Cy5 Fluc mRNA with N1-Me-pUTP (5’CAP)

Cy5 Fluc mRNA with N1-Me-pUTP (5'CAP) is a luciferase mRNA transcribed in vitro by simulating mRNA processing in eukaryotes. Cy5 Fluc mRNA with N1-Me-pUTP (5'CAP) carries Cy5-UTP modification, N1-Me-pUTP modification (Cy5-UTP: N1-Me-pUTP=3:1 (molar ratio)), Cap 1 cap structure, and poly (A) tail, which enhances mRNA stability and translation efficiency^[1]. N1-Me-pUTP is a methyl modification of naturally occurring pseudouridine pUTP, catalyzed by N1 specific pseudouridine methyltransferase Nepl present in archaea and eukaryotes^[2]. This product uses N1-Me-pUTP instead of UTP, effectively enhancing RNA stability while reducing anti RNA immune response^[2]. Cy5 Fluc mRNA with N1-Me-pUTP (5'CAP) can be used as a standard to detect the transfection efficiency of different transfection reagents, and can also be used as a control to study the transfection, localization, and expression of fluorescent proteins in mammalian cells.

The luciferase reporter gene (Fluc) can detect gene expression extremely sensitively and efficiently, and is therefore commonly used as a bioluminescence reporter gene for gene regulation and functional research. Cy5 Fluc mRNA with N1-Me-pUTP (5'CAP) can directly express proteins in the cytoplasm without relying on promoters, with a faster protein expression rate than transfected DNA. The protein expression level is directly related to the mRNA transfection level, and there is no risk of gene integration. Firefly luciferase catalyzes the spontaneous fluorescence of luciferin or fatty aldehydes, producing chemiluminescence at a wavelength of approximately 550-570nm^[3]. Cy5 is a commonly used cyanine fluorescent dye, with maximum excitation/emission wavelengths of 650/670nm, respectively.

Firefly luciferase can be used to detect promoter activity or double fluorescent molecule complementarity experiments. Firefly luciferase and sea kidney luciferase can catalyze their respective substrates to emit fluorescence of different colors, and the two light absorption wavelengths are different, so they do not interfere with each other in detection. Therefore, they can be used simultaneously as dual luciferase reporter gene systems in the same chemical reaction system^[4].

References:

[1]. 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.

[2]. 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.

[3]. 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.

[4]. Jemielity J, Fowler T, Zuberek J, et al. Novel "anti-reverse" cap analogs with superior translational properties. RNA. 2003;9(9):1108-1122.

Cy5 Fluc mRNA with N1-Me-pUTP (5’CAP)是通过模拟真核生物中mRNA加工过程在体外转录产生的荧光素酶mRNA，携带Cy5-UTP修饰、N1-Me-pUTP修饰（Cy5-UTP：N1-Me-pUTP=3:1（摩尔比））、Cap 1帽结构和poly(A)尾，增加了mRNA的稳定性和翻译效率^[1]。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^[2]。本产品使用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^[2]。Cy5 Fluc mRNA with N1-Me-pUTP (5’CAP)能够作为标准品检测不同转染试剂的转染效率，也能够作为对照研究哺乳动物细胞中荧光蛋白的转染、定位和表达。

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。Cy5 Fluc mRNA with N1-Me-pUTP (5’CAP)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。萤火虫荧光素酶催化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^[3]。Cy5是一种常用的花青类荧光染料，最大激发/发射光波长分别为650/670nm。

萤火虫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^[4]。