Synthetic messenger RNA (mRNA)−based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS-CoV-2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5′-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap^® AG co-transcriptional capping results in 5 mg/ml of IVT with 94% 5′-cap 1 structure. This is highly efficient compared to first-generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co-transcriptional capping using TriLink Biotechnology's CleanCap^® AG in IVT. © 2021 The Authors.

This article was corrected on 22 November 2021 and 26 July 2022 . See the end of the full text for details.

Basic Protocol 1 : IVT with CleanCap

Basic Protocol 2 : mRNA purification and analysis

Synthetic messenger RNA (mRNA) has shown great potential as a therapeutic in various clinical applications such as vaccines and genetic disease treatments, i.e., protein replacement therapies, stem cell reprograming, and gene editing (Sahin, Karikó, & Türeci, 2014; Warren et al., 2010). Transient expression of synthetic mRNA is desirable in such applications because it avoids the risk of genomic insertion events presented by viral systems. Advances in mRNA manufacturing, scalability, and delivery have expanded mRNA's utility as a therapeutic (Kowalski, Rudra, Miao, & Anderson, 2019). Currently, multiple mRNA vaccines against SARS-CoV-2 are in clinical trials, resulting in some of the fastest drug development programs in modern biotechnology history (Kis et al., 2020; Krammer, 2020). Two mRNA vaccines are now authorized by U.S. FDA for emergency use (Haynes, 2020), one of which uses CleanCap technology (Sahin et al., 2020).

mRNA is transcribed from DNA by RNA polymerase enzymes via a process called transcription. RNA polymerase binds to the promoter region of DNA and forms a transcription initiation bubble. Nucleotides complementary to the DNA are added in the 5′-to-3′ direction by the formation of phosphodiester bonds. In vitro, the same process is achieved by combining a DNA template, RNA polymerase, and nucleotide triphosphates (NTPs) with magnesium-containing buffer, RNase inhibitor, and inorganic pyrophosphatase (Chamberlin & Ring, 1973).

Most mature eukaryotic mRNA consists of a 5′-cap, 5′-untranslated region (UTR), coding sequence or open reading frame (ORF), 3′-UTR, and poly(A) tail, as shown in Figure 1. The 5′-cap is vital for mRNA stability, translation, and self- versus non-self identification, but several variations of this structure exist in nature (Furuichi, 2015; Wang et al., 2019). A cap 1 structure is critical for successful expression in cells (Ramanathan, Robb, & Chan, 2016). Therefore, creating this structure is a vital part of the synthetic process.

Traditional T7 RNA polymerase reactions will initiate transcription with guanosine triphosphate, forming a 5′-triphosphate RNA. Generating a cap structure then requires a second enzymatic treatment (Ensinger, Martin, Paoletti, & Moss, 1975; Moss, Gershowitz, Wei, & Boone, 1976). Alternatively, capped transcripts can be produced by using a cap analog during the in vitro transcription (IVT) reaction (Hadas et al., 2019; Ishikawa et al., 2009; Sikorski et al., 2020; Stepinski et al., 2001). Termed co-transcriptional capping, the cap analog is introduced into the IVT reaction along with NTPs, DNA plasmid, and RNA polymerase in a single reaction vessel, resulting in mature mRNA. This “one-pot” reaction decreases the number of manufacturing steps, which reduces overall handling time, purification steps, and number of enzymes required. Such reductions are critical for reducing the cost and complexity of mRNA manufacturing. First-generation cap analogs resulted in cap 0 structures with low reaction yields and capping efficiency. Therefore, a new cap analog resulting in cap 1 structure incorporated by the RNA polymerase was developed. Co-transcriptional capping with CleanCap AG trimer as shown in Figure 2A produces 94% (or higher) cap 1 mRNA in a one-pot synthesis, resulting in mRNA yields of approximately 5 mg mRNA per ml of IVT reaction. Transcription initiates downstream of the T7 promoter sequence when the CleanCap trimer binds to the +1 and +2 nucleotides of the template through complementary base pairing followed by incorporation of the complementary NTP at the +3 position, as shown in Figure 2B.

