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ARCA (Anti Reverse Cap Analog)

描述:体外实验的初始阶段,绝大多数真核生物 mRNA 5' 端 m7G 帽结构可促进翻译。对于绝大多数 RNA,帽结构都可提高 RNA 的稳定性,降低其对核酸外切酶降解的敏感性,并促进 mRNA 起始复合体的形成。一些具有 5' 端帽结构的原核 mRNA 也可以像真核 mRNA 一样,能在真核生物无细胞蛋白合成体系中获得高效翻译。另外,还发现在真核生物靶 RNA 的剪切过程同样需要帽结构。

更新时间:2018-03-03
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详情介绍

*也被称为 Anti-reverse Cap Analog (ARCA) ,即抗-反向帽类似物。

概述

体外实验的初始阶段,绝大多数真核生物 mRNA 5' 端 m7G 帽结构可促进翻译。对于绝大多数 RNA,帽结构都可提高 RNA 的稳定性,降低其对核酸外切酶降解的敏感性,并促进 mRNA 起始复合体的形成。一些具有 5' 端帽结构的原核 mRNA 也可以像真核 mRNA 一样,能在真核生物无细胞蛋白合成体系中获得高效翻译。另外,还发现在真核生物靶 RNA 的剪切过程同样需要帽结构。
 

用 7 甲基 G 帽结构类似物,m7G(5' )ppp(5' )G (NEB#S1404) 作为引物能在 SP6 RNA 聚合酶、T7 RNA 聚合酶和 T3 RNA 聚合酶作用下,转录出更多的带帽RNA。因为转录时,T7 RNA 聚合酶掺入的*个碱基是 G,所以用 #S1405 或 #S1406 转录出带有 A-帽结构的 RNA,就不能够翻译,但这对于转染和显微注射实验有很好的稳定性。Contrease 等已经建立了一套方法,以 m7G(5' )ppp(5' )G (NEB #S1404)或 m7G(5' )ppp(5' )A (NEB #S1405) 为引物,利用 E. coli RNA 聚合酶,在体外有效合成带帽 RNA。


帽类似物有两个3' 羟基基团使得它们可从任一方向结合到 RNA 上。而抗-反向帽类似物(ARCA)3' OMethyl-m7G(5' )ppp(5' )G (NEB #S1411) 的一个羟基被甲基化,只能以 m7G 作为*个碱基与 RNA 结合,而这个方向可以增强体外翻译。
 

 N-7003
ARCA (Anti Reverse Cap Analog)

 

key step in cellular mRNA processing is the addition of a 5’ cap structure, which is a 5'-5' triphosphate linkage between the 5' end of the RNA and a guanosine nucleotide. The cap is methylated enzymatically at the N-7 position of the guanosine to form mature mCAP.

 
When preparing synthetic mRNA, the cap is often added prior to use in order to stabilize the mRNA and significantly enhance translation. Using a 4:1 mixture of a cap analog to GTP in transcription reactions will cap 80% of the resulting mRNAs. If mCAP (pre-methylated CAP) is used, only half of the asymmetrical mCAP will insert in the functional orientation. ARCA can only insert in the correct orientation, resulting in mRNAs that are translated twice as efficiently. TriLink is the least expensive source of high quality ARCA. We also offer CAP and mCAP for more traditional capping reactions.
 
 

Anderson, B.R., Muramatsu, H., Jha, B.K., Silverman, R.H., Weissman, D., Kariko, K. Nucleoside modifications in RNA limit activation of 2'-5'-oligoadenylate synthetase and increase resistance to cleavage by RNase L (2011) Nucleic Acids Research, EPub Aug
         
Warren et al., Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA, Cell Stem Cell (2010), doi:10.1016/j.stem.2010.08.012.
         
Anderson, B., Muramatsu, H., Nallagatla, S.R., Bevilacqua, P.C., Sansing, L.H., Weissman, D. & Kariko, K. Incorporation of pseudouridine into mRNA enhances translation by dimishing PKR activation (2010) Nucleic Acids Research, 38(17): 5884-5892.
         
Kariko, K., Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modifiation and the Evolutionary Origin of RNA (2005). Immunity, 23(2), 165-175.
         
Peng ZH, Sharma V, Singleton SF, Gershon PD. Synthesis and application of a chain-terminating dinucleotide mRNA cap analog. (2002) Organic Letters. 4(2):161-4.
         
Konarska MM, Padgett RA, Sharp PA. Recognition of cap structure in splicing in vitro of mRNA precursors. (1984) Cell. 38(3):731-6.
         
Miura K. The cap structure in eukaryotic messenger RNA as a mark of a strand carrying protein information. (1981) Adv Biophys. 14:205-38.
         
Banerjee AK. 5'-terminal cap structure in eucaryotic messenger ribonucleic acids. (1980) Microbiol Rev. 44(2):175-205.
         
Rosenberg M, Paterson BM. Efficient cap-dependent translation of polycistronic prokaryotic mRNAs is restricted to the first gene in the operon. (1979) Nature. 21;279(5715):696-701.
         
Filipowicz W. Functions of the 5,-terminal m7G cap in eukaryotic mRNA. (1978) FEBS Lett. 96(1):1-11.
         
Zan-Kowalczewska M, Bretner M, Sierakowska H, Szczesna E, Filipowicz W, Shatkin AJ. Removal of 5'-terminal m7G from eukaryotic mRNAs by potato nucleotide pyrophosphatase and its effect on translation. (1977) Nucleic Acids Res. 4(9):3065-81.

 

N-7003-1 ARCA 1umole
N-7003-10 ARCA 10umoles
N-7003-5 ARCA 5umoles

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