The present disclosure relates, according to some embodiments, to compositions, methods, and/or kits for producing vaccinia capping enzyme. For example, active, heterodimers of vaccinia capping enzyme may be produced as fusions comprising D1 and D12 subunits. Vaccinia capping enzyme fusion proteins may further comprise a linker.

Disclosed herein is an engineered fusion enzyme composed of a prokaryotic phage RNA polymerase (T7 RNAP) and a viral capping enzyme (NP868R) for the generation of RNA transcripts containing a 5 7-methylguanylate cap.

Provided are a method for large-scale synthesis of a long-chain RNA and a method for site-specific modification of the long-chain RNA. The method for large-scale synthesis of a long-chain RNA comprises: designing short RNA fragments and splint DNA fragments; ligating; capping; and removing the splint DNA fragments and other steps. A large number of short RNA fragments and different splint DNA fragments are chemically synthesized, and then the different short RNA fragments are ligated by a biological method so as to form a target long-chain RNA. The product long-chain RNA has a low mutation rate, a plurality of the short RNA fragments can be assembled in a single reaction, and the long-chain RNA can be synthesized at a high throughput so as to fulfill the large-scale production of the long-chain RNA. In addition, by chemical modification of the short RNA fragments, the site-specific modification of the long-chain RNA can be realized.

A method for the production of proteins used in the in vitro transcription (IVT) of messenger RNA (mRNA), wherein the proteins are evaluated for purity and efficacy by the efficiency with which mRNA synthetically derived therefrom, subsequently transfects cells and produces encoded proteins.

The present invention provides, among other things, methods and compositions for large-scale production of capped mRNA using SUMO-Guanylyl Transferase fusion protein.