This method describes co-transcriptional capping using CleanCap AG reagent with optional DNase I treatment for synthesis of 5 mg/ml cap 1 mRNA through IVT reaction (Basic Protocol 1). Following IVT, the mRNA is purified and analyzed by UV-vis spectrophotometry and denaturing gel electrophoresis, as described in Basic Protocol 2.

DNA Template Design

DNA template design is an integral part of successful transcription. IVT of an mRNA requires a DNA template coding the desired message with an upstream RNA polymerase promoter site. Templates can be in the form of a linearized plasmid or a PCR product. Templates must contain the T7 RNA polymerase promoter sequence, a 5′-UTR, ORF, and 3′-UTR. Sequence optimization of the ORF using bioinformatic software may improve therapeutic applications (Vaidyanathan et al., 2018). A poly(A) tail is required for functional translation of an mRNA. The poly(A) tail can be included in the plasmid, added via PCR, or added post-transcriptionally by enzymatic polyadenylation. Of note, enzymatic polyadenylation results in heterogenous tail lengths. Optimal poly(A) tail length may vary by sequence and application (Jalkanen, Coleman, & Wilusz, 2014). Plasmid designs must include a unique restriction enzyme cut site for linearization downstream of the desired 3′-end, as shown in Figure 3A. A PCR template may be preferred if you require a longer poly(A) than can be maintained within the plasmid and/or if your current plasmid does not contain the T7 promoter region needed for transcription (Fig. 3B).

While it is likely that CleanCap would function with SP6 or T3 RNA polymerases, to the best of our knowledge CleanCap AG has only been tested with T7 RNA polymerase. Figure 2B shows the correct T7 promoter sequence (underlined) and initiator sequence for CleanCap AG.

Following restriction digest or PCR, the DNA template must be purified to remove enzymes and reaction components. Commercially available column purification such as QIAGEN's Plasmid Plus kits (cat no. 12941, or similar), or phenol/chloroform extraction followed by ethanol precipitation (see Current Protocols article: Dowhan, 2012), are common methods for DNA cleanup. If the template is prepared by phenol/chloroform extraction, all residual phenol must be removed from DNA template prior to IVT, as enzymes may be denatured.

RNase-free environment

RNases in an IVT reaction will degrade an mRNA product. It is essential that all reagents be rigorously RNase free. When possible, use dedicated RNase-free pipettes with filter tips.

Post IVT

Determine if any additional enzymatic reactions will be required after transcription. For example, DNase I treatment is employed to degrade the template, and is essential when using a purification method that does not readily separate RNA from DNA. Purification and analysis methods should be determined prior to beginning IVT. The quality of IVT products is negatively impacted during long periods of being kept in the crude reaction mixture. Make sure all equipment and reagents for downstream steps are available. Refer to Figure 4 for workflow options.

This procedure describes IVT of a DNA template containing a T7 promoter site and poly(A) track, with a co-transcriptional CleanCap AG analog, to produce a cap 1 mRNA. An optional DNase I treatment step is described. Reaction components will be mixed and incubated at 37°C for an extended time to allow RNA polymerization to occur. Reaction size may be scaled to achieve desired amounts of capped mRNA as necessary. The basic protocol below yields 5 mg of capped RNA per ml of reaction on average; however, template length and quality may affect actual results. Higher reaction yields may be possible with optimization of the synthesis protocol. If desired, an aliquot of crude mRNA can be analyzed by gel electrophoresis prior to purification, to verify reaction success (step 12). Purification of IVT product should immediately follow (see Basic Protocol 2).

Materials

• 10% bleach solution

• RNase- and DNase-free water

• RNaseZAP™ (Ambion, cat. no. AM9782) or similar decontaminant solution

• 70% ethanol solution

• ATP, 100 mM (Thermo Fisher, cat no. R0481 or similar)

• CTP, 100 mM (Thermo Fisher, cat no. R0481 or similar)

• GTP, 100 mM (Thermo Fisher, cat no. R0481 or similar)

• UTP, 100 mM (Thermo Fisher, cat no. R0481 or similar

• CleanCap AG^®, 100 mM (TriLink Biotechnologies, cat. no. N-7113)

• 10× in vitro transcription (IVT) buffer (see recipe)

