title: Efficient preparation and properties of mRNAs containing a fluorescent cap analog: Anthraniloyl-m(7)GpppG.
abstract: A method has been developed for synthesising fluorescently labeled capped mRNA. The method incorporates a single fluorescent molecule as part of the 5'-mRNA or oligonucleotide cap site. The fluorescent molecule, Ant-m(7)GTP is specifically incorporated into the cap site to yield Ant-m(7)GpppG-capped mRNA or oligonucleotide. Efficient capping was observed with 60-100% of the RNA transcripts capped with the fluorescent molecule. The Ant-m(7)G derivative, which has been previously shown to interact with the eukaryotic cap binding protein eIF4E, is shown in this paper to be a substrate for the Vaccinia capping enzyme and the DCP2 decapping enzyme from Arabidopsis. Further, the Ant-m(7)GTP-capped RNA is readily translated. This Ant-m(7)GTP-capped RNA provides an important tool for monitoring capping reactions, translation, and biophysical studies.
authors.affiliation: Department of Biochemistry and Molecular Biology; Bio21 Molecular Science and Biotechnology Institute; University of Melbourne, Parkville; Victoria, Australia; Present address: Department of Botany; University of Sri Jayewardenepura; Soratha Mawatha, Nugegoda, Sri Lanka.
authors.forename: Dilantha
authors.initials: D
authors.lastname: Gunawardana
authors.affiliation: Department of Sciences; John Jay College of Criminal Justice; City University of New York ; New York, NY USA.
authors.forename: Artem V
authors.initials: AV
authors.lastname: Domashevskiy
authors.affiliation: Department of Biochemistry and Molecular Biology; Bio21 Molecular Science and Biotechnology Institute; University of Melbourne, Parkville ; Victoria, Australia.
authors.forename: Ken R
authors.initials: KR
authors.lastname: Gayler
authors.affiliation: Department of Chemistry and Graduate Center; Hunter College; City University of New York ; New York, NY USA.
authors.forename: Dixie J
authors.initials: DJ
authors.lastname: Goss
keywords: Fluorescence; RNA capping; capped mRNA; decapping assay; imaging