208 results about "RNA polymerase" patented technology

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology, the enzyme specific activity of the variant is obviously improved compared with that of a wild type, and the addition amount of polymerase is reduced, so that the cost is saved. Meanwhile, the mutant also effectively reduces the generation of incomplete fragment impurities and improves the integrity. In addition, the invention further provides a method for generating RNA through in-vitro transcription.

The present disclosure provides compositions and methods for the targeted modification of RNA molecules by RNA prime editing. The compositions and methods may be conducted invitro or in vivo within cells (e.g., human cells) for the therapeutic correction of disease-causing mutations and / or installation of motifs or mutations in RNA molecules of interest as a tool for scientific research. The disclosure provides compositions and methods for conducting RNA prime editing of a target RNA molecule (e.g., an RNA transcript) that enables the incorporation of one or more nucleotide changes and / or targeted mutagenesis of a target RNA molecule. The nucleotide change can include a single-nucleotide change, an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides a variety of configurations of the RNA prime editors each comprising a nucleic acid programmable RNA binding proteins (napRNAbp), such as Cas13, and an RNA-dependent RNA polymerase (RDRP), which are provided as fusion proteins or which can be separately provided in trans. The RNA prime editors are guided to a target RNA site by a guide RNA, which can be a rpegRNA that includes a template region for the synthesis of an RNA sequence to be installed on the RNA molecule attached to an available 3′ terminus. In others embodiments, the RNA template can be provided in trans.

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. Wild type T7 RNA polymerase is modified to obtain the T7 RNA polymerase variant, the enzyme specific activity of the T7 RNA polymerase variant is remarkably improved compared with that of the wild type T7 RNA polymerase, the addition amount of the polymerase in the in-vitro transcription reaction process can be reduced, and the cost is saved; in addition, the mutant also effectively reduces the generation of incomplete fragment impurities, and the mRNA integrity is significantly improved to 99%. The invention further provides a method for preparing RNA through in-vitro transcription.

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology, the enzyme specific activity of the variant is obviously improved compared with that of a wild type, and the addition amount of polymerase is reduced, so that the cost is saved. Meanwhile, the mutant also effectively reduces the generation of incomplete fragment impurities and improves the integrity. In addition, the invention further provides a method for generating RNA through in-vitro transcription.

Provided is a variant of an RNA polymerase, the variant being characterized in that an amino acid corresponding to arginine at position 386 in the amino acid sequence of a wild-type T7 RNA polymerase represented by SEQ ID NO: 1 is substituted with an aromatic amino acid.

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a genetically engineered bacterium for stably and efficiently producing L-isoleucine as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes E. coliTHRD as a host and contains an RNA (Ribonucleic Acid) polymerase beta-subunit mutant coding gene rpoBM; compared with a parent RNA polymerase beta-subunit, the RNA polymerase beta-subunit mutant has the advantage that the amino acid residue at the 618th site of the amino acid sequence is sequentially replaced with leucine from the N terminal to the C terminal. The genetically engineered bacterium disclosed by the invention can tolerate L-isoleucine and accumulate less alpha-ketobutyric acid, and has a very good industrial application prospect.

The invention discloses an oil palm U6 promoter gene and application thereof, and belongs to the technical field of biology. The nucleotide sequence of the promoter gene is shown as SEQ ID NO.1. The oil palm RNA polymerase III type promoter gene, namely the oil palm endogenous U6 promoter gene EgU6, is obtained by cloning in an oil palm genome for the first time, and the promoter gene has high transcriptional activity and can drive downstream fluorescent protein mNeonGreen expression. The candidate oil palm endogenous U6 promoter gene can be provided for subsequently establishing a high-efficiency oil palm gene editing technology system based on a CRISPR / cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / Cas9) system.

