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

Abstract

The eukaryotic mRNA 5 ′ cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and highresolution NMR spectroscopy. In combination with 13 C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.

Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

“…1D). These RNA standards were prepared by in vitro transcription and were capped using a recombinant Vaccinia virus capping enzyme (37,38).…”

The S. pombe mRNA decapping complex recruits cofactors and an Edc1-like activator through a single dynamic surface

“…To that end, we used a capped and fluorescently labeled RNA (Fuchs et al 2016), to which we added increasing amounts of Dcp1: Dcp2 RD + CD . We performed those experiments in the absence of Edc1 and in the presence of saturating amounts of Dcp1 is a dynamic hub for decapping regulators www.rnajournal.org 1367 Edc1 or Edc1 ΔYAG (Fig.…”

“…Both RNAs were purified by denaturing anion exchange chromatography using a DNAPac PA100 column (22 × 250 mm, Dionex) at 80°C (Buffer A: 20 mM, Tris pH 8, 5 M Urea, Buffer B: as A plus 2 M NaCl). Afterward, the N7-methylguanosine cap was added to the 5 ′ end of the RNAs using vaccinia virus capping enzyme according to a recently published protocol (Fuchs et al 2016). The 30mer RNA was fluorescently labeled after capping using 5-(iodoacetamido)-fluorescein (Ramos and Varani 1998).…”

Nanomedicines to Deliver mRNA: State of the Art and Future Perspectives

“…A strategy to reduce the stimulation of the immune system is the removal of the triphosphates of the 5 end of uncapped IVT mRNA by using a phosphatase [9,28]. On the other hand, there is the risk of reverse orientation of the cap analogues, which impede the binding to the CBP and the posterior translation [11,24,29]. As an alternative, an anti-reverse cap analogue (ARCA) has been developed [11,24].…”