• Purified, linearized DNA transcription template (500-1000 μg/ml)

• 10× DNase I reaction buffer (optional; NEB, cat no. B0303, or similar)

• DNase I, RNase-free (optional, NEB, cat no. M0303, or similar)

• RNase inhibitor, murine, 40 U/µl (NEB, cat no. M0314, or similar)

• Inorganic pyrophosphatase, yeast, 0.1 U/µl (NEB, cat. no. M2403, or similar)

• T7 RNA polymerase, 50 U/µl (NEB, cat. no. M0251 or similar)

• 37°C water bath or heat block

• Microcentrifuge tubes, nuclease-free (VWR, cat. no. 87003-294, or similar)

• Benchtop vortex

• Microcentrifuge

• Micropipettes

• Cryosafe enzyme cooler or ice bucket

• Parafilm

Prepare reagents

1.Clean the work area prior to starting the reaction to avoid common lab contaminants. Treat surfaces and pipettes with 10% bleach to ensure no nucleic acid cross contamination. Allow bleach to rest on surfaces for 10 min. Wipe all bleached surfaces and pipettes with RNase-free water followed by RNaseZap and 70% ethanol.

2.Equilibrate water bath to 37°C.

3.Thaw NTPs, 10× IVT buffer, and linearized plasmid transcription template at room temperature. Vortex NTPs and 10× IVT buffer until homogenous. Spin tubes briefly to collect any liquid from caps.

Prepare reaction

4.Combine the components in an RNase-free tube. For a 1-ml reaction, use volumes listed in Table 1.Reactions may be scaled up or down using these ratios depending on desired final mRNA yield, based on approximately 5 mg mRNA per 1 ml reaction. Add transcription components in the order listed in the table.

5.Mix total reaction well by gentle pipetting until homogenous. Seal tube with parafilm.

Incubate reaction

6.Incubate the reaction at 37°C in a water bath or heat block.

DNase I treatment (optional)

7.Thaw 10× DNase I reaction buffer and vortex to mix.

8.Add the 10× buffer to the finished IVT reaction mixture. Volumes in Table 2 may be scaled up or down to match IVT volume used.

9.Add DNase I enzyme to the finished IVT reaction mixture.

10.Gently mix new reaction until homogenous.

11.Incubate DNase reaction at 37°C for at least 15 min.

Retain crude sample for analysis

12.Save 20 µl of crude IVT reaction at 4°C for troubleshooting purposes.

Purify IVT product

13.Take remaining IVT reaction forward to purification (Basic Protocol 2, step 3).

Several options for purification of capped mRNA are available. Methods utilized for uncapped, enzymatically capped, or ARCA-capped mRNA purification are appropriate for use with CleanCap AG mRNA. This protocol describes lithium chloride precipitation of IVT material, similar to Dowhan (2012) or Walker and Lorsch (2013), with helpful tips for successful mRNA recovery. Lithium chloride precipitation employs elevated concentrations of lithium cations to selectively precipitate RNA. The precipitated mRNA is pelleted and the supernatant containing IVT salts, free NTPs, proteins, and DNA is discarded. DNase I treatment post-transcription is optional with this RNA purification method, but will ensure complete template DNA removal.

Following purification, the mRNA product should be measured and visualized to assess reaction yield and quality. The most common visualization technique is gel electrophoresis. Electrophoretic techniques achieve size-based separation of RNA molecules by exploiting their constant mass-to-charge ratios. Electrophoresis is commonly performed by means of agarose gel electrophoresis, which uses the porosity of an agarose slab cast at a sample-specific concentration to separate nucleic acids by size alongside known size markers. Voltage is applied to a sample solution, and the negatively charged RNA molecules are forced to migrate toward an anode through a size-selective matrix. Under denaturing conditions RNA migration rates through the matrix are determined nearly exclusively by their length (Armstrong & Shultz, 2008; Goda & Minton, 1995). This protocol provides steps to denature the RNA using a glyoxal loading dye (Rio, 2015), which improves sizing accuracy by eliminating the effects of secondary structure on gel migration.