The invention provides a kind of RNA polymerase variants and application thereof, the variants have improved catalytic activity compared with wild type T7 RNA polymerase, and the capping rate of mRNA products in the in vitro transcription (co-transcription capping) process can be increased. In addition, the disclosure also provides a method for preparing RNA by using the variant, and by using the method to prepare RNA molecules, more capped mRNA can be obtained.

The invention provides an RNA polymerase variant, the enzyme specific activity of the variant is obviously improved compared with that of a wild type, and the addition amount of polymerase is reduced, so that the cost is saved. In addition, the invention further provides a method for generating RNA through in-vitro transcription.

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. The RNA polymerase variant provided by the invention has the performance of remarkably improving the integrity of a super-long fragment mRNA transcription product, can be used for preparing mRNA through in-vitro transcription, particularly can improve the integrity of the product when preparing saRNA exceeding 10000 nt, and has important significance for industrial production of saRNA.

The invention discloses a T7-RNA polymerase mutant and application thereof, and relates to the field of nucleic acid tool enzymes and nucleic acid biology. The T7-RNA polymerase mutant is obtained by mutating glycine at the 47th site from the N end of a full-length amino acid sequence of wild type T7-RNA polymerase into phenylalanine, tryptophan or tyrosine; or, the amino acid sequence of the T7-RNA polymerase mutant and the amino acid sequence of the wild type T7-RNA polymerase have at least 80% homology, and glycine in a peptide fragment as shown in SEQ ID NO.10 in the amino acid sequence is mutated into phenylalanine, tryptophan or tyrosine; the full-length amino acid sequence of the wild type T7-RNA polymerase is as shown in SEQ ID NO. 1. The T7-RNA polymerase mutant is suitable for enzymatic synthesis of RNA, and has strong application potential in the aspects of in-vitro transcription, RNA synthesis, RNA drug synthesis, RNA vaccine manufacturing, gene editing, in-vivo protein expression or in-vitro translation systems and the like.

The invention provides a T7RNA polymerase variant and application thereof, the amino acid sequence of the T7RNA polymerase variant comprises substitution or deletion of at least one of the following amino acid sites relative to SEQ ID NO: 1: R34, K172, Y178, R386, D388, Q435, N437 and D438. Compared with wild type T7RNA polymerase, the variant has improved catalytic activity, and can improve the capping rate of mRNA products in the in vitro transcription (co-transcription capping) process. In addition, the invention also provides a method for preparing RNA by using the variant, and more capped mRNA can be obtained by adopting the method for preparing RNA molecules.

The invention belongs to the field of genetic engineering and biological treatment, and provides an eukaryotic expression vector, gram-negative bacteria containing the eukaryotic expression vector and application of the eukaryotic expression vector in preparation of tumor drugs. By selecting and combining a phage-derived promoter, a specific phage RNA polymerase gene, a membrane rupture protein gene and an eukaryotic expression drug protein gene, an eukaryotic expression vector is divided into three modules: (1) a phage-derived RNA polymerase gene transcription module; (2) a membrane rupture protein gene expression module; and (3) a eukaryotic drug protein gene expression module. Through organic combination of the three modules, the eukaryotic drug delivery system is constructed, and the system and method for stably and efficiently performing eukaryotic drug protein expression, drug delivery and drug killing are realized.

The invention provides a kind of RNA polymerase variants and application thereof, the variants have improved catalytic activity compared with wild type T7RNA polymerase, and the capping rate of mRNA products in the in vitro transcription (co-transcription capping) process can be increased. In addition, the disclosure also provides a method for preparing RNA by using the variant, and by using the method to prepare RNA molecules, more capped mRNA can be obtained.

The invention provides an RNA (Ribonucleic Acid) polymerase variant and a preparation method thereof, and an RNA product with low dsRNA pollution can be obtained by applying the RNA polymerase variant disclosed by the invention to in-vitro transcription. In addition, the invention further provides a method for preparing RNA through in-vitro transcription.

