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Capped RNA that Gives Higher Protein Yields mMESSAGE mMACHINE™ T7 Ultra
- 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)
The mMESSAGE mMACHINE T7 Ultra Kit produces high quality, efficiently capped in vitro transcribed mRNA that translates with much greater efficiency than RNA synthesized with conventional cap analog. Ensuring that cap is added to RNA transcripts only in the correct orientation increases translation efficiency during both in vitro and in vivo translation experiments. Ambion's Poly(A) Tailing Kit is incorporated into the mMESSAGE mMACHINE T7 Ultra Kit for production of stable RNA specifically for microinjection and transfection.
ARCA-capped RNA is Translationally More Active
Incorporating a new cap analog, Anti-Reverse Cap Analog (ARCA) (Figure 1), into RNA transcripts has a strong stimulatory effect on subsequent translation (Figures 2 and 3). A base modification in this cap analog generates RNAs with ARCA incorporated only in the functional, translatable orientation; conventional cap analog is incorporated in both a functional and nonfunctional orientation (see "Proper Capping of in Vitro Transcribed RNA" at right). Substitution of conventional cap analog with ARCA yields capped RNAs that are 100% translatable. The level of Translation is further enhanced by adding a poly(A) tail to the transcript. Figures 2 and 3 present protein synthesis data comparing ARCA and ARCA/poly(A) tailed transcripts to conventional cap analog and cap analog/poly(A) tailed transcripts in both transfection and microinjection experiments, respectively. All of the experiments indicated higher levels of protein synthesis with ARCA capped RNA.
Figure 1. Schematic of ARCA Molecule.
Figure 2. Transcripts Containing ARCA Are More Highly Translated In Transfected Cells. Comparison of protein expression from standard and ARCA capped luciferase RNAs, plus and minus poly(A) tail, at different time points after transfection. A. Either standard cap analog-capped or ARCA-capped luciferase in vitro transcribed RNA (1 µg) was transfected into HeLa cells. Cells were harvested and lysed at 8 hr, 10 hr, 12 hr, 24 hr and 48 hr post-transfection. Luciferase activity was measured and plotted against transfection time. B. Poly(A)-tailed luciferase RNA (1 µg) prepared in either a standard mMESSAGE mMACHINE™ or a mMESSAGE mMACHINE Ultra reaction was transfected as above. Luciferase activity was measured and plotted against time following transfection.
Figure 3. Transcripts Containing ARCA Are More Highly Translated In Microinjected Embryos. Comparison of protein expression, from standard Cap Analog and ARCA capped luciferase RNA at different time points after microinjection. Either standard cap analog-capped or ARCA-capped luciferase RNA (100 pg) was microinjected into Xenopus embryos. Embryos were harvested and lysed at 8 hr, 24 hr, 50 hr, and 70 hr post injection. Luciferase activity was measured and plotted against time since microinjection.
The mMESSAGE mMACHINE T7 Ultra Kit combines ARCA and reagents for poly(A) tailing with Ambion's patented high yield transcription technology. The incorporation of ARCA into RNA transcripts is efficient and does not compromise RNA yield (see below). In addition, RNA transcribed with the mMESSAGE mMACHINE T7 Ultra Kit is more stable and more efficiently translated than RNA synthesized by traditional transcription kits. mMESSAGE mMACHINE T7 Ultra transcripts are ideal for both in vitro and in vivo translation experiments.
Figure 1. Schematic of ARCA Molecule.
Figure 2. Transcripts Containing ARCA Are More Highly Translated In Transfected Cells. Comparison of protein expression from standard and ARCA capped luciferase RNAs, plus and minus poly(A) tail, at different time points after transfection. A. Either standard cap analog-capped or ARCA-capped luciferase in vitro transcribed RNA (1 µg) was transfected into HeLa cells. Cells were harvested and lysed at 8 hr, 10 hr, 12 hr, 24 hr and 48 hr post-transfection. Luciferase activity was measured and plotted against transfection time. B. Poly(A)-tailed luciferase RNA (1 µg) prepared in either a standard mMESSAGE mMACHINE™ or a mMESSAGE mMACHINE Ultra reaction was transfected as above. Luciferase activity was measured and plotted against time following transfection.