Materials

• 70% (v/v) ethanol

• Crude mRNA (Basic Protocol 1, step 13)

• 7.5 M lithium chloride solution (Thermo Fisher, cat. no. AM9480 or similar)

• Resuspension buffer of choice: e.g., RNase-free water, 1× TE buffer, pH 8.0, 1 mM sodium citrate pH 6.4

• Agarose

• NorthernMax™-Gly Gel Prep/Running Buffer, 10× (Thermo Fisher, cat. no. AM8678)

• NorthernMax™-Gly Sample Loading Dye (Thermo Fisher, cat. no. AM8551, or similar)

• Ethidium bromide

• Millennium™ RNA Marker (Thermo Fisher, cat. no. AM7150, or similar )

• Refrigerated centrifuge and rotor appropriate for centrifugation at 18,500 × g

• Centrifuge tubes of appropriate volume and strength

• NanoDrop (Thermo Fisher) or UV-vis spectrophotometer with cuvette

• 50°C water bath or heat block

• Gel rig with power supply

• Gel imager

• Additional reagents and equipment for agarose gel electrophoresis (see Current Protocols article: Armstrong & Schulz, 2008)

Prepare reagents

1.Prepare 70% ethanol and chill at −20°C for at least 1 hr before use.

2.Begin chilling centrifuge to 4°C.

Precipitate sample

3.Transfer crude mRNA from Basic Protocol 1, step 13, to a centrifuge tube that can tolerate at least 18,500 × g with a capacity of 1.5× its current volume.

4.Add LiCl to 2.5 M final concentration.

5.Mix by gentle inversion or swirling. Do not vortex or shake.

6.Chill at −20°C for at least 30 min.

7.Centrifuge 30 min at 18,500 × g , 4°C.

8.Discard the supernatant, taking care to retain the pellet and, potentially, any smaller pellet fragments.

9.Wash the pellet with the cold 70% ethanol prepared in step 1.Use maximum volume allowed by the precipitation tube. Pipet the ethanol over the pellet until it dislodges from the wall of the tube, and allow the pellet to move gently through the wash.

10.Centrifuge the pellet in 70% ethanol 5 min at 12,000 × g , 4°C.

11.Discard the ethanol supernatant. Use a pipet to remove as much of the residual wash as possible without disturbing the pellet.

12.Repeat steps 9-11 to perform a second wash.

13.Dry the pellet.

Resuspend pelleted mRNA sample

14.Resuspend the pellet in a buffer of choice.

Measure mRNA concentration

15.Use NanoDrop per manufacturer's instructions to read the absorbance of the purified mRNA at 260 nm.

Visualize mRNA by gel electrophoresis

Refer to Current Protocols article Armstrong and Schulz (2008) for general guidelines.

16.Melt agarose in 1× NothernMax™-Gly Running Buffer.

17.Allow agarose to cool 10 min. Add 1 µl of ethidium bromide per 100 ml of melted agarose. Swirl to mix. Pour agarose into the rig and allow to set for 30 min. Once gel is set, fill chambers with 1× running buffer.

18.Prepare RNA sample(s) with NorthernMax™-Gly Sample Loading Dye. Incubate samples for 30 min at 50°C.

19.Load size marker and denatured RNA samples in gel wells and apply voltage.

20.Image gel.

Aliquot sample

21.After purification, aliquot the capped mRNA into single-use vials and store long term at −80°C.

In vitro transcription (IVT) buffer, 10×

• 400 mM Tris·HCl pH 8.0
• 165 mM magnesium acetate
• 100 mM dithiothreitol (DTT)
• 20 mM spermidine
• 0.02% (v/v) Triton X-100

Fresh transcription buffer is crucial for optimal polymerase activity, as the DTT may oxidize over time. Store at −20°C in single-use aliquots or mix 10× buffer fresh before use.

