Eukaryotic messenger RNAs (mRNAs) are characterized by a 5’-end m7G cap, a structure involved in nuclear export, mRNA stability and translation. Capping is generated by the addition of a guanine nucleoside methylated at N7 that is joined by a 5’-5’ triphosphate, bound to the end of primary RNA. The formation of the mRNA capping by eukaryotic cells classically involves a series of three enzymatic reactions: RNA triphosphatase that removes the γ phosphate residue from the 5’ triphosphate end of nascent pre-mRNA leaving a diphosphate, RNA guanylyltransferase that transfers guanosine monophosphate from guanosine-5’-triphosphate to the diphosphate nascent RNA terminus, and RNA N7-guanine methyltransferase that adds a methyl residue to N7 of guanine to the GpppN cap.[1,2]
Current methods to study capping and decapping of RNA are limited to the tedious use of radiolabeled guanine nucleotides followed by one-dimensional and two-dimensional thin-layer chromatography,[4-6] or denaturing gel electrophoresis.[7,8] These techniques require expensive commercial kits and specific laboratory equipment. Therefore, there is a need for a simple alternative non-radioactive assay to quantitatively assess mRNAcapping or decapping. We present a novel non-radioactive assay allowing a semi-quantitative monitoring of capped RNAs, which fulfils these requirements. This new method, which is based on differential digestion of enzymatic combinations on mRNA, shows robust performance.
The principle of the reaction assay [Figure 1] is to remove the γ and β phosphate residues from the 5’ end of the non-capped RNAs by the RNA 5’ polyphosphatase (RP8092H; Epicentre, Madison, WI); the capped RNAs being resistant to this enzyme. The 5’ monophosphate RNAs (the original non-capped RNA with α phosphate residue) are then digested by the action of the Terminator™ 5´-Phosphate-Dependent Exonuclease (TER51020; Epicentre), leaving only the capped-RNAs. As a positive control, Tobacco Acid Pyrophosphatase (T19050; Epicentre) is used to release the cap nucleoside and/or both γ and β phosphates from the 5’ end, leaving only 5’ monophosphated RNA, which will be degraded by the Terminator™ 5´-Phosphate-Dependent Exonuclease.
Figure 1: Experimental scheme of the enzymatic reaction. (1) In the first step, the γ and β phosphate residues are removed by the RNA 5' polyphosphatase leaving non-capped RNA with a single α phosphate residue at their 5'-ends, whereas capped RNA are resistant to this enzymatic digestion; (2) in the second step, the 5' α-monophosphated RNA is subjected to enzymatic degradation by the Terminator 5' phosphate dependent exonuclease, whereas capped RNA are mostly resistant to the enzymatic digestionClick here to view
A ~2.1kb synthetic firefly luciferase RNA, was produced from a linearized plasmid by SP6 RNA polymerase in vitro transcription in a final volume of 100 µL and using manufacturer conditions (New England Biolabs, Ipswich, MA). The produced RNA was purified on RNA clean up columns (Macherey-Nagel, Düren, Germany) following manual instruction. Purified RNA was verified by electrophoresis and quantified using a NanoDrop Spectrophotometer.
Ten µg of ~2.1kb luciferase RNA were capped using the Vaccinia capping system under manufacturer conditions (New England Biolabs), precipitated by LiCl 2.5 M, resuspended in RNAse free water and quantified with a NanoDrop.
Five hundred ng of luciferase RNA were incubated with 5 units of RNA 5’ polyphosphatase or 2.5 units of Tobacco Acid Pyrophosphatase in a final volume of 10 µL at 37 °C for 45 min. The RNAs were then subjected to a digestion by 0.25 units of the Terminator 5’ phosphate dependent exonuclease in a final volume of 20 µL (leading to 0.5 × RNA 5’ polyphosphatase buffer and 1 × Terminator 5’ phosphate dependent exonuclease buffer A) for 45 min at 30 °C. As controls, single digestions (5’ polyphosphatase alone and Terminator 5’ phosphate dependent exonuclease alone) as well as a non-digested condition are performed. RNA is then analyzed on a 1% agarose gel and band intensities are calculated by ImageJ analysis FIJI.
