In Vitro Transcribed mRNA: Structure, Synthesis, Purification, and Analysis

Messenger RNA (mRNA) is a type of RNA molecule that carries genetic information from DNA to the sites of protein synthesis in the cytoplasm, namely the ribosomes. The structure and function of mRNA are essential for the expression and regulation of genes in all living organisms. However, the natural mRNA molecules are often unstable, degraded, or immunogenic, limiting their potential applications in research and therapeutics. Therefore, there is a need to synthesize mRNA molecules in vitro, using DNA templates and RNA polymerases, to produce customized and modified RNA molecules that can overcome these challenges. In vitro transcribed (IVT) mRNA has been widely used for various purposes, such as vaccines, protein replacement, induced pluripotent stem cells, and cancer immunotherapy.

Structure of IVT-mRNA

The structure of IVT-mRNA resembles endogenous mRNA, consisting of a 5' cap, 5' untranslated region (5' UTR), open reading frame (ORF), 3' untranslated region (3' UTR), and poly A tail structure in the 5' to 3' direction. The 5' cap is a modified guanine nucleotide that protects the mRNA from degradation and facilitates its recognition by the ribosome. The 5' UTR contains regulatory elements that affect mRNA stability, translation efficiency, and subcellular localization. The ORF encodes the desired protein sequence, which can be optimized for codon usage, GC content, and secondary structure formation. The 3' UTR also contains regulatory elements that influence mRNA stability, translation efficiency, and subcellular localization. The poly A tail is a stretch of adenine nucleotides that enhances mRNA stability, translation efficiency, and nuclear export.

Fig.1 Structural features of IVT mRNA. ¹

The IVT-mRNA can be modified by various methods to improve its performance and reduce its immunogenicity. For example, the 5' cap can be added by either a multi-step enzymatic reaction or a co-transcriptional method, resulting in different cap structures (Cap 0 and Cap 1) that have different effects on the mRNA properties. The NTPs can be substituted by modified nucleotides, such as pseudouridine or 1-methylpseudouridine, that reduce the recognition of the mRNA by the innate immune system. The 5' and 3' UTRs can be designed by using synthetic or natural sequences or by incorporating RNA elements, such as internal ribosome entry sites (IRES), stem-loops, or microRNA target sites, that modulate the mRNA function. The poly A tail can be varied in length and composition, affecting the mRNA stability and translation.

Synthesis of IVT-mRNA

The synthesis of IVT-mRNA involves the transcription of the DNA template into RNA molecules by RNA polymerases. The DNA template can be a plasmid, a cDNA, or a PCR product, which consists of a promoter, a 5' UTR, a cDNA, a 3' UTR, a poly A tail, and a unique cleavage site. The RNA polymerases can be T7, T3, or SP6, which recognize different promoters and have different transcription rates and fidelity. The transcription reaction requires the following components:

• DNA template: The DNA template provides the sequence of interest to be transcribed into RNA. The DNA template should be linearized by a restriction enzyme or PCR amplification and purified by gel extraction or column purification. The DNA template should be free of contaminants, such as proteins, salts, or organic solvents, that may inhibit the transcription reaction. The DNA template should be quantified by a spectrophotometer or a fluorometer and diluted to an appropriate concentration for the transcription reaction.
• RNA polymerase: The RNA polymerase is the enzyme that catalyzes the synthesis of RNA from the DNA template. The RNA polymerase should be compatible with the promoter of the DNA template and have a high specificity and activity for the transcription reaction. The RNA polymerase should be stored at -20°C and thawed on ice before use. The RNA polymerase should be added to the transcription reaction at an optimal concentration, depending on the type of polymerase and the length of the DNA template.
• NTPs: The NTPs are the substrates that provide the nucleotides for RNA synthesis. The NTPs should be of high purity and quality, and free of contaminants, such as RNases, DNases, or pyrophosphates, that may degrade or inhibit RNA synthesis. The NTPs should be stored at -20°C and thawed on ice before use. The NTPs should be added to the transcription reaction at an optimal concentration, depending on the type of polymerase and the length of the DNA template. The NTPs can be modified by adding cap analogs or modified nucleotides, such as pseudouridine or 1-methylpseudouridine, that can improve the stability and functionality of the IVT-mRNA.
• RNase inhibitor: The RNase inhibitor is a protein that prevents the degradation of newly synthesized RNA by RNases. The RNase inhibitor should be of high purity and quality and free of contaminants, such as RNases, DNases, or pyrophosphates, that may degrade or inhibit the RNA synthesis. The RNase inhibitor should be stored at -20°C and thawed on ice before use. The RNase inhibitor should be added to the transcription reaction at an optimal concentration, depending on the type of polymerase and the length of the DNA template.
• Pyrophosphatase: The pyrophosphatase is an enzyme that hydrolyzes the pyrophosphates that are released during RNA synthesis. The pyrophosphates can bind to the free magnesium ions and inhibit RNA polymerization. The pyrophosphatase should be of high purity and quality and free of contaminants, such as RNases, DNases, or pyrophosphates, that may degrade or inhibit the RNA synthesis. The pyrophosphatase should be stored at -20°C and thawed on ice before use. The pyrophosphatase should be added to the transcription reaction at an optimal concentration, depending on the type of polymerase and the length of the DNA template.
• IVT buffer: The IVT buffer is a solution that provides the optimal pH and salt conditions for RNA synthesis. The IVT buffer should be of high purity and quality and free of contaminants, such as RNases, DNases, or pyrophosphates, that may degrade or inhibit RNA synthesis. The IVT buffer should be stored at -20°C and thawed on ice before use. The IVT buffer should be added to the transcription reaction at an optimal volume, depending on the type of polymerase and the length of the DNA template. The IVT buffer should contain an optimal concentration of magnesium ions, which are essential for RNA polymerization.

Fig.2 Synthesis of IVT-mRNA. ²

The transcription reaction should be performed in a sterile and RNase-free environment, using RNase-free tubes, pipettes, and tips. The transcription reaction should be mixed well by vortexing or pipetting and incubated at an optimal temperature and time, depending on the type of polymerase and the length of the DNA template. The transcription reaction should be stopped by adding EDTA or heating and purified by a gel extraction, a column purification, or a chromatography method. The transcription reaction should be analyzed by gel electrophoresis, a spectrophotometer, or a fluorometer to determine the yield, quality, and functionality of the IVT-mRNA.

Purification of IVT-mRNA

The purification of IVT-mRNA is the process of removing impurities from the transcription reaction, such as DNA template, RNA polymerase, NTPs, RNases, proteins, salts, and truncated transcripts. The purification of IVT-mRNA is essential for ensuring the quality, functionality, and safety of the mRNA product. The purification methods can be classified into three categories: gel-based, column-based, and chromatography-based.

• Gel-based purification: Gel-based purification separates the IVT-mRNA from the impurities based on their size and charge, using a gel electrophoresis system. The IVT-mRNA is loaded onto a denaturing polyacrylamide gel, which is subjected to an electric field. The IVT-mRNA migrates through the gel according to its molecular weight, while the impurities are retained or migrate at different rates. The IVT-mRNA band is visualized by staining or UV illumination and excised from the gel. The IVT-mRNA is then eluted from the gel slice by using a buffer or a solvent and precipitated by adding ethanol or isopropanol. Gel-based purification can achieve high purity and resolution of IVT-mRNA, but it has some drawbacks, such as low recovery, high risk of contamination, long processing time, and limited scalability.
• Column-based purification: Column-based purification separates the IVT-mRNA from the impurities based on their affinity, using a spin column or a filter device. The IVT-mRNA is loaded onto the column, which contains a solid phase that binds to the IVT-mRNA or the impurities. The column is then washed with a buffer or a solvent, which removes the unbound or weakly bound impurities. The IVT-mRNA is then eluted from the column by using a buffer or a solvent, which disrupts the binding between the IVT-mRNA and the solid phase. Column-based purification can achieve high recovery and speed of IVT-mRNA, but it has some drawbacks, such as low purity, variable size cut-offs, and limited loading capacities.
• Chromatography-based purification: Chromatography-based purification separates the IVT-mRNA from the impurities based on their physical or chemical properties, using a chromatography system. The IVT-mRNA is loaded onto a column, which contains a stationary phase that interacts with the IVT-mRNA or the impurities. The column is then eluted with a mobile phase, which changes the interaction between the IVT-mRNA and the stationary phase. The IVT-mRNA and the impurities are separated by their different retention times or elution profiles and collected by a detector or a fraction collector. Chromatography-based purification can achieve high purity, resolution, and scalability of IVT-mRNA, but it has some drawbacks, such as high cost, complexity, and optimization.

