描述:體外實(shí)驗(yàn)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。
體外實(shí)驗(yàn)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)。
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?yàn)檗D(zhuǎn)錄時(shí),T7 RNA 聚合酶摻入的*個(gè)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(shí)驗(yàn)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(gè)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(gè)羥基被甲基化,只能以 m7G 作為*個(gè)堿基與 RNA 結(jié)合,而這個(gè)方向可以增強(qiáng)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.
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 |