Abstract:
A method has been developed for synthesising fluorescentiy labeled capped mRNA. The method incorporates a single fluorescent molecule as part of the 5'-mRNA or oligonucleotide cap site. The fluorescent molecule, Ant-m7GTP is specifically incorporated into the cap site to yield Ant-m7GpppG-capped mRNA or oligonucleotide. Efficient capping was observed with 60-100% of the RNA transcripts capped with the fluorescent molecule. The Ant-m7G derivative, which has been previously shown to interact with the eukaryotic cap binding protein elF4E, is shown in this paper to be a substrate for the Vaccinia capping enzyme and the DCP2 decapping enzyme from Arabidopsis. Further, the Antm7GTP- capped RNA is readily translated. This Ant-m7GTP-capped RNA provides an important tool for monitoring capping reactions, translation, and biophysical studies.
