• Synthesize more protein from in vitro transcribed RNA
  
  
• Express RNA transcripts more efficiently both in vitro and in vivo
  
  
• Stablize transcripts in vivo with reagents for poly(A) tailing (included)
  
  

ARCA-capped RNA is Translationally More Active
The 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 when translated in vitro or in vivo, result in much higher protein yields. Incorporating ARCA into RNA transcripts has a strong stimulatory effect on subsequent translation. A base modification in this cap analog generates RNAs with ARCA incorporated only in the functional, translatable orientation (see "Proper Capping of In Vitro Transcribed RNA", below); traditional cap analog can be incorporated in both a functional and nonfunctional orientation. Therefore, substitution of traditional cap analog with ARCA results in the synthesis of capped RNAs that are 100% translatable. The increased translation efficiency provided by ARCA is further enhanced by the addition of a poly(A) tail in the transcript. 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 (see figures on this page).

Complete Kit Including Poly(A) Tailing Reagents
The mMESSAGE mMACHINE T7 Ultra Kit combines ARCA with Ambion's patented high yield transcription technology. The kit provides sufficient reagents to perform ten 20 µl transcription reactions. Also included are reagents for poly(A) tailing for the production of poly(A) tailed RNA specifically for microinjection and transfection experiments.

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, unlike transcripts synthesized with conventional cap analog, 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 reticulocyte lysate and wheat germ in vitro translation systems. While "cap", or N⁷-methylguanosine, is present on the 5' end of most naturally occurring eukaryotic mRNAs and many viral RNAs, the dinucleotide, m⁷G(5')ppp(5')G, or 'standard' cap analog, is routinely added onto synthetic transcripts generated during in vitro transcription. However, cap analog is incorporated into the RNA in both the forward [m⁷G(5')pppG(pg)] and reverse orientation [G(5')pppm⁷G(pN)] leading to the synthesis of two isomeric populations of RNA of approximately equal proportion (Pasquinelli et al., (1995) RNA 1:957­–967). RNA capped with reverse 5' caps cannot be translated, resulting in a 50% reduction in expected yield of protein.

The use of ARCA (Stepinski J, et al. (2001) RNA 7:1486­–1495, Peng Z-H, et al. (2002) Organic Letters 4:161­–164) prevents incorporation of cap analog in the reverse orientation and maximizes translation efficiency. In ARCA, one of the 3' OH groups (closer to m⁷G) is replaced with ­OCH₃ (see schematic). This modification restricts transcription initiation to the remaining ­OH group, 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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