Background Information

As the intermediate code of gene expression, messenger RNA can be harnessed for several research and therapeutic purposes. By directly delivering a synthetic mRNA to a cell, almost any protein of interest can be transiently expressed. In vitro mRNA production should be optimized for high purity and intact molecular structure. This can become time consuming and costly if the appropriate tools are missing. Purity and structure of the IVT product become especially critical during delivery to the cell. Pattern recognition receptors (PRR) detect self- from non-self RNA and trigger the downstream release of interferons, interleukins, and pro-inflammatory cytokines upon binding double-stranded RNA (dsRNA), uncapped RNA, or partially capped RNA (Decroly, Ferron, Lescar, & Canard, 2011). To address dsRNA-induced immunogenicity, modified NTPs such as pseudouridine (Ѱ), N1-methyl-Ѱ, 5-methoxyuridine, and others have been substituted for uridine bases to evade PRR activation (Karikó, Buckstein, Ni, & Weissman, 2005; Vaidyanathan et al., 2018). Alternatively, ion-pairing reversed-phase (IP-RP) HPLC methods (Karikó, Muramatsu, Ludwig, & Weissman, 2011) and/or cellulose chromatography (Baiersdörfer et al., 2019) can be used to clear dsRNA contaminants post IVT. To address the 5′-cap, the only structural element not directly derived from template DNA, two main approaches have been developed: multi-step enzymatic capping or co-transcriptional incorporation of cap analogs.

The 5′-cap structure consists of an N7-methylated guanosine (m7G) connected by a 5′- to 5′-O -triphosphate bridge to the first nucleotide (known as cap 0). An optional 2′-O -methylation of the +1 nucleotide's ribose sugar results in a cap 1 structure (^m7GpppN_2′OmeN), and 2′-O -methylation of the +1 and +2 ribose sugars results in a cap 2 structure (Topisirovic, Svitkin, Sonenberg, & Shatkin, 2011). Enzymatic reactions using the vaccinia capping enzyme can efficiently produce cap 0, which can be converted to cap 1 with an additional enzyme, 2′-O -methyltransferase. These reactions require the temperature-sensitive methyl donor S -adenosylmethionine, and occur after the T7 RNA polymerase IVT reaction (Ensinger et al., 1975). This second enzymatic reaction requires more product handling and longer incubation time at elevated temperature to denature any structure at the 5′-end of the molecule for enzyme access, which can lead to mRNA degradation.

Co-transcriptional capping with ARCA dimer produces a cap 0 structure and requires a limited GTP concentration in IVT to allow for the cap analog to outcompete the single nucleotide for initiation of the transcript (Stepinski, Waddell, Stolarski, Darzynkiewicz, & Rhoads, 2001). This results in lower reaction yield (approximately 3 mg/ml of reaction volume), lower mRNA quality (truncation of full-length product as limited NTP runs out), and inefficient 5′-capping that requires additional purifications to eliminate immunogenic 5′-triphosphate species from the final mRNA product (Hadas et al., 2019). In mammalian in vivo use, cap 1 typically results in higher gene expression (Furiuchi, 2015), while cap 2 mRNAs are still widely unstudied, without appropriate research tools (Werner et al., 2011). Thus, there is a need for scalable co-transcriptional cap 1 mRNA production in a single step.

Extending chemically synthesized cap analogs from dimers to trimers allows for direct cap 1 production with more flexibility for the +1 nucleotide base composition (U.S. Patent No. 20180273576; Hogrefe, Lebedev, McCaffrey, & Shin, 2018). Similarly, extending the cap analog from trimer to tetramer allows for the possibility of cap 2−initiated 5′-ends during IVT. CleanCap AG trimer allows for 94% or higher cap 1 mRNA synthesis in a one-pot reaction, with increased RNA yield from 3 to 5 mg/ml of IVT. With no limitation in NTP concentrations during transcription, the quality for full-length mRNA also increases compared to other methods, which often show truncated products as GTP substrates run out (Hadas et al., 2019).

Recent work from Sikorski et al. (2020) supports the concept of co-transcriptional trimeric cap analogs, with results showing increased gene expression by cap 1 versus cap 0 in certain cell types. However, template sequences were not appropriately adjusted for trimer hybridization and cap analogs incorporated at −1 and/or +2 positions relative to the “TATA” box. We find that cap efficiency improves when the DNA template is designed to hybridize with the cap analog at both the +1 and +2 positions (Fig. 2B).

