Most eukaryotic mRNAs require a 7-methyl guanosine (m7G) cap structure at the 5´-end and a poly(A) tail at the 3′-end for efficient translation. The HiScribe T7 mRNA Kit with CleanCap Reagent AG utilizes an optimized RNA synthesis formulation and trinucleotide cap analog technology for co-transcriptionally capping mRNAs that contain a natural Cap-1 structure in a single simplified reaction without compromising RNA yield. By using a DNA template with a T7 promoter sequence followed by an AG initiation sequence and an encoded poly(A) tail, mRNAs can be transcribed with a 5´-m7G Cap-1 structure that is polyadenylated, translationally competent and able to evade the cellular innate immune response.
The HiScribe T7 mRNA Kit with CleanCap Reagent AG is formatted with individual vials of NTPs and CleanCap Reagent AG to allow for partial or complete substitution of modified NTPs, with a total kit yield of 1.8 mg of mRNA. Cap-1 mRNA synthesized from this kit is suitable for many applications, including transfections, microinjections, in vitro translation, preclinical mRNA therapeutic mRNA studies as well as RNA structure and function analysis.
This kit contains sufficient reagents for 20 reactions (20 µl each). Each standard reaction yields ≥ 90 µg of RNA from 1 µg CLuc AG Control Template DNA. Each kit can yield ≥ 1.8 mg RNA.
Figure: 1: CleanCap Reagent AG
FIgure 2: Schematic of CleanCap Reagent AG Promoter and Initiation Sequence
Figure 3: CleanCap Reagent AG results in higher mRNA synthesis yield than the ARCA analog
All reactions were performed with 5 mM CTP, 5 mM UTP and 6 mM ATP. Standard IVT reactions included 5 mM GTP and no cap analog. ARCA reactions contained a 4:1 ratio of ARCA:GTP (4mM:1mM). IVT with CleanCap Reagent AG contained 5 mM GTP and 4 mM CleanCap Reagent AG and was performed according to recommended protocol (Standard mRNA Synthesis, HiScribe T7 mRNA Kit with CleanCap Reagent AG). Reactions were incubated for 2 hours at 37°C, purified and quantified by NanoDrop®.
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