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

• Published in: Translation (Austin, Tex.) Vol. 3; no. 1
• Main Authors: Gunawardana, Dilantha, Domashevskiy, Artem V, Gayler, Ken R, Goss, Dixie J
• Format: Journal Article
• Language: English
• Published: Taylor & Francis 02-02-2015
• Subjects: Ant, Anthraniloyl; Ant-GTP, Anthraniloyl guanosine triphosphate; Ant-m; capped mRNA; decapping assay; Fluorescence; GMP, Anthraniloyl-7-methyl guanosine monophosphate; GTP, Anthraniloyl-7-methyl guanosine triphosphate; imaging; IVT, In vitro translation; Methods; OMP, Outer Membrane Protein; RNA capping; SAM, S-Adenosyl Methionine
• ISSN: 2169-0731
• DOI: 10.4161/21690731.2014.988538

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
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GTP is specifically incorporated into the cap site to yield Ant-m
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GpppG-capped mRNA or oligonucleotide. Efficient capping was observed with 60-100% of the RNA transcripts capped with the fluorescent molecule. The Ant-m
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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
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GTP-capped RNA is readily translated. This Ant-m
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GTP-capped RNA provides an important tool for monitoring capping reactions, translation, and biophysical studies.