251 results about "RNA polymerase" patented technology

The invention relates to the field of nucleic acid detection, in particular to a multifunctional dumbbell type nucleic acid probe, a kit, a target detection method and application thereof. The multifunctional dumbbell type nucleic acid probe provided by the invention comprises two or more of the following functional areas: a promoter area used for being combined with RNA polymerase; the target recognition area is used for recognizing and combining a target sequence and opening the dumbbell type nucleic acid probe; the dumbbell type nucleic acid probe can be transcribed and combined with a rolling ring, a specific sequence is subjected to mass transcription, amplification of the specific sequence is achieved, finally, signal amplification is achieved during detection, and the dumbbell type nucleic acid probe has the advantages of being simple in reaction system, low in background signal and high in detection specificity; and the dumbbell type nucleic acid probe can be designed according to different target sequences, so that the controllable detection of different targets can be realized.

The invention provides an RNA polymerase variant, and relates to the technical field of biology. The RNA polymerase variant provided by the invention can be used for remarkably reducing the generation of dsRNA impurities in an in-vitro transcription process and remarkably improving the integrity of an RNA transcription product, especially in transcription of long-fragment RNA. In addition, the invention provides an application of the RNA polymerase variant in in-vitro transcription, and also provides a method for preparing ultra-long saRNA (greater than 10000 nt).

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.

The present invention provides methods of reducing oncogene transcription, or of reducing phosphorylated RNA polymerase 2, in a subject in need thereof by administering to the subject of Compound (I), or a pharmaceutically acceptable salt thereof.

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 a targeted mutation system based on orthogonal bacteriophage RNA polymerase and an orthogonal transcription mutant. According to the orthogonal transcription mutants, phage RNA polymerase and deaminase are fused, and a large number of mutations can be rapidly and efficiently generated on a target gene. The bacteriophage RNA polymerase part of the orthogonal transcription mutants can specifically recognize corresponding promoters and transcribe downstream genes, and the deaminase part of the bacteriophage RNA polymerase part of the orthogonal transcription mutants can generate mutation in the transcription process. The optimized orthogonal transcription mutants can simultaneously generate mutation from C: G to T: A and mutation from A: T to G: C on a target gene. According to the method, mutation and screening can be carried out on target genes on plasmids or genomes or genes on metabolic pathways, the evolution efficiency of enzymes or other proteins can be greatly improved, and time and cost are saved.

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 T7-RNA polymerase mutant capable of reducing generation of dsRNA and application of the T7-RNA polymerase mutant, tryptophan at the 328th site from the N terminal of an amino acid sequence of wild type T7-RNA polymerase is substituted by methionine, phenylalanine, asparagine, alanine or lysine, and a by-product dsRNA generated in an in-vitro transcription process can be reduced. Through computer simulation, a large amount of screening work is reduced, the enzyme evolution speed is increased, and the cost and time of enzyme modification are greatly reduced; the T7-RNA polymerase mutant maintains efficient transcription, reduces the complexity of later purification work, reduces the purification cost of RNA production, can quickly prepare RNA drugs meeting the use requirements, and provides an efficient tool enzyme for large-scale production of the RNA drugs.

Disclosed are compositions, systems, kits, and methods for detecting an analyte or target molecule 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 (ATT), wherein the ATT binds an analyte or target molecule as a ligand; (c) 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 ATT. The promoter sequence and operator sequence are operably linked to a sequence encoding an RNA, wherein the ATT modulates transcription of the encoded RNA when the ATT binds the analyte or target molecule as a ligand, wherein the transcribed RNA generates a detectable signal in conjunction with a reporter molecule.

The invention discloses a T7 RNA (Ribonucleic Acid) polymerase mutant, and the T7 RNA polymerase mutant comprises any one or more of the following amino acid mutation sites: W797L, P657K, Y846R, M306K, N601E, P476E, S606V, K642G, P742G, I117L, Q786L, S430P, L446F or T375K, and the T7 RNA polymerase mutant has RNA polymerase activity. The invention also discloses an application of the T7 RNA polymerase mutant in in-vitro transcription, non-coding RNA synthesis, RNA drug synthesis, in-vivo protein expression or cell-free protein expression.

The present invention comprises compositions comprising at least two RNA replicons (self-amplifying RNA vector (saRNA or rRNA)) that can be replicated by a replicase of a self-replicating virus (e.g., a replicase of alphavirus origin). In the at least two replicons, at least one of the replicons optionally comprises an open reading frame encoding an RNA-dependent RNA polymerase or replicase, the RNA-dependent RNA polymerase or replicase being capable of replicating each of the at least two replicons. Furthermore, each replicon comprises an open reading frame encoding a different antigen of interest, such as a different antigen derived from the same or different pathogenic organism, such as glycoproteins and nucleoproteins of Ebola virus.

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 discloses a method for preparing RNA (Ribonucleic Acid) through in-vitro transcription and application. The method comprises the following steps: (1) preparing a linearized DNA template; (2) constructing an in-vitro transcription reaction system, and performing in-vitro transcription under a reaction condition suitable for transcription; and (3) purifying the prepared mRNA. According to the method, RNA polymerase can reduce 3 '-extension and non-specific transcription initiation of an IVT product under the condition that the temperature is increased, and an appropriate amount of L-arginine or salt thereof is added into a reaction system, so that the RNA structure can be effectively prevented from being damaged while the mRNA yield is not reduced, and the generation of dsRNA is further reduced. In addition, the reduction of dsRNA can significantly reduce the immunogenicity of mRNA.

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.

The invention discloses a BCG1059c gene knock-down recombinant bacillus calmette guerin vaccine strain, and the BCG1059c gene of the strain is knocked down. A CRISPRi / dCas9th1 technology is utilized to block RNA polymerase movement in space through a compound formed by crRNA, dCas9 protein and sgRNA at a transcriptional level, so that the expression of the BCG1059c gene in the BCG vaccine is inhibited. The strain has the characteristics of low-abundance polar fat and high-abundance fat arabinomannan compared with wild BCG (Bacillus Calmette Guerin); in an in-vitro experiment, compared with a wild type BCG, the macrophage treated by the BCGkd can secrete IL-2 and TNF-alpha at a higher level, and shows a stronger bactericidal ability on escherichia coli; in an in-vivo experiment, after intravenous injection of a mouse, the survival time of the gene knock-down strain BCGkd in the lung can reach more than 16 weeks, and a more lasting anti-tuberculosis capability can be provided.

Provided herein is a nucleic acid detection system comprising: a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind to corresponding target molecules; an RNA-based masking construct; and optionally, nucleic acid amplification reagents to amplify target RNA molecules in a sample. In another aspect, the embodiments provide a polypeptide detection system comprising: a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind a trigger RNA, an RNA-based masking construct; and one or more detection aptamers comprising a masked RNA polymerase promoter binding site or a masked primer binding site. In some embodiments, the system may be used to detect viruses in samples.

The invention provides an RNA polymerase variant capable of improving the RNA capping rate in in-vitro transcription, and relates to the technical field of biology. According to the present invention, the wild type T7RNA polymerase is modified to obtain the T7RNA polymerase variant, and the capping rate of the RNA product can be substantially improved compared with the wild type T7RNA polymerase;

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.