Cy5 Fluc-eGFP mRNA with N1-Me-pUTP (5'CAP) is a luciferase green fluorescent protein mRNA transcribed in vitro by simulating mRNA processing in eukaryotes. Cy5 Fluc-eGFP 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, increasing 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-eGFP 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.
Fluc-eGFP fluorescent protein is a fluorescent reporter gene commonly used in molecular biology research. This product connects firefly luciferase mRNA and green fluorescent protein eGFP mRNA through Linker and can be used for the detection of two reporter gene experiments. Cy5 Fluc-eGFP 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 transfection with deoxyribonucleotides. The protein expression level is directly related to the mRNA transfection level, and there is no risk of gene integration.
After transfection with N1-Me-pUTP (5'CAP), Cy5 Fluc-eGFP mRNA can express strong and bright green fluorescent protein eGFP and firefly luciferase protein in cells. The excitation/emission wavelengths of eGFP are 488/509nm, respectively; firefly luciferase catalyzes the spontaneous fluorescence and chemiluminescence of luciferin or fatty aldehydes, with wavelengths of approximately 550-570nm[3]. Cy5 is a commonly used cyanine fluorescent dye, with maximum excitation/emission wavelengths of 650/670nm, respectively.
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.
Cy5 Fluc-eGFP 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-绿色荧光蛋白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-eGFP 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。
Fluc-eGFP荧光蛋白是一种常用于分子生物学研究的荧光报告基因。本产品通过Linker连接萤火虫荧光素酶mRNA和绿色荧光蛋白eGFP mRNA，可用于两种报告基因实验的检测。Cy5 Fluc-eGFP 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脱氧核糖核苷酸更快，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。
Cy5 Fluc-eGFP mRNA with N1-Me-pUTP(5’CAP)转染细胞后能够表达强烈且明亮的绿色荧光蛋白eGFP和萤火虫荧光素酶蛋白。eGFP激发/发射光波长分别为488/509nm；萤火虫荧光素酶催化荧光素或脂肪醛产生自发荧光和化学发光，波长约为550-570nm[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。