In Vitro Transcriptional Synthesis of mRNA

Summary

In vitro transcription (IVT) is the synthesis of RNA molecules, allowing researchers to customize the synthesis processes and introduce modifications to produce transcripts. It is worth spending some time optimizing mRNA template design, IVT, and other process parameters to simplify downstream processing and ensure a proper balance of yield and secondary structure formation. The synthetic mRNA should contain four important structural elements: 5'-cap, untranslated regions (UTRs), open reading frame (ORF), and poly (A) tail at the 3'-end, they play essential roles in mRNA stability, subcellular localization, and translational efficiency.

The use of mRNA synthesized in vitro has played an important role in the development of RNA vaccines and CRISPR/Cas9 genome editing tools, the generation of pluripotent stem cells, and the development of diagnostic techniques based on RNA amplification as well as gene therapy.

Enzymatic Method

1. Prepare plasmid DNA (pDNA) or circular DNA templates for IVT

Synthetic mRNAs can be prepared by IVT of circular DNA templates or plasmid DNA (pDNA) using bacteriophage RNA polymerases (T7, SP6, T3). The design and construction of a DNA template is the first step in the preparation of IVT mRNA. A complete DNA template for IVT requires at least four elements: the bacteriophage promoter, UTR, ORF, and poly (T) sequence. The IVT reaction solution contains four types of nucleoside triphosphates (NTPs), RNA polymerase, DNA template, RNase inhibitor (prevents degradation of newly formed mRNA), and pyrophosphatase (pyrophosphatases must be added to catalyze the hydrolysis of pyrophosphates into inactive single orthophosphate ions that don't bind magnesium ions, which increases the yield of mRNA). For co-transcription capping, the IVT reaction solution also contains cap analogs.

2. mRNA In vitro transcription and capping

Bacteriophage RNA polymerase is normally used to transcribe linearized plasmid DNA. The pDNA is first linearized with the selected unique restriction site enzyme. After digestion, the linearized DNA may be purified using methods such as the phenol-chloroform protocol. For large-scale purification, tangential flow filtration (TFF) is often advisable, as it can be easily scaled up. Following linearization, the templates are subsequently amplified by PCR, in this step, poly (A) tail is added by PCR using primers with poly (T). IVT and capping processes are performed in a mixed solution of recombinant RNA polymerase (T7, T3, or SP6) and nucleoside triphosphates, plus a cap analog.

Appropriate cap analogs and chemically modified/unmodified nucleotides were added during IVT. RNA polymerase (T7, T3, or SP6) transcribes DNA templates using added nucleotides and cap analogs. Capping and chemical modification of IVT mRNA can be easily modulated by the addition of different components and ratios of nucleotides. Following IVT, the original DNA template was digested by DNase treatment and the 5' triphosphate group of mRNA was removed using heat-labile antarctic phosphatase.

3. Purification and analysis of mRNA

The first step in mRNA purification is to remove the linearized DNA template using deoxyribonuclease, or DNase. The resulting mRNA is treated with heat-labile antarctic phosphatase to remove the 5'-triphosphate since phosphatase treatment prevents re-circularization in a self-ligation reaction and reduces the possible immune response. In addition, cellulose-based purification using cellulose fibers can be applied for the removal of double-stranded RNA, a transcriptional by-product that is a major concomitant of mRNA synthesis.

Agarose gel electrophoresis can check for any potential degradation of mRNA, while concentration can be determined by fluorescence-based, RNA-specific assays.

Fig. 1 Preparation of synthetic mRNA by in vitro transcription (IVT). (Kwon H, 2018)

Chemical Synthesis Coupled with Enzymatic Modification

1. Chemical synthesis of the mRNA sequence in the solid phase with 2-cyanoethoxymethyl (CEM) as the 2'-hydroxy protecting group
2. Pyrophosphorylation of the synthetic RNA at the 5'-end on the solid support
3. Cleavage of the 5'-pyrophosphorylated RNA from the solid support followed by enzymatic capping
4. Enzymatic polyadenylation of the 5'-capped RNA at the 3'-end. A 130-nt mRNA sequence synthesized by this method is biologically active that can encode a 33-amino acid peptide including the sequence of glucagon-like peptide-1 (GLP-1)

Fig. 2 Enzymatic conversion of 130-mer to artificial mRNA. (Nagata S, 2010)