CleanCap® AU FLuc saRNA (5mC) - (L-7902)
This construct expresses a firefly luciferase (FLuc) protein, originally isolated from the firefly Photinus pyralis, as self-amplifying RNA (saRNA). FLuc is commonly used in mammalian cell culture to measure both gene expression and cell viability. It emits bioluminescence in the presence of the substrate, luciferin.
- Capped using CleanCap® Reagent AU, mimicking the authentic 5′ alphavirus end while providing an optimal Cap-1 structure with >95% capping efficiency, for efficient expression in mammalian cells.
- Synthesized using CleanScribe™ RNA Polymerase, reducing unwanted double-stranded RNA (dsRNA) by-products by up to 85%, for lower inflammatory responses and enhanced functionality.
- Manufactured using TriLink’s proprietary CleanScript® saRNA in vitro transcription (IVT) method, improving saRNA yield and integrity while also lowering dsRNA formation, for enhanced protein expression.
- Modified with 5-methylcytidine (5mC), which can increase nuclease stability, reduce interaction of innate immune receptors, and enhance translational efficiency of saRNAs (McGee et al. (2024)).
Our catalog saRNA construct employs alphaviral replication machinery from Venezuelan equine encephalitis virus (VEEV) that encodes for the non-structural proteins (nsP1-4). It is polyadenylated (26A) and optimized for mammalian systems.
| Catalog No | L-7902 |
|---|---|
| Purity | Passes Agarose Gel Mobility |
| Length | 9377 nucleotides |
| Base Composition | Fully substituted with 5-methylcytidine |
| Concentration | 1.0 mg/mL |
| Buffer | 1 mM Sodium Citrate pH 6.4 |
| Conversion Factor | 40 µg/OD260 |
| Recommended Storage | At or below -40°C |
| Application | Reporter Genes |
| Cap | AU Start, Cap 1, CleanCap |
While both saRNA and mRNA deliver instructions for protein production within cells, they have key structural and functional differences:
Length and structure: saRNA is generally longer than mRNA. This is because saRNA includes the coding sequence for alphavirus nonstructural proteins nsP1-4 to self-replicate within the cell. mRNA, on the other hand, does not contain such structure.
5′ cap: saRNAs are typically capped with AU to preserve the authentic alphavirus 5′ end whereas AG and GG caps are common for mRNAs.
Modified nucleotides: Both saRNA and mRNA often incorporate modified nucleotides to improve their functionality. saRNA frequently uses 5-methylcytidines (5mC or m5C), while N1-methylpseudouridine is a common modification in mRNA.
Protein expression: saRNA, due to its self-amplifying nature, generally results in sustained protein expression over a longer period. mRNA, while often providing a stronger initial burst of protein production, typically leads to shorter-lived expression as the RNA is not replicated.
The sequence reported is just the ORF, start codon to stop codon, for our catalog saRNAs. It does not include any of the saRNA vector. For full saRNA length and the length of the ORF please see the corresponding product insert.
Our catalog saRNAs are intended for research use and manufactured with procedures in place to minimize endotoxin exposure. However, they are manufactured outside of a cleanroom and thus are not released with an endotoxin specification. If you need saRNA released with an endotoxin specification or a higher grade of material, please contact support@trilinkbiotech.com.
We recommend storing the saRNAs at -40° C to -80 C. To minimize freeze-thaw cycles, aliquot the sample into single-use quantities on the first usage.
For saRNA capping, the use of an AU cap is recommended to preserve the authentic alphavirus 5′ end. The AU dinucleotide at the very terminus of the 5′ UTR is required for replication and may be needed for binding of the viral RNA dependent RNA polymerase complex (RdRp) (nsP4) to the 3′ end of the negative-sense strand for initiation of (+)strand synthesis. For details, please see Shirako et al. (2003).
CleanCap Reagent AU was specifically designed for sARNAs based on the genomes of positive sense strand RNA viruses which start with a 5’ AU structure. Learn more about CleanCap Reagent AU in this tech note.
It is TriLink’s proprietary in vitro transcription method that produces high-quality, high-yield saRNAs from a broad range of sequences. It has been optimized to minimize dsRNA formation, improve saRNA integrity, and improve protein expression from the resulting saRNAs.
Modifications of saRNAs with 5-methylcytidines (5mC or m5C) have been reported to improve transfection efficiency, improve protein expression, and reduce inflammatory responses (Komori et al. (2023), Aboshi et al. (2024), McGee et al. (2024)).
TriLink's premade saRNAs are built on a freedom-to-operate (FTO) Venezuelan equine encephalitis virus (VEEV) vector, which encodes necessary saRNA components such as 5′ and 3′ UTRs, non-structural proteins (nsP1-4), a subgenomic promoter, and a 26-nt poly(A) tail.
Our catalog saRNAs are purified through DNase treatment to remove DNA templates, diafiltration to remove salts and small molecules, and oligo dT capture to remove impurities and retain species with poly(A) tails.
We run an residual dsRNA by dot blot, which is a qualitative test to determine the relative amount of dsRNA present in a sample. Generally, this test is performed to assess dsRNA levels in saRNAs.
We minimize the dsRNA level in our ready-to-use saRNAs by incorporating stringent processes that consist of:
- In vitro transcription using our proprietary CleanScript® saRNA IVT method
CleanScribe™ RNA polymerase, a novel enzyme designed to reduce dsRNA, replacing wild-type T7 RNA polymerase in IVT
We also assess dsRNA level in the sample by dot blot as part of quality analysis.
We look for a single main band running to approximately the correct length to pass the gel result. Some factors such as modified NTPs can make a sample run slightly lower than the expected size. Sometimes, sequence-related factors such as highly repetitive or UTP-rich regions can result in additional bands. We take into account of all these factors to confirm that the saRNA was manufactured appropriately and the band is sequence specific before passing the results.
The fragment analyzer reports the percent of smear with a chromatogram. The value reported corresponds to the full-length integrity of an saRNA sample.
We use 40 as the extinction coefficient for our saRNAs. Assigning a sequence-specific extinction coefficient for saRNA can be problematic due to its dependence on length and sequence composition. Factors like final buffer and temperature can also impact results. Thus, it is standard to use 40 for all saRNA species and not to calculate a coefficient for each sequence as you would with an oligonucleotide.
CleanCap capping technology For Research Use Only. Not for use in humans. Not for use in diagnostic or therapeutic purposes. For additional licensing restrictions, please see the license agreement at trilinkbiotech.com/cleancap-research-license. Patents and patent pending, see trilinkbiotech.com/legal-notices.