The experimental design was validated on an etalon curve with a mix of Vaccinia capped/ non-capped RNA from 0% to 100% capped in 25% increments. The positive control, Tobacco acid pyrophosphatase followed by Terminator 5’ phosphate dependent exonuclease, allows the digestion of the totality of the RNAs [Figure 2A]. The negative control 5’ RNA polyphosphatase alone does not alter RNA concentration. However, the negative control Terminator exonuclease alone shows reduced RNA concentration, demonstrating a non-specific RNAse activity of the enzyme on SP6 transcripts, which are known to contain 5’-triphosphate ends. This non-specific activity, stated in the enzyme documentation, appears variable between experiments even within the batch of enzyme and we weren’t able to pinpoint the cause of variation. In order to estimate the capping of the RNA, we used the ratio of 5’ RNA phosphatase followed by Terminator 5’ phosphate dependent exonuclease over the negative control Terminator 5’ phosphate dependent exonuclease alone. Using this method, we estimated the capped RNAs in the mixes of the etalon curve and obtained a linear regression (Figure 2B; R2 = 0.95) between the measured capping and the actual capped RNAs proportion in the mixes. The slope of the linear regression estimates Vaccinia capping system efficiency at 86%. The measurement was performedtwelve times on the same Vaccinia capped RNA, leading to a measured capping of 82.4 ± 5.9% (variation coefficient of 7.2%).
Figure 2: Experimental validation of the enzymatic digestion process. (A) RNA electrophoresis of digestions of the vaccinia capped/non-capped RNA etalon mixes according to experimental scheme. Non-capped RNA is mostly digested by 5' polyphosphatase (5' RNA PolyPase) and Terminator 5' phosphate dependent exonuclease (TerExo), whereas capped RNA is mostly resistant; (B) RNA bands were quantified using the area under the curve on ImageJ and measured capping was plotted against the vaccinia capped ratio in the mixes. The ratio of RNA capping was estimated as the 5' RNA phosphatase followed by Terminator 5' phosphate dependent exonuclease over the negative control Terminator 5' phosphate dependent exonuclease alone (boxed RNA bands)Click here to view
After the validation of the method, we estimated the efficiency of capping protocols based on dinucleotide cap analog incorporation using commercial kits mMessagemMACHINE® SP6 transcription kit (Ambion; Austin, TX) and AmplicapTM SP6 High Yield Message maker kit (Cellscript; Madison, WI), as well as cap analog incorporation during “in house” SP6 RNA synthesis (New England Biolabs), G(5’)ppp(5’)G; S1407S; m7G(5’)ppp(5’)G; 3’-O-Me-m7G(5’)ppp(5’)G. After digestions, RNA electrophoresis and quantifications, capping using dinucleotide cap analogs show efficiencies of 43.7 ± 5.5% for G(5’)ppp(5’)G, 37.3 ± 7.1% for m7G(5’)ppp(5’)G and 43.7 ± 9% for 3’-O-Me-m7G(5’)ppp(5’)G analogs. Commercial kits increase the capping efficiency, reaching 61 ± 7% and 62 ± 7.8% respectively for mMessage and Amplicap, which are both optimized for the incorporation of m7G(5’)ppp(5’)G analog.
mRNA is arising kind of biologics for gene compensation,[11-13] vaccines,[14,15] immunotherapy, regenerative and cellular medicine. Despite its rapidly growing uses, analysis of the capping rate of synthetic mRNA remains complex and difficult. Therefore, we have developed the present semi-quantitative analysis of capping which can be performed with common laboratory equipment without radio-nucleotide incorporation.
Financial support and sponsorship
This work was supported by Eukarÿs SAS.
Conflicts of interest
There are no conflicts of interest.
There is no patient involved.
This letter is waived for ethical approval.
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