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Cap Analogs – Enhance mRNA stability and translation efficiency

Many eukaryotic and viral mRNAs are modified at their 5' ends by addition of 7-Methylguanosine (N7-methyl guanosine or m7G), known as "Cap". "Capping" of the mRNA structure plays a crucial role in a variety of cellular processes which include translation initiation[1], splicing[2], intracellular transport[3] and turnover[4].

Consistently, successful downstream application of in vitro transcribed mRNAs strongly depends on the 5' Cap structure. Capped mRNAs are generally more efficiently translated in wheat germ and reticulocyte in vitro translation systems[5], and they are less susceptible to exonuclease degradation during microinjection experiments compared to uncapped mRNAs[6].

In vitro synthesis of capped mRNAs is performed by bacteriophage RNA polymerase (T7, SP6 or T3)-mediated in vitro transcription that co-transcriptionally incorporate cap analogs at the 5'-end of the transcripts (Fig. 1).

Reactions with traditional cap analogs (GpppG, m7GpppG or m2,2,7GpppG) routinely yield a mixture of mRNAs containing the cap analog incorporated both in a correct or reverse orientation[7]. Thus about 50 % of the resulting capped mRNAs are recognized by the translational machinery. Translational efficiency however, can be markedly increased by usage of the "anti-reverse" cap analog (ARCA, m7,3'-OGpppG)[8]. This is due to substitution of the 3'-OH of the m7 guanine moiety by a 3'-O-methyl group that forces ARCA incorporation in the correct orientation and subsequently results in a 100 % translatable mRNA population.


Figure 1: Cap analogs are enzymatically incorporated at the RNA 5'-end by bacteriophage RNA Polymerase-mediated in vitro Transcription.

Selected References

[1] Gingras et al. (1999) eIF4 initiation factors: Effectors of mRNA recruitment to ribosomes and regulators of translation. Annu. Rev. Biochem. 68:913.
[2] Izaurralde et al. (1994) A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell 78:657.
[3] Izaurralde et al. (1992) A cap binding protein that may mediate nuclear export of RNA polymerase II-transcribed RNAs. J. Cell Biol. 118:1287.
[4] Beelman et al. (1998) An essential component of the decapping enzyme required for normal rates of mRNA turnover. Nature 382:642.
[5] Paterson et al. (1979) Efficient translation of prokaryotic mRNAs in a eukaryotic cell-free system requires addition of a cap structure. Nature 279:692.
[6] Drummond et al. (1985) The effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger RNAs in Xenopus oocytes. Nucl. Acids Res. 13:375.
[7] Pasquinelli et al. (1995) Reverse 5' caps in RNAs made in vitro by phage RNA polymerases. RNA 1:957.
[8] Grudzien et al. (2007) Synthesis of Anti-Reverse Cap Analogs (ARCAs) and their Applications in mRNA Translation and Stability. Methods Enzymol. 431:203.