[9] Specific Labeling of 3′ Termini of RNA with T4 RNA Ligase

This chapter deals with specific labeling of 3' termini of RNA with T4 RNA ligase. T4 RNA ligase catalyzes the formation of an internucleotide phosphodiester bond between a 5' terminal phosphate and a 3' terminal hydroxyl of oligo- or polyribonucleotides with the accompanying hydrolysis of ATE. If the two termini are present on the same molecule, a cyclic RNA product is formed. However, if the oligoribonucleotide with the Y-phosphate (the donor) is too short to cyclize or has a blocked 3' terminus, it will join with another oligoribonucleotide with a free 3'- hydroxyl (the acceptor) to form an intermolecular ligation product. One important application of the RNA ligase reaction is the use of an RNA molecule as an acceptor and a [5'-³²P]nucleoside 3',5'-bisphosphate as a donor to form a product RNA one nucleotide longer with a 3' terminal phosphate and a 3∼P-phosphate in the last internucleotide linkage. The chapter also explains the preparation of [5'-³²P] nucleoside 3',5'-bisphosphates.