Standard RNA ladders such as the DynaMarker® for large or small RNA or double-stranded size determination are routinely used by researchers wishing to confirm the molecular weight of an in vitro transcribed or chemically-synthesized RNA. Due to the altered gel mobility of pseudouridine-containing RNAs, a PseudoU-RNA Molecular Weight Marker can be used to more accurately assess the molecular weight of these modified RNAs.
Pseudouridine-5′-Triphosphate is a naturally occurring base used by researchers to decrease nuclease activity and toll-like receptor (TLR) activation. Genome editing researchers might use, for example a Cas9 mRNA produced with pseudouridine (abbreviated: Ψ or Psi) rather than standard uridine. Cas9 mRNA (Ψ) can then be transfected into cells and will be translated into Cas9 protein without worry of a Cas9 expression plasmid incorporating into the genome. Further optimization of innate immune response reduction can be achieved by replacing cytidine with 5-Methylcytidine-5′-Triphosphate (abbreviated: 5meC). tebu-bio offers a large catalog offer of 5meC/Ψ mRNAs carrying both pseudouridine and 5-methylcytidine substitutions. These mRNAs encode induced pluripotent stem cell programming factors (Oct4, Klf4, SOX2, c-Myc, Lin28), reporter genes (EGFP, YFP, beta-gal, luciferase, mCherry and hAAT), and Cas9 carrying a nuclear localization signal. This NLS-Cas9 5meC/Ψ mRNA, similar to NLS-Cre recombinase mRNA will be translated into a protein targeted to the nucleus of cells for genome editing.
Users at the preliminary phases of working with chemically-modified mRNAs might opt for Cyanine 5-labeled mRNAs that allow visualization of the mRNA by fluorescence microscopy. These mRNAs encoding EGFP for luciferase then allow researchers to evaluate transfection efficiency and mRNA translation independently.
Interested in custom mRNA synthesis? tebu-bio supplies chemically-synthesized RNA with nearly any modification up to 100 nucleotides in length. Long RNAs can be made by in vitro transcription. Contact the specialists there using this form.
Some of the most popular kits for in vitro mRNA transcription include the various CellScript Kits. These kits allow for in vitro T7 or SP6 transcription and allow users to produced capped and poly(A) mRNAs. The MessageMAX™ T7 ARCA-Capped Message Transcription Kit has the advantage that it caps mRNAs with the anti-reverse cap analog (ARCA). Because ARCA contains a 3′-O-methyl group on the m7G nucleotide, it can only be incorporated in the correct orientation at the 5′ end of the RNA. This is not true for the standard cap analog (m7GTP). Thus, ARCA incorporation results in the synthesis of capped RNA that is more efficiently translated than standard cap analog.
Our recommendation for mRNA synthesis experts ready to produce their own chemically-modified mRNAs:
- 5-Methylcytidine-5′-Triphosphate (1.0 umole)
- Pseudouridine-5′-Triphosphate (1.0 umole)
- ARCA (Anti Reverse Cap Analog) (2 x 1.0 umole)