Fluc mRNA with N1-Me-pUTP (5’CAP)

Luciferase(Fluc) can detect gene expression extremely sensitively and efficiently, so it is often used as a bioluminescent reporter gene for gene regulation and functional studies. Fluc mRNA can express proteins directly in the cytoplasm without relying on a promoter. The protein expression speed is faster than transfected DNA. The protein expression level is directly related to the transfection amount of mRNA, and there is no risk of gene integration. Firefly luciferase protein catalyzes intracellular luciferin or fatty aldehyde to produce autofluorescence, producing chemiluminescence at a wavelength of about 550-570nm[1].

Luciferase can be used to detect promoter activity or dual fluorescent molecule complementation experiments. Firefly luciferase and Renilla luciferase can respectively catalyze their respective substrates to emit fluorescence of different colors. The two light absorption wavelengths are different and the detection does not interfere with each other. Therefore, they can be used as dual luciferases in the same chemical reaction system. Reporter gene systems are used simultaneously[2].

By simulating the mRNA processing process in eukaryotes, the product has a Cap 1 cap structure at the 5' end and a poly(A) tail at the 3' end, which increases the stability and translation efficiency of the mRNA[3]. N1-Me-pUTP is a methyl modification of the naturally occurring pseudouridine pUTP, which is catalyzed by the N1-specific pseudouridine methyltransferase Nepl that exists in archaea and eukaryotes[4]. This product uses N1-Me-pUTP instead of UTP, which effectively enhances RNA stability and reduces anti-RNA immune responses[5].

References:
[1]. JoÃo M M LeitÃo, Joaquim C G Esteves da Silva. Firefly luciferase inhibition. 2010 Oct 5;101(1):1-8. doi: 10.1016/j.jphotobiol.2010.06.015. Epub 2010 Jul 3.
[2]. Jemielity J, Fowler T, Zuberek J, et al. Novel "anti-reverse" cap analogs with superior translational properties. RNA. 2003;9(9):1108-1122
[3]. Callum J C Parr, et al.N 1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells. 2020 Apr 6;48(6):e35. doi: 10.1093/nar/gkaa070.
[4]. Pedro Morais, Hironori Adachi, Yi-Tao Yu.The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines. 2021 Nov 4;9:789427. doi: 10.3389/fcell.2021.789427.
[5]. Pedro Morais, Hironori Adachi, Yi-Tao Yu.The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines. 2021 Nov 4;9:789427. doi: 10.3389/fcell.2021.789427.

荧光素酶报告基因(Luciferase,Fluc)能够极其灵敏、高效地检测基因的表达,故而通常用作基因调控和功能研究的生物发光报告基因。Fluc mRNA能够在不依赖于启动子的情况下直接在细胞质中表达蛋白,蛋白表达速度比转染DNA更快,蛋白表达量与mRNA的转染量直接相关,并且没有基因整合的风险。萤火虫荧光素酶蛋白催化胞内的荧光素或者脂肪醛产生自发荧光,在约550-570nm波长处产生化学发光[1]。

荧光素酶可用于检测启动子活性或双荧光分子互补实验。萤火虫荧光素酶和海肾荧光素酶可分别催化各自的底物发出不同颜色的荧光,且两种光吸收波长不同,检测互不干扰,因此可在同一个化学反应体系中作为双荧光素酶报告基因系统同时使用[2]。

通过模拟真核生物中mRNA加工过程,该产品的5'端具有Cap 1帽结构,3'端具有poly(A)尾,增加了mRNA的稳定性和翻译效率[3]。N1-Me-pUTP是天然存在的假尿苷pUTP的甲基修饰物,由存在于古细菌和真核生物中的N1特异性假尿苷甲基转移酶Nepl催化生成[4]。该产品使用N1-Me-pUTP替代UTP,有效增强了RNA稳定性,同时降低抗RNA免疫应答[5]。