Biotin and Fluorescent Labeling of Ribonucleic Acids Using T4 Rna Ligase (Rna Modification, Rna-Dna Hybridization)

212 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1984.The biotin, fluroescein, and tetramethylrhodamine derivatives of P('1)-(6-aminohex-1-yl)-P('2)-(5'adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3'-hydroxyl of an RNA molecule (acceptor) to form a phosphodiester bond and the AMP portion is released. E. coli and D. melanogaster 5S RNA, yeast tRNA('Phe), (Ap)(,3)C, and (Ap)(,3)A serve as acceptors with yields of products varying from 50 to 100%. Analysis of the oligoribonucleotide (Ap)(,3)C reactions with RNase A and PNase A plus bacterial alkaline phosphatase demonstrated that a phosphodiester bond linked the oligomer and functional group in the product. Biotin-labeled oligoribonucleotides are bound selectively and quantitatively to avidin-bound agarose and may be eluted with 6M guanidine hydrochloride, pH 2.5. The fluorescein and tetramethylrhodamine-labeled oligoribonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. Analysis of the tRNA and 5S RNA reactions by polyacrylamide gel electrophoresis demonstrated the incorporation of ('14)C-labeled biotin or a fluorescent dye into the RNA molecules. The major advantage of modifying RNA with Ado-5'PP-X compounds and T4 RNA ligase rather than by direct chemical methods derives from the highly specific nature of enzyme catalyzed reactions. The required organic chemistry is carried out at the level of the ADP derivative and this product can be highly purified before it is brought into contact with the RNA.Fluorescent or biotin-labeled RNA molecules may be useful as probes to detect complementary DNA sequences that have been immobilized on nitrocellulose. Using tetramethylrhodamine-labeled D. melanogaster 5S RNA as a hybridization probe to detect 5S DNA sequences in a plasmid, 80 to 120 femptomoles of 5S genes were readily detected on a Southern blot. When the RNA was labeled with biotin and detected by means of streptavidin and biotinylated horseradish peroxidase, 20 to 30 femptomoles of 5S genes were detected. These techniques offer a potentially attractive alternative to methods requiring radioactively labeled RNA and the ability to form the requisite tagged RNA molecules by the methods described may facilitate their development.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD