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

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.

Provided herein is a method for capping RNA in vitro. In some embodiments the capping reaction may be done using an RNA guanylyltransferase capping enzyme from a Tupanviruses virus, and in particular a capping enzyme from Tupanvirus soda lake virus or Tupanvirus deep ocean virus, or a fragment or variant thereof. Compositions and kits for capping RNA in vitro are also provided. Also included are methods, compositions and kits for the manufacturer and capping of therapeutic RNA oligonucleotides, such as RNA-based vaccines and therapeutics.

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 invention relates to compositions and methods for the design, evolution, preparation, and/or manufacture of enzymes for use with polynucleotides, primary transcripts and mmRNA molecules.

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

The invention relates to compositions and methods for the design, evolution, preparation, and/or manufacture of enzymes for use with polynucleotides, primary transcripts and mmRNA molecules.

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.