體外實驗的初始階段，絕大多數(shù)真核生物 mRNA 5' 端 m7G 帽結(jié)構(gòu)可促進(jìn)翻譯。對于絕大多數(shù) RNA，帽結(jié)構(gòu)都可提高 RNA 的穩(wěn)定性，降低其對核酸外切酶降解的敏感性，并促進(jìn) mRNA 起始復(fù)合體的形成。一些具有 5' 端帽結(jié)構(gòu)的原核 mRNA 也可以像真核 mRNA 一樣，能在真核生物無細(xì)胞蛋白合成體系中獲得高效翻譯。另外，還發(fā)現(xiàn)在真核生物靶 RNA 的剪切過程同樣需要帽結(jié)構(gòu)。

*也被稱為 Anti-reverse Cap Analog （ARCA），即抗-反向帽類似物。

概述

用 7 甲基 G 帽結(jié)構(gòu)類似物，m⁷G（5' ）ppp（5' ）G （NEB#S1404）作為引物能在 SP6 RNA 聚合酶、T7 RNA 聚合酶和 T3 RNA 聚合酶作用下，轉(zhuǎn)錄出更多的帶帽RNA。因為轉(zhuǎn)錄時，T7 RNA 聚合酶摻入的*個堿基是 G，所以用 #S1405 或 #S1406 轉(zhuǎn)錄出帶有 A-帽結(jié)構(gòu)的 RNA，就不能夠翻譯，但這對于轉(zhuǎn)染和顯微注射實驗有很好的穩(wěn)定性。Contrease 等已經(jīng)建立了一套方法，以 m⁷G（5' ）ppp（5' ）G （NEB #S1404）或 m⁷G（5' ）ppp（5' ）A （NEB #S1405）為引物，利用 E. coli RNA 聚合酶，在體外有效合成帶帽 RNA。

帽類似物有兩個3' 羥基基團(tuán)使得它們可從任一方向結(jié)合到 RNA 上。而抗-反向帽類似物（ARCA）3' OMethyl-m⁷G（5' ）ppp（5' ）G （NEB #S1411）的一個羥基被甲基化，只能以 m⁷G 作為*個堿基與 RNA 結(jié)合，而這個方向可以增強體外翻譯。

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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