Critical Parameters and Troubleshooting

Low product yield or quality

Component mixing and setup are critical for all IVTs to ensure reaction completion. Low mRNA yields and/or quality may occur if the reaction is not homogenized, a component is not homogenized before use, or a component is omitted. Of note, the reaction buffer requires fresh DTT for optimal enzyme activity. Oxidized buffer may contribute to low yields and/or low mRNA quality with increased production of truncated transcripts. Incomplete heat transfer may also contribute to low reaction yields; water baths with mixing are optimal. Avoid reaction inhibitors such as RNase and EDTA. Limit the amount of NaCl in IVT to less than 100 mM. Maintain pH at or below 8 for the duration of the reaction as well as any downstream processes to avoid alkaline hydrolysis of mRNA. Addition of DNase enzyme will immediately degrade the DNA template substrate for T7 RNA polymerase and should only be done after the allotted IVT reaction time.

The ratio of CleanCap reagent to NTPs during IVT is critical for complete capping as well as reaction yield of mRNA. Incomplete capping may occur if the reaction setup includes each NTP at a high molar excess over CleanCap, allowing NTPs to outcompete the CleanCap trimer for initiation (hybridization to template at the +1 and +2 positions). The appropriate ratio in Basic Protocol 1 is 5 mM each NTP to 4 mM cap analog.

DNA template sequence and quality will also influence mRNA quality. The template DNA sequence must include the full T7 promoter consensus sequence (underlined), followed by 5′-AG (TAATACGACTCACTATAAG). The 3′-A in the “TATA” box does not serve as the initiation site. Legacy transcription templates designed for enzymatic or ARCA capping must be mutagenized to initiate with a 5′-AG. Should a highly structured sequence such as a T7 termination site or thermodynamically favored hairpin exist in the template, creating a block in polymerization, truncation of the mRNA may occur. Optimal template design may need to be determined empirically. Confirm that the A ₂₆₀/A ₂₈₀ purity ratio of DNA is close to 1.8. A lower ratio may indicate residual phenol, which absorbs maximally at 280 nm. If a longer- or shorter-than-expected RNA molecule is observed after IVT, ensure that the DNA template is of the appropriate size. Incomplete linearization may contribute to longer RNA molecules, as the T7 RNA polymerase will continue to read through any undigested DNA template available. Shorter-than-expected molecules may arise from truncated DNA templates inadvertently formed by multiple cut sites in a plasmid digest, or multiple primer-binding sites in PCR.

RNA integrity

RNA can be difficult to work with, as it is less stable than DNA and readily degraded by ribonucleases, commonly referred to as RNases. RNases can be introduced through reagents, tips, tubes, bottles, surfaces, and human contact. As discussed in Strategic Planning, only use RNase-free consumables. Always wear gloves during setup, transcription, and handling of mRNA product. Change gloves often. Surfaces and pipettes can be wiped down with RNaseZap or equivalent RNase cleaning solution to destroy RNases.

Planning for necessary purifications and aliquotting is critical to maintaining integrity of the molecule. It is critical to limit handling and hold times throughout this process. Reduce the number of processing steps and handling time of the mRNA following IVT. Avoid freeze-thaws and prepare single-use aliquots for storage of final product.

mRNA purification

It is essential that the transcription reaction be quenched and purified to obtain mRNA free of proteins, DNA, NTPs, and buffer components such as magnesium, which may cause phosphodiester cleavage at elevated temperatures. Residual DNA and proteins may interfere with downstream applications of the mRNA, while residual free NTPs will result in inflated quantity measurements by UV spectrometry. Removal of dsRNA contaminants is essential for in vivo use, to prevent immunostimulatory reactions (Baiersdörfer et al., 2019; Karikó et al., 2011).

