Efficient preparation and properties of mRNAs containing a fluorescent cap analog: Anthraniloyl-m7GpppG

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.