RNA, circularisation of RNA and DNA oligonucleotides and in cloning full-length cDNA. The enzyme may be inactivated by heating at 65 °C for 15 minutes.

One unit catalyzes the formation of 1 nmol of [5'-³²P]rA14–20 into a phosphatase-resistant form in 30 minutes at 37 °C in a total volume of 30 μl. The reaction conditions are: 50 mM Tris-HCl (pH 7,8 at 25 °C), 10 mM MgCl₂, 5 mM DTT, 1 mM ATP and 10 μM [5'-³²P]rA₁₄-₂₀.