Precipitations are a manual purification method that may require a practiced technique to avoid loss of material. Alternative purification methods include silica membrane columns, preparative HPLC, or oligo(dT) capture of poly(A) tails. Silica membrane purification kits such as RNeasy (Qiagen, cat no. 75162) treat RNA solutions with ethanol and chaotropic salt to selectively bind nucleic acids, allowing salts, free NTPs, and proteins to fall through in a shorter protocol than lithium chloride precipitation (see Current Protocols article: Dowhan, 2012). Such membranes will retain intact template DNA; removal requires DNase I treatment post-transcription. Immobilized oligo(dT) will bind the poly(A) tail of mRNA. This method results in high purity of full-length samples, with most truncated mRNA molecules washed away. Typically, immobilized oligo(dT) resin and mRNA samples are equilibrated in high-salt buffer (∼0.5 M NaCl) to prevent repulsion between negatively charged phosphate backbones. mRNA is applied to the resin, and its poly(A) tails form hydrogen bonds to the oligo(dT) for immobilization via canonical base pairing. An intermediate salt wash (∼0.1 M NaCl) removes unbound impurities, and purified mRNA is eluted with low-salt buffer or water (Aviv & Leder, 1972; Green & Sambrook, 2019). Commercial kits suitable for small-scale oligo(dT) purifications using spin columns or magnetic beads are widely available (for example, Qiagen, cat no. 70022, NEB, cat no. E7490, or Thermo Fisher, cat no. AM1922.)

mRNA analysis

Depending on the application, additional or more sensitive analysis of IVT-produced mRNA beyond what is presented in this article may be necessary. These may include cap efficiency, sequence verification, residual solvents, residual DNA, dsRNA content, and/or poly(A) tail length confirmation. Capping efficiency may be assayed in various ways. However, the most sensitive methods employ mass spectrometry (Efimov et al., 2001; Galloway et al., 2020; Hengesbach, Meusburger, Lyko, & Helm, 2008; Sikorski et al., 2020; Wang et al., 2019).

Alternative techniques for interrogating mRNA length and quality with improved sensitivity over gels include capillary electrophoresis and analytical HPLC. Capillary gel electrophoresis requires more elaborate instrumentation such as a Bioanalyzer or Fragment Analyzer (Agilent part number G2939BA or M5310AA, respectively). The RNA sample is injected into fused silica capillaries filled with a sieving gel, generally polymer solutions such as linear polyacrylamide or hydroxyethylcellulose. Voltage is applied along the length of the capillaries, and the RNA migrates to the anode through the medium. Entanglement between polymers impedes RNA migration, effecting size-based separation. Intercalating fluorophores present in the gel allow quantitative detection when RNA molecules reach a detector positioned near the capillary ends. The result is a plot of fluorescence signal against time, termed an electropherogram. RNA ladders are run alongside samples to establish a relationship between migration time and RNA size, enabling quantification of test sample fragment sizes. mRNA quality can be quantified by comparing fluorescence signal from full-length transcripts to that of degradation products or other contaminants (Todorov, de Carmejane, Walter, & Morris, 2001).

In principle, analytical HPLC is achievable by any chromatographic technique capable of resolving mRNAs by size. The two most established techniques are IP-RP HPLC and size-exclusion chromatography (SEC). IP-RP binds mRNA samples to a hydrophobic resin, including polymers such as polystyrene-divinylbenzene, C18 or other hydrophobic ligands on silica supports, or a combination of the two. The mobile phase contains an ion-pairing reagent, usually an alkylamine such as triethylamine or n -hexylamine, which mediates interaction between the RNA phosphate backbone and the hydrophobic stationary phase. A gradient of organic solvent such as acetonitrile will then elute the RNA in order of increasing size. SEC applies RNA samples to a column packed with inert porous beads. Smaller RNA exhibits slower linear flow down the length of the column because it diffuses into the beads more readily. SEC and IP-RP generally employ UV-vis detectors measuring absorbance at 260 nm (Azarani & Hecker, 2001; Fujii et al., 2014).

Understanding Results

UV-vis measurement of RNA

The expected reaction yield (mass of RNA per volume of IVT) of purified CleanCap AG mRNA using this protocol is 5 mg/ml, with an absorbance maximum at 260 nm and a value of 2.0-2.2 for A ₂₆₀/A ₂₈₀ ratio. Figure 5 shows a typical IVT sample post purification with peak absorbance at 260 nm and purity ratio of 2.15 (A ₂₆₀/A ₂₈₀). If no absorbance peak is observed at 260 nm, there is little or no RNA in the sample. If traditional cuvette measurements are done in lieu of a NanoDrop, the optimal dilution factor will vary by sample concentration and cuvette pathlength. Refer to the instrumentation manual to ensure an accurate reading within the instrument's quantitative range. If a large dilution factor is used for cuvette reading, RNA concentration may be below the instrument's limit of detection. Try measuring more samples to validate the original reading.

