EZ Cap^TM Cy5 Firefly Luciferase mRNA (5-moUTP) will express luciferase protein once entering cells which is initially extracted from firefly Photinus pyralis. This enzyme catalyzes ATP-dependent D-luciferin oxidation and lead to yield chemiluminescence at about 560 nm wavelength. Firefly Luciferase is a frequently used bioluminescent reporter for gene regulation and function study. It is applicable in assays for mRNA delivery, translation efficiency, cell viability and in vivo imaging etc.

EZ Cap^TM Cy5 Firefly Luciferase mRNA (5-moUTP) is provided at a concentration of ~1 mg/ml with Cap1 structure. There are currently two ways to cap mRNA: One is co-transcription method, by adding Cap analogues into the transcription process. The other is enzymatic Capping. After transcription, Cap0 capping is performed by Vaccinia virus Capping Enzyme (VCE), GTP and S-adenosylmethionine (SAM). The Cap0 is then generated into the Cap1 through 2´-O-Methyltransferase and SAM. Cap1 Capping can also be performed by adding VCE, 2´-O-Methyltransferase, GTP and SAM in a one-step process. Cap 1 structure is more ideal for mammalian systems and possess higher transcription efficiency than Cap 0 structure. The addition of 5-moUTP and poly(A) tail suppress RNA-mediated innate immune activation and increase the stability and lifetime of the mRNA in vitro and in vivo. Poly(A) tail also plays an important role in enhancing the efficiency of translation initiation. Cy5 is a synthetic red fluorescent dye with maximum excitation and emission wavelengths of 650 nm and 670 nm, respectively. Cy5-UTP and 5-moUTP is used in a ratio of 1:3 when transcribed. Substitution in this ratio results in mRNA that is easy to visualize and still can be translated in cell culture. In the rough, there is an anti-correlation between translation efficiency and Cy5-UTP substitution.