This invention relates to in vitro production of nucleic acids, particularly RNAs and specifically messenger RNAs (mRNA).

Turning now to the drawings, systems and methods to enhance RNA transcription in a cost-effective manner and uses thereof are provided. One of the most common enzymes for RNA transcription is T7 RNA polymerase. Many embodiments increase RNA yield in transcription reactions by adding ribonucleoside vanadyl complex (VRC) to the transcription reaction. Various embodiments use VRC at low concentrations in an RNA transcription reaction. Reactions in accordance with many embodiments are capable of increasing RNA yield by approximately 2-fold or more.

This specification discloses compositions of matter and processes that allow the detection of RNA from coronaviruses and other RNA viruses, in particular, compositions and processes that have the capacity to detect in multiplexed form many RNA targets within individual viruses, targets from multiple viruses, and other RNA molecules that can be used as positive controls.

The present invention concerns a method for expressing a recombinant DNA molecule in a eukaryotic host cell, comprising the steps of: (a) expressing or introducing at least one chimeric protein, in said host cell, wherein said chimeric protein comprises: (i) at least one catalytic domain of a capping enzyme, in particular selected in the group consisting of cap-0 canonical capping enzymes, cap-0 non-canonical capping enzymes, cap-1 capping enzymes and cap-2 capping enzymes; and (ii) at least one catalytic domain of a DNA-dependent RNA polymerase, in particular a bacteriophage DNA-dependent RNA polymerase, (b) constitutively or transiently downregulating the phosphorylation level of subunit a of translation initiation factor eIF2 (eIF2α) in said host cell.

The invention also concerns an isolated nucleic acid molecule or a set of nucleic acid molecules, comprising or consisting of (1) at least one nucleic acid sequence encoding a chimeric protein comprising at least one catalytic domain of a capping enzyme; and at least one catalytic domain of a DNA-dependent RNA polymerase; and (2) at least one nucleic acid sequence downregulating the phosphorylation level of eIF2α in a eukaryotic host cell or encoding a polypeptide downregulating said phosphorylation level; and (3) optionally, at least one nucleic acid sequence encoding a poly(A) polymerase, as well as vectors, kits and cells comprising said nucleic acid molecule or set, and different uses and applications thereof.

Described herein are synthetic methods for producing sequence-specific RNA oligonucleotides that eliminate impurities produced in prior art methods. In one aspect, an end-protected capture DNA complementary to a portion of the product RNA is employed. In another aspect, the template DNA is covalently or noncovalently linked to the RNA polymerase, either directly or through the use of a nontemplate DNA. In a third aspect, a flow chamber is employed. All of the methods can be used in combination.

Disclosed herein, inter alia, are novel strand-displacing polymerases and methods of use thereof.

The present invention provides, among other things, methods for large-scale production of a composition comprising full-length messenger RNA product that is substantially free of double-stranded RNA, and compositions produced using such methods and uses thereof. The present invention is based, in part, on the surprising discovery that mRNA product produced by in vitro transcription using an SP6 RNA polymerase is substantially free of double-stranded RNA. In one aspect, the present invention provides methods of generating large-scale mRNA product for mRNA therapy without need for a chromatography step.

The present invention relates to a novel variant RNA polymerase sigma factor 70 (δ.sup.70) polypeptide, a polynucleotide encoding the same, a microorganism containing the polypeptide, and a method for producing L-threonine by using the microorganism.

The present disclosure is generally related to modified Gram positive bacterial cells producing increased amounts of one or more protein(s) of interest and modified Gram positive bacterial cells having increased genetic competency. Thus, certain embodiments of the disclosure are directed to modified Gram positive bacterial cells expressing an increased amount of a protein of interest, relative to an unmodified (parental) Gram positive bacterial cell expressing the same protein of interest, wherein the modified bacterial cell comprises at least one mutation in a rpoC gene encoding a variant RNA-polymerase (RNAP) β′-subunit polypeptide. In certain embodiments, the rpoC gene encoding the variant β′-subunit polypeptide is integrated into the chromosome of the modified cell. In other embodiments, the rpoC gene encoding the variant β′-subunit polypeptide is comprised on an extrachromosomal plasmid introduced into the modified cell. In other embodiments, the disclosure is directed to competent Bacillus host cells comprising at least one copy of a nucleic acid construct encoding a modified rpoC polypeptide comprising 90% sequence identity to SEQ ID NO: 8 and an aspartic acid to glycine substitution at position 796 of SEQ ID NO: 8, wherein the polynucleotide encoding the rpoC polypeptide is foreign to the Bacillus host cell that was non-competent prior to the introduction of the first nucleic acid construct.

The present disclosure provides, in some aspects, variant RNA polymerases, the use of which increases transcription efficiency while reducing the number of double-stranded RNA contaminates and run-on transcripts produced during an in vitro transcription reaction.