Cap Analog, Methylated
Cap Analog, Methylated
Most naturally occurring eukaryotic mRNAs and many viral RNAs have a modified nucleotide structure known as a “cap” on their 5′ terminus. In vivo, the cap structure is involved in the initiation of protein synthesis and in mRNA processing and stability.1 Many viral RNAs are infectious only when capped, and uncapped RNAs introduced into cells via transfection or microinjection are rapidly degraded by cellular RNases.2,3 Therefore,RNA repared for microinjection intooocytes or for transfection into eukaryotic cells should be capped. In vitro, capped mRNAs are translated more efficiently than uncapped transcripts in rabbit reticulocyte lysate or wheat germ translation systems.4 The trimethylated cap is involved in RNA transport5,6 and RNA translation in nematodes.7 Transcripts synthesized for use in in vitro RNA splicing experiments may also need to be capped.8 RNA Cap Analogs Cat. Nos. C32010, C61025, C06005, and C50110 Anti-reverse cap analogs (ARCAs)*, such as m2 7,3′–OG[5′]ppp[5′]G, were found to result in synthesis of transcripts that were more efficiently translated in vitro since the 3′-O-methyl group only permitted them to be incorporated in the correct orientation9,10,11 (see Figure 1). Capping does reduce RNA yields relative to transcription reactions without diguanosine triphosphate, but with the right conditions and high-quality reagents, a large proportion of the transcripts that are made will be capped. For optimum yields of monomethylated, capped in vitro transcripts, we recommend using the AmpliCap-Max™ T7 and T3 High Yield Message Maker Kits. EPICENTRE also offers the MessageMAX™ T7 ARCA-Capped Message Transcription Kit which is optimized to produce the highest yield of RNA having an ARCA on the 5′ end. These kits contain a convenient Cap/NTP PreMix and yield up to 60 μg of RNA per μg of Control Template in a standard 30-minute reaction. Capping efficiencies are up to 80%. EPICENTRE’s RNA Cap Analog preparations can also be used with the AmpliScribe™ T7, T3, and SP6 High Yield Transcription Kits. Capping reactions with these kits typically yield about 20-40 μg of total RNA with ~80% capping efficiency. Protocols for the synthesis of
capped RNA using AmpliScribe or conventional transcription reagents are given on page 3.