High Efficiency Synthetic mRNAs

Synthesis of custom-designed, highly modified mRNAs

Applications

• High efficiency protein synthesis in vitro by translation in rabbit reticulocyte lysate
• High efficiency of protein synthesis in living cells, plus a bypass of innate antiviral responses

Modified mRNAs as templates for in vitro translation

Ψ-containing mRNA is translated more efficiently than its unmodified counterpart, which results in higher protein expression.
In addition to the well-known benefit of 5‘-capped mRNAs for mammalian in vitro translation systems, the internal base modifications have also significant effects.

Please note: not all base modifications are beneficial.

The translational yield of mRNAs with m5U or m5C is similar to the unmodified transcripts, while mRNAs containing s2U are translated poorly. The presence of m6A in mRNAs has completely abolished their translatability.
Further more, the Ψ-mediated translational enhancement is not universal for all translational systems.
In wheat germ extracts, ~50% less protein is produced from the Ψ-containing mRNAs compared with unmodified mRNAs, while in bacterial cell lysates, mRNAs with Ψ-modifications are not translated.

Reference: Karikó et al. (2008) Incorporation of Pseudouridine (Ψ) Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability. Mol Ther. 16(11): 1833-1840.

Efficient reprogramming of human cells with synthetic, highly modified mRNAs

For more information visit our synthetic mRNA for directed stem cell differentiation page.

The Full Service includes: Synthesis of synthetic DNA, plasmid cloning, purification, full DNA sequencing documentation, PCR amplification of insert, purification and characterisation with subsequent RNA synthesis by in vitro transcription, purification and characterisation.
The customer provides text files with the desired sequence data.

Production steps:

1. Chemical synthesis of dsDNA fragments
2. Cloning of the DNA fragments in a plasmid
3. Quality control by sequencing of plasmid inserts
4. Synthesis of PCR primers, based on the plasmid sequence, for PCR amplification of the cloned insert sequence. If desired, a 3’-Poly(A) tail can be included.
5. PCR amplification of the cloned insert with the primers described in step 4
6. Purification of the PCR products with spin columns
7. Quality control of PCR products by capillary electrophoresis (Agilent Bioanalyzer)
   
   Electropherogram of DNA template with Agilent Bioanalyzer
   Peaks at 50 bp and at 10,380 bp are internal reference size markers. The template DNA is a homogeneous product.






8. PCR templates for production of ssRNA by in vitro transcription with T7 RNA polymerase
9. DNase treatment for removal of the DNA template
10. Purification of ssRNA with spin columns
11. Quality control of the in vitro transcripts by capillary electrophoresis (Agilent Bioanalyzer)
    
    Electropherogram of RNA products with Agilent Bioanalyzer Peak at 25 nt is an internal reference size marker. Red RNA profile: unmodified RNA transcript Blue RNA profile: fully modified RNA transcript with 5‘ terminal cap and internal base modifications, m5C (100%) and Ψ (100%). A distinct mobility shift is visible.

Please contact us at info@amsbio.com for more information.