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

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Richardson, Ross William

• Chemistry, Biochemistry

Identifier

(UMI)AAI8502281

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/70539