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: Dilantha Gunawardana, Artem V Domashevskiy, Ken R Gayler, Dixie J Goss
• Journal: Translation (Austin, Tex.) (Translation (Austin)) 2015 Jan-Jun Vol. 3 Issue 1 Pg. e988538 ISSN: 2169-074X [Print] United States
• PMID: 26779415 (Publication Type: Journal Article)