Studying the mechanism by which the cap structure performs its numerous biological roles in mRNA metabolism by NMR spectroscopy requires the large- scale production of isotopically-labeled capped RNA. We present an efficient method for production of short RNA containing the cap structure m7GpppA, by combining chemical synthesis with enzymatic synthesis using a DNA primase. This method was employed to synthesize three capped RNA molecules, m7Gppp([13C,15N])A, m7Gppp([13C, 15N])ACC, and m7GpppA([13C,15N])C([13C,15N])C, that contain selective 13C,15N- labeled adenosine or cytosine nucleotides. This selective labeling technique enabled us to obtain the chemical shift assignments of these oligonucleotides in complex with the eukaryotic translation initiation factor 4E (eIF4E). Furthermore, we were able to observed intermolecular NOE interactions between the RNA and protein.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry