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The Ambion® mMESSAGE mMACHINE® T7 Ultra Kit combines a new cap analog – Anti-Reverse Cap Analog (ARCA) – with Ambion's patented high-yield transcription technology, to generate RNA transcripts that produce higher protein yields compared to other transcripts upon translation. The kit includes sufficient reagents for 10 reactions.
• Synthesize more protein from in vitro transcribed RNA • Express RNA transcripts more efficiently both in vitro and in vivo • Stabilize transcripts in vivo with included reagents for poly(A) tailing
ARCA-capped RNA is Translationally More Active: A base modification in ARCA results in its incorporation in the functional, translatable orientation only; traditional cap analog can be incorporated in both functional and nonfunctional orientations. As a result, incorporating ARCA into transcription reactions yields capped RNAs that are 100% translatable. This is further enhanced by the inclusion of poly(A) tails in mMESSAGE mMACHINE® T7 Ultra transcripts. Experiments comparing ARCA and ARCA/poly(A) tailed transcripts to cap analog and cap analog/poly(A) tailed transcripts indicate higher levels of protein synthesis with ARCA capped RNA .
What Is ARCA? Anti-Reverse Cap Analog (ARCA) is a modified cap analog in which the 3' OH group (closer to m7G) is replaced with OCH3 (see schematic). Because of this substitution, the RNA polymerase can only initiate transcription with the remaining hydroxyl group, thus forcing ARCA incorporation in the forward orientation. As a result, 100% of the transcripts synthesized with ARCA at the 5' end are translatable, leading to a strong stimulatory effect on translation.
Proper Capping of In Vitro Transcribed RNA: Proper capping of RNA promotes correct initiation of protein synthesis, as well as stability and processing of mRNA in vivo. Uncapped RNA is rapidly degraded by cellular RNases after microinjection or transfection into cells. Capped RNA is also typically translated more efficiently in in vitro translation systems, generating RNAs with cap analog incorporated only in the functional orientation. Therefore, substitution of traditional cap analog with ARCA results in the synthesis of capped RNAs that are 100% translatable.
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