Biotin and fluorescent labeling of RNA using T4 RNA ligase
Open Access
- 24 September 1983
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 11 (18), 6167-6184
- https://doi.org/10.1093/nar/11.18.6167
Abstract
Biotin, fluorescein, and tetramethylrhodamlne derivatives of P1-(6-aminohex-l-yl)-P2-(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 acceptor to form a phosphodiester bond and the AMP portion is released. E. coli and D. melanogaster 5S RNA, yeast tRNAphe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100Z. Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochlorlde, pH 2.5. Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3′-terminus of RNA molecules.Keywords
This publication has 33 references indexed in Scilit:
- Electron microscopic visualization of tRNA genes with ferritin-avidin: biotin labelsNucleic Acids Research, 1978
- Dinucleoside pyrophosphate are substrates for T4-induced RNA ligase.Proceedings of the National Academy of Sciences, 1977
- Equimolar addition of oligoribonucleotides with T4 RNA ligaseNucleic Acids Research, 1977
- Selective retrieval of biotin-labeled cells using immobilized avidinExperimental Cell Research, 1976
- Preparation of adenosine nucleotide derivatives suitable for affinity chromatographyBiochemical Journal, 1974
- Structural Studies on Phenylalanine Transfer Ribonucleic Acid from Yeast with the Spectroscopic Label FormycinEuropean Journal of Biochemistry, 1974
- [56] Nucleoside phosphates attached to agaroseMethods in Enzymology, 1974
- [20] Insolubilized biotin for the purification of avidinMethods in Enzymology, 1974
- Covalent attachment of fluorescent groups to the 5′-end of transfer RNAArchives of Biochemistry and Biophysics, 1973
- Agarose Derivatives of Uridine Diphosphate and N-Acetylglucosamine for the Purification of a GalactosyltransferaseJournal of Biological Chemistry, 1972