Provided are an RNA polymerase variant and a preparation method therefor. The RNA polymerase variant is used in in vitro transcription to obtain an RNA product having low dsRNA contamination. In addition, provided is a method for preparing an RNA by means of in vitro transcription.

This invention generally relates to a novel RNA / mRNA production and amplification method using viral RNA replicase and / or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA / mRNA sequences carrying at least an RdRp-binding site in the 5′- or 3′-end, or both. The RNA / mRNA so obtained is useful for not only producing mRNA vaccines and / or RNA-based medicines but also for generating the mRNA-associated proteins, peptides, and / or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase-mediated RNA / mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and / or bacteriophage RNA-dependent RNA polymerases (RdRp), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.

Disclosed are compositions, systems, kits, and methods for detecting an analyte in a sample by regulated in vitro transcription. The compositions, systems, kits, and methods typically comprise and / or utilize one or more components selected from: (a) an RNA polymerase; (b) an allosteric transcription factor (ATF), wherein the ATF binds an analyte or target molecule as a ligand; (c) a reporter molecule, and (d) an engineered transcription template; and / or any combination thereof. The engineered transcription template typically comprises a promoter sequence for the RNA polymerase and an operator sequence for the ATF. The promoter sequence and operator sequence are operably linked to a sequence encoding an RNA, wherein the ATF modulates transcription of the encoded RNA when the ATF binds the analyte or target molecule as a ligand, wherein the transcribed RNA generates or interferes with a detectable signal in conjunction with the reporter molecule.

The invention belongs to the technical field of biology, and relates to an RNA polymerase variant, and when the RNA polymerase variant is applied to in-vitro transcription, an RNA product with low dsRNA pollution can be obtained. In addition, the invention further provides a method for generating RNA through in-vitro transcription.

The present disclosure relates to compounds which exhibit activity in the inhibition of RNA polymerase as well as pharmaceutical compositions comprising these compounds and methods of treatment of viral infections by administration of these compounds or the pharmaceutical compositions.

The present invention provides a pharmaceutical composition comprising one or more RNA polymerase primary σ70 (encoded by gene rpoD) targeting polynucleotides capable of targeting rpoD. In an embodiment, the one or more rpoD targeting polynucleotides is encapsulated in a nanoparticle such as exosomes or LNP. The present invention also provides a method of treatment of rpoD-associated disease of a subject comprising administration of a therapeutically effective amount of the pharmaceutical composition of the present invention to the subject.

The invention discloses a construction method and application of a rabies virus RNA polymerase dual-luciferase report system, and belongs to the technical field of biology. In order to solve the technical problem that a report system for detecting the RABV RNA polymerase activity needs to be developed urgently, a Firefly luciferase fragment and a vector pcDNA3.1-SRV9-M-G obtained by amplifying M protein and G protein-deleted rabies virus SRV9 strain full-length plasmid are seamlessly connected, a recombinant luciferase report plasmid is constructed, the recombinant luciferase report plasmid and an auxiliary plasmid are combined, and the recombinant luciferase RNA polymerase activity detection system is constructed. And the RABV RNA polymerase dual-luciferase report system is formed by the Renilla luciferase report plasmid and the T7 promoter plasmid, and the RABV RNA polymerase dual-luciferase report system comprises the Renilla luciferase report plasmid and the T7 promoter plasmid. The system can accurately and efficiently detect the activity of RABV polymerase, and by virtue of the function, effective antiviral targets and therapeutic drugs can be screened out in an assisted manner.

The present disclosure provides variant RNA polymerases with amino acid substitutions, and their use of which increases transcription yield, RNA integrity, capping efficiency, and reduces dsRNA formation during an in vitro transcription reaction.

