Efficient Synthesis of ¹³C,¹⁵N-Labeled RNA Containing the Cap Structure m⁷GpppA

Abstract

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.

Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein

“…www.rnajournal.org tween the second adenosine and the protein were also observed by NOE measurements for yeast eIF4E bound with 13 C/ 15 N double-labeled adenosine containing m 7 GpppA (Matsuo et al 2000). Moreover, no NOE contacts with the yeast protein were detected for the double-labeled cytosines of m 7 GpppACC (Matsuo et al 2000), and the Gibbs free energy change (⌬⌬G) due to additional interactions of the pUm 2Ј pAm 2Ј fragment with murine eIF4E is −5.24 kJ/mole (calculated from the K as values in Table 1).…”

“…Moreover, no NOE contacts with the yeast protein were detected for the double-labeled cytosines of m 7 GpppACC (Matsuo et al 2000), and the Gibbs free energy change (⌬⌬G) due to additional interactions of the pUm 2Ј pAm 2Ј fragment with murine eIF4E is −5.24 kJ/mole (calculated from the K as values in Table 1). Our results point to some stabilizing, electrostatic interactions between the unphosphorylated protein and the RNA chain, which are diminished by +1.7 kJ/mole due to the introduction of an additional negative charge at the protein surface by the phosphorylation.…”

Novel “anti-reverse” cap analogs with superior translational properties

“…If one takes into account some favorable effect of a smaller stacked population (Table 1) and the unfavorable entropic effect of the phosphate chain extension, which partially cancel each other out, the more negative ⌬G°for the pentaphosphate might be caused by an additional stabilizing contact between the negatively charged chain and the positively charged protein surface, that is, Lys 159. It is clear that groups in capped mRNA "downstream" of the m 7 Guo interact with eIF4E, because (1) capped oligonucleotides bind more strongly than cap dinucleotides (Carberry et al 1992), and (2) the Ado moiety in m 7 Gp 3 A is stabilized by two H-bonds to Thr 205 and Thr 211 and van der Walls contacts at the surface of full-length human eIF4E (Tomoo et al 2002) and at the surface of yeast eIF4E (Matsuo et al 2000). On the other hand, no such interactions were observed in the latter study for m 7 Gp 3 ACC.…”

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

“…To prevent this reverse cap orientation, "anti-reverse" cap analogs can be used in the transcription reaction, where the 3 ′ OH group of the m 7 G nucleotide is modified (Grudzien-Nogalska et al 2007), which results in the incorporation of a non-natural cap-like structure in the RNA. For very short RNA sequences (<9 nt), the incorporation of a reverse cap can also be prevented by the use of gene 4 primase (Matsuo et al 2000) or a fragment thereof (Peyrane et al 2007) that incorporates cap analogs solely in the correct orientation.…”