If reaction yield is truly lower than expected, compare crude IVT (sample retained in Basic Protocol 1, step 12) to purified mRNA by gel electrophoresis. This comparison can be used to determine if the RNA was lost during purification or if the IVT reaction was unsuccessful. For gel-loading purposes, assume a crude IVT yield of 5 mg/ml and load equal masses for crude and purified samples. In the gel image following electrophoresis, the band intensity correlates with the mass load. If a dark band is observed in crude sample but not in purified sample, then the RNA may have been lost during precipitation. If a band of lower intensity is observed in crude IVT, then a suboptimal reaction likely occurred. See Troubleshooting guidelines above to improve IVT reaction.

An A ₂₆₀/A ₂₈₀ ratio of 1.8 or lower may indicate residual DNA or protein. The mRNA may not be pure enough for downstream applications. Another round of purification or an alternative purification method is recommended.

Interpretation of gel image

Relative to an appropriate size marker, purified RNA in a glyoxal gel will migrate close to true size (length in nucleotides). A gel band at unexpected size can indicate a variety of issues, such as contamination, incomplete template digestion, or incomplete IVT reaction. A typical glyoxal gel of CleanCap mRNA is shown in Figure 6.

A gel band at expected size with lower-molecular-weight smears indicates degradation of the RNA sample, while higher-molecular-weight smearing can occur in unpurified samples due to residual salts. Lower-molecular-weight smearing is also observed when band intensity is increased by gel overloading. Longer RNA molecules are generally less stable, because the probability of at least one phosphodiester bond break increases with the number of bonds, i.e., RNA length. Thus, lower-molecular-weight smearing is more prevalent for longer RNAs. Significant lower- and higher-molecular-weight smearing is observed in the crude samples shown in Figure 6, lanes 4-6. Following sample purification, these smears are typically reduced, and sample analysis shows a clean single band as shown in Figure 6, lanes 10 (purified sample shown in lane 5) and 11 (purified sample shown in lane 6).

A lack of denaturation, i.e., analysis of RNA by native gels, will result in relatively higher mobility, producing bands that run at lower-than-expected size and/or multiple bands.

Time Considerations

IVT, purification, and analysis can be done in 1-2 days. IVT setup will take approximately 1 hr, including reagent thaw time. The IVT reaction will take 2-3 hr, and optional DNase I treatment will take approximately 30 min. Purification by LiCl may be split over 2 days, with samples incubating overnight at −20°C. UV measurements are completed in 10 min or less, and gel analysis may range from 30 to 90 min depending on gel size and precast gel options.

Acknowledgments

We would like to thank and acknowledge Drs. Richard Hogrefe, Alexandre Lebedev, Anton McCaffrey, and Dongwon Shin for their contributions to the invention of CleanCap^®. We thank Dr. Krist Azizian and Sabrina Shore for their previous contributions toward the development of this procedure.

Author Contributions

Jordana M. Henderson : Investigation; methodology; visualization; writing-original draft; writing-review & editing. Andrew Ujita : Investigation; validation; writing-original draft; writing-review & editing. Elizabeth Hill : Visualization; writing-original draft. Sally Yousif-Rosales : writing-original draft. Cory Smith : writing-original draft. Nicholas Ko : writing-original draft. Taylor McReynolds : writing-original draft. Charles R. Cabral : Supervision. Julienne R. Escamilla-Powers : Data curation; methodology; resources; supervision; validation; writing-review & editing. Michael E. Houston : Resources; supervision; writing-review & editing.

Conflict of Interest

The authors are employees of TriLink Biotechnologies, LLC.

Data Availability Statement

Data sharing not applicable–no new data generated.

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In this publication, the following corrections have been made:

An error was corrected for the concentration of Triton X–100 in the recipe for 10× in vitro transcription (IVT) buffer in the Reagents and Solutions section, which now reads 0.02% (not 0.2%).

Author's conflict of interest and data availability statement have been added.

The current version online now includes this correction and may be considered the authoritative version of record.

Number of times cited according to CrossRef: 112

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