Figure 3. Transcripts Containing ARCA Are More Highly Translated In Microinjected Embryos. Comparison of protein expression, from standard Cap Analog and ARCA capped luciferase RNA at different time points after microinjection. Either standard cap analog-capped or ARCA-capped luciferase RNA (100 pg) was microinjected into Xenopus embryos. Embryos were harvested and lysed at 8 hr, 24 hr, 50 hr, and 70 hr post injection. Luciferase activity was measured and plotted against time since microinjection.
The mMESSAGE mMACHINE T7 Ultra Kit combines ARCA and reagents for poly(A) tailing with Ambion's patented high yield transcription technology. The incorporation of ARCA into RNA transcripts is efficient and does not compromise RNA yield (see below). In addition, RNA transcribed with the mMESSAGE mMACHINE T7 Ultra Kit is more stable and more efficiently translated than RNA synthesized by traditional transcription kits. mMESSAGE mMACHINE T7 Ultra transcripts are ideal for both in vitro and in vivo translation experiments.
Capping RNA Transcripts with ARCA
ARCA was incorporated in an in vitro transcription system using short (6 nt) and long (1.8 kb) transcripts, each beginning with a G residue. Cap analogs such as ARCA compete with GTP for incorporation into the first nucleotide position. As the ratio of the cap analog to GTP increases in the reaction, the ratio of capped RNA to uncapped RNA increases proportionally. The 6 nt transcripts were trace labeled during synthesis and analyzed by denaturing polyacrylamide gel electrophoresis (Figure 4). Capped RNA migrated more slowly than uncapped RNA. These data confirm that ARCA is efficiently incorporated into in vitro transcripts and that the yield of synthetic RNA in the presence of ARCA is comparable to the yield in the presence of m
7GpppG.
Figure 4. Ratio of Cap Analog:GTP to Maximize Yield of ARCA Capped Transcripts. ARCA was incorporated along with GTP during in vitro transcription system using short (6 nt) and long (1.8 kb) transcripts containing a "G" as the first nucleotide. Cap analogs such as ARCA compete with GTP for incorporation into the first nucleotide position. As the ratio of the cap analog to GTP increases in the reaction, the ratio of capped RNA to uncapped RNA increases proportionally. The short RNA transcripts were resolved on a 20% denaturing polyacrylamide/8 M urea gel and synthesized products were detected by autoradiography.
Figure 4. Ratio of Cap Analog:GTP to Maximize Yield of ARCA Capped Transcripts. ARCA was incorporated along with GTP during in vitro transcription system using short (6 nt) and long (1.8 kb) transcripts containing a "G" as the first nucleotide. Cap analogs such as ARCA compete with GTP for incorporation into the first nucleotide position. As the ratio of the cap analog to GTP increases in the reaction, the ratio of capped RNA to uncapped RNA increases proportionally. The short RNA transcripts were resolved on a 20% denaturing polyacrylamide/8 M urea gel and synthesized products were detected by autoradiography.
Complete Kit Including Poly(A) Tailing Reagents
The mMessage mMachine T7 Ultra Kit produces high quality, ARCA capped RNA that translates with much greater efficiency than RNA synthesized with conventional cap analog. 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 microinjections and transfections.
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. One way to prevent incorporation of cap analog in the reverse orientation and to maximize translation efficiency is to use the recently described Anti-Reverse Cap Analog (ARCA) (Stepinski J, et al. (2001) RNA
7:1486 1495, Peng, Z-H, et al. (2002) Organic Letters
4:161 164). In ARCA, one of the 3' OH groups (closer to 7mG) is replaced with OCH
3(Figure 1). 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.