CleanCap® AU EGFP saRNA

While both saRNA and mRNA deliver instructions for protein production within cells, they have key structural and functional differences:

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.

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)).

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:

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.

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.

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).

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.