Efficient synthesis of C-13,N-15-labeled RNA containing the cap structure m(7)GpppA

Studying the mechanism by which the cap structure performs its numerous bio logical roles in mRNA metabolism by NMR spectroscopy requires the large-sca le production of isotopically labeled capped RNA. We present an efficient m ethod for production of short RNA containing the cap structure m(7)GpppA, b y combining chemical synthesis with enzymatic synthesis using a DNA primase . This method was employed to synthesize three capped RNA molecules, m(7)Gp pp([13C,15N])A, m(7)Gppp([13C,15N])ACC, and m(7)GpppA([13C,15N])C([13C,15N] )C, that contain selective C-13,N-15-labeled adenosine or cytosine nucleoti des. This selective labeling technique enabled us to obtain the chemical sh ift assignments of these oligonucleotides in complex with the eukaryotic tr anslation initiation factor 4E (eIF4E). Furthermore, we were able to observ ed intermolecular NOE interactions between the RNA and protein.