Pyrophosphatase

The recombinant plasmids encoding ALAB and four single genes (Sin1 to Sin4) were extracted and linearized by restriction endonuclease BspQ I cleavage followed by plasmid recovery and purification. In vitro transcription reaction (IVT in 50 μL) was performed at 37 °C for 3 h after vortexing of the mixture containing linearized plasmid template, ATP (100 mM), GTP (100 mM), CTP (100 mM), UTP (100 mM), Cap analogue (100 mM, Vazyme, Nanjing, China), T7 RNA polymerase (200 U/μL, Vazyme), 10X T7 Reaction Solution (Vazyme), RNase enzyme inhibitor (40 U/μL, Vazyme), pyrophosphatase (0.1 U/μL, Vazyme), and RNase Free H₂O (Invitrogen, Carlsbad, CA, USA, 10977015). After the IVT, 170 μL of Rnase-free H₂O, 5 μL of DNaseI enzyme (NEB, Beverly, MA, USA, M0303L), and 25 μL of 10× DNaseI enzyme reaction solution (NEB, M0303L) were added to the mRNA and incubated at 37 °C for 20 min. Purified mRNA was obtained using OligoDT beads (Vazyme, N401). The mRNA integrity and purity were analyzed using Agarose Gel (Bio-Rad, Hercules, CA, USA) and HPLC-SEC (Thermo Fisher Scientific, Waltham, MA, USA).

The pQE-30 plasmid and Escherichia coli BL21(DE3) were purchased from Novagen Inc. (Madison, WI, USA) for T7 RNAP heterologous expression. The site-directed mutagenesis kit and dsRNA quantitative kit were supplied by Vazyme Co., Ltd. (Nanjing, China). ATP, GTP, CTP, UTP, pyrophosphatase, and murine RNase inhibitor were purchased from Vazyme Co., Ltd. (Nanjing, China). MgCl₂, glycerol, dithiothreitol (DTT), and other chemicals were purchased from Aladdin Industrial Inc. (Shanghai, China) and other commercial sources.