To provide T7 RNA polymerase variants.SOLUTION: The present invention provides engineered T7 RNA polymerase variants and a composition thereof. The variants have been devised in order to selectively incorporate a symmetrically capped GTP analogue exceeding GTP in starting in-vitro transcription. The present invention further provides a method for using a variant provided in the specification. The present invention further provides a use of a composition provided in the specification. The engineered RNA polymerase variant can include at least one substitution or substitution set in a polypeptide sequence.SELECTED DRAWING: None

The invention provides a preparation method and application of a preparation for RNA cell-free synthesis and belongs to the technical field of molecular biology, the preparation method of the preparation for RNA cell-free synthesis mainly comprises the steps that escherichia coli serves as a chassis strain, a pET-28a carrier is utilized, a recombinant strain for expressing T7RNA polymerase is constructed, and the T7RNA polymerase has enzyme activity at the temperature of 50-60 DEG C; inducing, culturing and collecting thalli, carrying out ultrasonic lysis, and treating a lysis solution to prepare a preparation for RNA cell-free synthesis; the preparation provided by the invention naturally contains various cofactors, and RNA polymerase does not need to be extracted and purified, so that the production cost is reduced.

The present application provides a class of T7 RNA polymerase variants and applications thereof, wherein the amino acid sequence of the T7 RNA polymerase variant comprises at least one substitution or deletion selected from the following amino acid sites relative to SEQ ID NO: 1: R34, K172, Y178, R386, D388, Q435, N437, D438. The variant has improved catalytic activity relative to the wild-type T7 RNA polymerase, and can increase the capping rate of mRNA products during in vitro transcription (co-transcription capping). In addition, the present application also provides a method for preparing RNA using such variants, and by using the method to prepare RNA molecules, more capped mRNA can be obtained.

The invention provides the RNA polymerase variant and the preparation method thereof, the RNA polymerase variant is applied to in-vitro transcription, an RNA product with low dsRNA pollution and high integrity can be obtained, meanwhile, the utilization efficiency of cap analogues in a co-transcription capping reaction system can be improved, and the cost is saved. In addition, the invention further provides a method for generating RNA through in-vitro transcription.

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. According to the present invention, the wild type T7 RNA polymerase is modified to obtain the T7 RNA polymerase variant, and the capping rate of the RNA product can be substantially improved compared with the wild type T7 RNA polymerase;

The invention provides an in-vitro transcription method based on RNA polymerase and a kit, which are suitable for the fields of mRNA synthesis, gene research and biological pharmacy. According to the method, the problems of low efficiency, poor stability and the like of traditional in-vitro transcription are solved through an optimization technology. The method comprises the following specific steps: providing a linear DNA template containing a T7 promoter; according to the method, site-directed mutagenesis or fusion protein modified high-activity T7RNA polymerase is combined with chemically modified NTPs and a spermine-BSA compound stabilizer to react under the conditions that the temperature is 37-42 DEG C and the pH is 7.5-8.5, and formation of double-stranded RNA is promoted through dynamic temperature control. A reaction system contains Mg < 2 + > / Mn < 2 + > and DTT, and enzyme activity and product integrity are optimized. The kit comprises an improved reagent and a standardized process, the transcription efficiency is improved by 2-5 times, the product yield is greater than or equal to 80%, and the activity is not lost after the kit is stored at 4 DEG C for 1 month. The method has the technical advantages that NTPs degradation resistance is achieved, template preparation is simplified, universality is high, the method is suitable for large-scale production of mRNA vaccines, gene therapy and the like, the production cost is remarkably reduced, and wide application of the in-vitro transcription technology is promoted.

This application provides an RNA polymerase variant and its application in in vitro transcription to prepare RNA, relating to the field of biotechnology. Using the RNA polymerase variant provided in this application for in vitro transcription not only reduces the generation of truncated or over-extended impurities in low-magnesium in vitro transcription reaction systems, thus improving the integrity of the transcribed RNA product, but also increases the yield and capping rate of the RNA product in low-magnesium in vitro transcription reaction systems. Furthermore, this application also provides the application of the RNA polymerase variant in in vitro transcription.