3′-Terminal labelling of RNA with T4 RNA ligase

Abstract

T4 RNA LIGASE catalyses the formation of an internucleotide phosphodiester bond between an oligonucleotide donor molecule with a 5′-terminal phosphate and an oligonucleotide acceptor molecule with a 3′-terminal hydroxyl^1–3. Although the minimal acceptor must be a trinucleoside diphosphate, dinucleoside pyrophosphates and mononucleoside 3′,5′-bisphosphates (pNps) are effective donors in the intermolecular reaction^4–6. We demonstrate here that various high molecular weight RNA molecules are acceptors in the RNA ligase reaction even when present in very low concentrations in the reaction mixture. One immediate consequence of this observation is that a convenient method for labelling the 3′ end of RNA molecules in vitro becomes available. By using a [5′-³²P]pNp as a donor and RNA as an acceptor, the product of the reaction is an RNA molecule one nucleotide longer, with a 3′-terminal phosphate and a ³²P-phosphate in the last internucleotide linkage. This reaction is therefore analogous to the in vitro labelling of the 5′ termini of RNA chains with polynucleotide kinase and [γ-³²P]ATP and can be used in situations where 5′ labelling is not possible. In addition, the ability to add various donors to an RNA molecule should allow the function of the 3′ terminus of the molecule to be investigated.