The present disclosure relates to compositions, kits, and methods of making RNA vaccines having an appropriate cap structure. Systems, apparatus, compositions, and/or methods may include and/or use, in some embodiments, non-naturally occurring single-chain RNA capping enzymes. In some embodiments, an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) one or more substitutions relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.

Aspects of the disclosure relate to production of vaccinia capping enzyme (VCE) in host cells. For example, host cells may comprise: a promoter; a ribosome binding site (RBS); a nucleic acid encoding a vaccinia capping enzyme (VCE) or VCE subunit; and a terminator.

An improved data intake and query system that can perform and display ingest-time and search-time field extraction, redaction, copy, and/or categorization is described herein. As described herein, ingest-time field extraction, redaction, copy, and/or categorization may refer to field or field value extraction, redaction, copy, and/or categorization that is performed by a log observer system of the data intake and query system on raw machine data as the raw machine data is ingested or received from a publisher. As described herein, search-time field extraction, redaction, copy, and/or categorization may refer to field or field value extraction, redaction, copy, and/or categorization that is performed by the log observer system and/or other components of the improved data intake and query system on historical raw machine data that has already been ingested and indexed by the improved data intake and query system.

The emergence of zoonotic pathogenic viruses has accentuated the need to develop broad-spectrum antivirals and vaccines. Highly modular yeast-based phenotypic platforms for characterization and targeting of RNA capping enzymes from emerging pathogens including coronaviruses, MPV, ASFV, and WNV, are disclosed herein. This platform can identify key amino acid residues and protein domains. Inactivation and attenuation mutations in viral enzymes are also disclosed herein. This platform is applied to vertebrate RNA capping enzymes, demonstrating use for high-throughput phenotypic screening. The disclosed platforms are highly modular and can be adapted for RNA capping enzymes from viruses and variants that emerge in the future.

The present invention relates to an immobilized capping enzyme, preferably an immobilized Vaccinia virus capping enzyme. Furthermore, the present invention relates to an immobilized cap-specific nucleoside 2-O-methyltransferase, preferably an immobilized Vaccinia virus cap-specific nucleoside 2-O-methyltransferase. Moreover, the present invention relates to a method for immobilizing said enzymes and to a method of using said enzymes for the addition of a 5-cap structure to RNAs. Moreover, the present invention relates to an enzyme reactor for performing the capping reaction using said immobilized enzymes and the subsequent separation of the 5-capped RNA product. In addition, the present invention relates to a kit comprising the capping enzyme and/or the cap-specific nucleoside 2-O-methyltransferase.

The present disclosure relates to compositions, kits, and methods of making RNA vaccines having an appropriate cap structure. Systems, apparatus, compositions, and/or methods may include and/or use, in some embodiments, non-naturally occurring single-chain RNA capping enzymes. In some embodiments, an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) one or more substitutions relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.

The present disclosure relates to compositions, kits, and methods of making RNA vaccines having an appropriate cap structure. Systems, apparatus, compositions, and/or methods may include and/or use, in some embodiments, non-naturally occurring single-chain RNA capping enzymes. In some embodiments, an RNA capping enzyme may include an FCE variant having (a) an amino acid sequence at least 90% identical to positions 1 to 878 of SEQ ID NO: 1, and/or (b) one or more substitutions relative to SEQ ID NO: 1 at a position selected from positions corresponding to positions 215, 337, 572, 648, and 833 (e.g., a position selected from positions corresponding to position 215, 337, and 572) of SEQ ID NO: 1.

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.