In vitro transcription (IVT)

A: Very clean template DNA ensures the best performance of an in vitro transcription reaction. If using a PCR product for the template, purify the desired product from the reaction to remove any remaining primers, primer-dimers and residual dNTPs. If you are using a plasmid for the template, in order to transcribe the desired RNA product, completely linearize the plasmid, leaving a blunt or 5′-overhanging end. Uncut plasmid serves as excellent template, but the RNA polymerase will transcribe past the desired transcription stop point and can continue around the plasmid several times before the reaction finally stops, creating an RNA that is far longer than desired and includes undesirable vector sequences.

A: Optimal conditions for in vitro transcription can vary depending on the specific requirements of your experiment, but here are some general guidelines:

1. DNA Template: Use a purified, linear DNA template with a promoter sequence (e.g., T7, T3, or SP6).
2. RNA Polymerase: Select the appropriate RNA polymerase based on your promoter sequence.
3. Nucleotide Mix: Include ribonucleotide triphosphates (NTPs) in your reaction mix. If using modified nucleotides, replace some or all of the canonical NTPs with the modified versions.
4. Buffer System: Use a buffer that provides the optimal pH (typically around 7.5-8.0), ionic strength, and necessary cofactors like magnesium ions (Mg²⁺). Dithiothreitol (DTT) is often included to maintain a reducing environment.
5. Reaction Temperature: Conduct the transcription reaction at 37°C, which is optimal for most RNA polymerases.
6. Incubation Time: Typically, the reaction is incubated for 1-2 hours. However, this can be adjusted based on the yield and length of RNA required.
7. Additional Components: Include ribonuclease inhibitors to protect the RNA from degradation and inorganic pyrophosphatase to prevent the accumulation of inhibitory pyrophosphate.
8. Purification: After transcription, purify the RNA to remove unincorporated nucleotides, enzymes, and other reaction components

A: Yes, RNA synthesis can be carried out with a mixture of modified nucleotides in place of the canonical mixture of A, G, C and U triphosphates. If using modified nucleotides, replace some or all of the canonical NTPs with the modified versions. CELLSCRIPT™ offers kits containing N1-methyl-pseudouridine, pseudouridine, and/or 5-methyl-cytidine in the place of U and C, respectively.

A: Using modified nucleotides in mRNA synthesis offers several advantages:

1. Increased Stability: Modified nucleotides can enhance the stability of mRNA, making it less prone to degradation. This is crucial for ensuring that the mRNA remains intact long enough to be translated into protein.
2. Reduced Immunogenicity: Unmodified mRNA can trigger immune responses, leading to inflammation and reduced protein production. Modifications can help mRNA evade the immune system, reducing these unwanted reactions.
3. Improved Translational Efficiency: Certain modifications can enhance the efficiency with which mRNA is translated into protein. This means that more protein can be produced from the same amount of mRNA.
4. Enhanced Therapeutic Potential: These modifications make mRNA a more viable option for vaccines and gene therapies, as seen with the success of mRNA-based COVID-19 vaccines.
5. Avoidance of Genomic Integration: Unlike DNA-based therapies, mRNA does not integrate into the genome, reducing the risk of insertional mutagenesis.

These benefits make modified mRNA a powerful tool in modern medicine, particularly for developing new vaccines and treatments for various diseases.

A: To be functional, mRNA needs modified 5’ and 3’ ends, along with a coding region (an open reading frame or ORF) that encodes the desired protein, flanked by untranslated regions (UTRs). The newly formed mRNA (pre-mRNA) undergoes two major modifications besides splicing.

• During its synthesis, a 7-methylguanylate structure, known as a “cap,” is added to the 5’-end of the pre-mRNA through a 5’-5’ pyrophosphate linkage. This cap protects the mature mRNA from degradation and plays a crucial role in its export from the nucleus and efficient translation.
• Addition of a poly(A) tail at the 3′-end of the transcript enhances the translation efficiency of the mRNA into proteins by facilitating the binding of ribosomes via poly(A) binding protein and eukaryotic translation initiation factors. It also helps in stability of the mRNA as well as its transportation to the cytoplasm.

A: For in vitro transcribed RNA to be translated efficiently in vivo, the mRNA requires:

• A poly(A) tail at the 3′-end of the transcript.
• A correctly orientated N7-methylated cap (Cap 0) at the 5′-end of the molecule. However, the addition of another methyl group onto the penultimate nucleotide from the 5′-end of the mRNA (producing a Cap 1 structure) will further boost translation.