Use of a 2-5A analogue probe for detecting RNA ligase and RNA ligase substrates in mammalian cell extracts.
The compound ppp(A2'p)3A3'[32P]pCp is a commercially available radioactive analogue of the 2',5' oligoadenylate series ppp(A2'p)nA, n greater than or equal to 2, commonly referred to as 2-5A. It is used as a probe for measuring concentrations in competition radiobinding and radioimmune assays. We have found that incubation of the probe with extracts from HeLa, CV1, or neuroblastoma cells results in its covalent attachment to two size classes of RNA: the first includes a major species with a molecular weight of approximately 350,000, the second is much smaller (40 +/- 5 nucleotides in length) and could represent tRNA half-molecules. Ligation is to the 3' end of the probe molecule with formation of a 3',5'-phosphodiester bond. Thus, probe ligation provides a sensitive and convenient assay for the detection not only of RNA ligase(s) but also of ligatable RNAs (such as the putative tRNA half-molecules) in mammalian cell extracts.
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007668:Kidney; D007700:Kinetics; D009447:Neuroblastoma; D009841:Oligonucleotides / Q000494:pharmacology*; D009843:Oligoribonucleotides / Q000494:pharmacology*; D010761:Phosphorus Radioisotopes; D011118:Polynucleotide Ligases / Q000378:metabolism*; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000302:isolation & purification
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D010761:Phosphorus Radioisotopes; D012334:RNA, Neoplasm; C042702:triadenylyl-(2'-3')-adenylyl-cytidylic acid; D011118:Polynucleotide Ligases; D011130:RNA Ligase (ATP)