Custom mRNA Synthesis
Polyadenylated and capped mRNA offers the distinct advantage of integration-free gene expression. A range of modified nucleotides for reduced immune stimulation, enhanced translation and increased nuclease stability are available. TriLink's custom mRNA transcription service offers some exceptional advantages including:
- Custom tailored support to meet specific application or program needs from template amplification to a wide variety of modifications, treatments and purification options.
- High quality capped mRNA at highly competitive prices due to in-house manufacturing of key raw materials.
- Wide range of transcription RNA synthesis scales yielding µgrams to hundreds of milligrams.
- Fully traceable documentation system.
In a transcription reaction, an RNA polymerase produces many copies of RNA by transcribing a DNA template which encodes the RNA. RNA transcript synthesis is an alternative to chemical RNA synthesis and is useful when a long and/or modified capped mRNA is needed.
Yields of chemically-synthesized RNA decrease exponentially with increasing length because coupling efficiencies at each step are 90-99% (depending on the type of incorporated base). Thus it is not feasible to synthesize very long RNA chemically. In contrast, bacteriophage polymerases, such as T7 RNA polymerase, are quite processive and it is therefore possible to obtain RNA by transcription that are thousands of bases long and migrate as a single band on a denaturing gel.
In cells, the ribosome translates mRNA into proteins. mRNA in eukaryotic cells has either a 5' Cap 1 or Cap 2 [m7G(5')ppp(5')G] structure which stabilizes the mRNA and enhances translation. A cap can be incorporated in the mRNA transcription by including a mixture of cap analog and NTPs. Greater than 90% of transcribed mRNA will possess a 5' cap, while the remaining mRNA will possess a 5' triphosphate. In some cases it is desirable to remove the 5' triphosphate with a phosphatase treatment.
The first cap analog to be introduced was mCAP [m7G(5')ppp(5')G]. 50% of the time, mCAP is inserted in the correct orientation to enhance translation. The other 50% of molecules are not substrates for efficient translation, reducing the specific activity of the RNA transcript. One of the drawbacks to mCAP is it results in a 5' Cap 0 structure. The second cap analog introduced was Anti-Reverse Cap Analog (ARCA) [3' O-Me-m7G(5')ppp(5')G](1). ARCA can only insert in one orientation, resulting in 70% capped mRNAs. The increased yield over mCAP was a benefit, but ARCA only resulted in a 5' Cap 0 structure.
The current cap analog used is CleanCap®. CleanCap enables high capping efficiencies (90%+) resulting in more biologically active mRNA per transcription. It also produces a natural Cap 1 structure, plus Cap 2 analogs will be available soon (other modifications are possible too, contact us).
Efficient translation of the mRNA into protein also requires a poly(A) tail. This can be efficiently introduced by including a poly(dT) stretch at the end of the transcription template. This can also be accomplished by an additional post-transcription PCR step that utilizes a primer containing the poly(dT) stretch.
An mRNA transcription template containing a bacteriophage promoter, or a plasmid or PCR product containing the sequence of interest may be submitted. TriLink will amplify the plasmid or PCR product and add the required elements (promoter and poly(A) tail, if required) to the PCR primers.
Scales & Yields
Synthesis scales yielding µgrams to hundreds of milligrams are available. Quotes are based on mRNA transcription starting synthesis scale due to the high degree of variability in transcription rna synthesis yields from template to template. TriLink will make a conservative estimate of the scale necessary to obtain the quantity required. All capped mRNA produced will be shipped.
A table of transcription RNA synthesis expected yields for a 1-2 kilobase transcript is below. Final yields are not guaranteed, variations can be caused by many factors including sequence, length and base composition. Incorporation of modified bases will also affect final yield. Some modifications, like 5-Methyl-C have little effect upon final yield while 2' Fluoro modifications can have a large effect.
|Transcription RNA Synthesis Scale*
|| Capped mRNA
||0.5 - 1.5 mg
||1 - 3 mg
||2 - 6 mg
||3 - 9 mg
||5 - 15 mg
*Please inquire for smaller scales.
Silica-gel Membrane Spin Column Purification: RNA can be bound to silica-gel membranes, washed and eluted in water or TE pH 7. This process removes most proteins, DNA and NTPs.
Additional Enzymatic Treatments
DNase Treatment: For many applications, it is desirable to remove the DNA transcription template following mRNA transcription synthesis. DNase treatment degrades the DNA template into smaller fragments that can then be removed in subsequent purification steps.
Phosphatase Treatment: Transcription leaves a 5′ triphosphate at the end of the RNA. RNA with a terminal triphosphate induces innate immune responses in mammalian cells by activating RIG-I. In other applications, it may be desirable to 5′ end label the RNA using T4 polynucleotide kinase. This requires removal of the 5′ triphosphate. Therefore, we offer RNA dephosphorylation by phosphatase treatment. Prior purification is required due to competition for the phosphatase by free NTPs in the transcription reaction.
Spectrophotometric Scan: A wavelength scan from 220-320 nm allows calculation of concentration and provides an estimate of contaminating protein, salt and phenol.
Gel Analysis: An analytical gel sample is treated with glyoxal to disrupt secondary structure and run on an agarose gel.
All RNA transcripts have fully traceable documentation and are supplied with a certificate of analysis.
Not for resale without express written permission. Not for use in humans. No license under any patent or patent pending is granted or implied by the purchase of any TriLink product. TriLink does not warrant that the use or sale of the products delivered hereunder will not infringe the claims of any United States or other patents or patents pending covering the use of the product alone or in combination with other products or in the operation of any process. All and any use of TriLink product is the purchaser’s sole responsibility.