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.
ASJC Scopus subject areas
- Molecular Biology