Table 1. Comparison of IVT-mRNA Purification Methods

Analysis of IVT-mRNA

The analysis of IVT-mRNA is the process of evaluating the quality and functionality of the purified mRNA product using various methods and techniques. The analysis of IVT-mRNA is important for ensuring the accuracy, efficiency, and safety of the mRNA applications. The analysis methods can be classified into four categories: concentration, size, integrity, and activity.

• Concentration: The concentration of IVT-mRNA is the amount of mRNA per unit volume, usually expressed in nanograms per microliter (ng/μL). The concentration of IVT-mRNA can be measured by fluorescence-based, RNA-specific assays which use fluorescent dyes that bind to RNA and emit signals proportional to the amount of RNA. The concentration of IVT-mRNA can also be estimated by spectrophotometry, which measures the absorbance of RNA at 260 nm and uses a conversion factor of 40 ng/μL per 1 OD260 unit. However, spectrophotometry is less accurate and sensitive than fluorescence-based assays and can be affected by contaminants such as proteins, salts, and organic solvents.
• Size: The size of IVT-mRNA is the length of the mRNA molecule, usually expressed in nucleotides (nt) or kilobases (kb). The size of IVT-mRNA can be determined by gel electrophoresis, which separates the mRNA molecules based on their size and charge using a denaturing polyacrylamide gel or an agarose gel. The size of IVT-mRNA can also be determined by capillary electrophoresis which separates the mRNA molecules based on their size and charge, using a capillary filled with a sieving matrix. Capillary electrophoresis is more sensitive, accurate, and reproducible than gel electrophoresis, and can analyze longer mRNA molecules up to 10 kb.
• Integrity: The integrity of IVT-mRNA is the degree of completeness and purity of the mRNA molecule, which reflects its stability and functionality. The integrity of IVT-mRNA can be assessed by gel electrophoresis or capillary electrophoresis, which can detect any potential degradation, truncation, or contamination of the mRNA molecule. The integrity of IVT-mRNA can also be assessed by nuclear magnetic resonance (NMR) or mass spectrometry (MS), which can detect any potential modification, such as capping, methylation, or pseudouridylation, of the mRNA molecule. NMR and MS are more sensitive, specific, and comprehensive than gel electrophoresis or capillary electrophoresis, but they are also more complex, costly, and time-consuming.
• Activity: The activity of IVT-mRNA is the ability of the mRNA molecule to be translated into the desired protein, which reflects its efficiency and safety. The activity of IVT-mRNA can be measured by in vitro translation assays, which use cell-free systems, such as rabbit reticulocyte lysate or wheat germ extract, to synthesize the protein from the mRNA. The activity of IVT-mRNA can also be measured by in vivo translation assays, which use living cells, such as bacteria, yeast, or mammalian cells, to express the protein from the mRNA. The activity of IVT-mRNA can be quantified by detecting the protein level using methods such as enzyme-linked immunosorbent assay (ELISA), western blot, or fluorescence microscopy.

References

1. Verbeke, Rein, et al. "Three decades of messenger RNA vaccine development." Nano Today 28 (2019): 100766.
2. Rouf, Nusrat Zahan, et al. "Demystifying mRNA vaccines: an emerging platform at the forefront of cryptic diseases." RNA biology 19.1 (2022): 386-410.