142 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 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 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 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.

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.

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 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 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.

The invention provides a phage T7RNA (Ribonucleic Acid) polymerase (T7RNAP) mutant with high performance. The phage T7RNA polymerase (T7RNAP) mutant has mutation at a 744th amino acid residue corresponding to a wild type T7RNA polymerase; further, the T7RNAP mutant comprises an amino acid residue mutation occurring at at least one site selected from the following groups: a 62 site, a 68 site, a 72 site, a 152 site, a 173 site, a 535 site, a 538 site, a 539 site, a 633 site, a 723 site, a 767 site, a 881 site and a 883 site. When the T7RNAP mutant is applied to an in-vitro transcription (IVT) reaction, the yield of a target product in the IVT reaction can be remarkably increased, and / or the formation of a double-stranded RNA (dsRNA) byproduct in the IVT reaction can be reduced, so that the T7RNAP mutant can provide selection and adaptive industrial application for the IVT reaction of mRNA, CircRNA or saRNA and other drug molecules.

The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.

Provided is an RNA polymerase mutant in which an amino acid residue selected from the group consisting of glutamic acid at position 48 and arginine at position 50 in the amino acid sequence of a wild-type T7 RNA polymerase set forth in SEQ ID NO: 1 is substituted with an aliphatic amino acid residue.

The invention relates to a Pseudomonas sp. strain for use in the production of a recombinant protein characterised in that said strain comprises a nucleotide sequence encoding a phi15 RNA polymerase. The invention further relates to a plasmid, capable of integrating or replicating in Pseudomonas sp., comprising a phi15 promoter sequence operably linked to a nucleotide comprising one or more restriction sites for the insertion of a nucleotide sequence encoding a recombinant protein, or operably linked to a nucleotide sequence encoding a recombinant protein.

This invention belongs to the field of biology, specifically relating to a composition and its use against foot-and-mouth disease virus (FMDV). This invention discovered that compounds Pasireotide and Oxyclozanide alone cannot significantly inhibit FMDV replication. Unexpectedly, the combined use of Pasireotide and Oxyclozanide significantly inhibits the expression of FMDV RNA polymerase 3D protein, reducing the half-maximal tissue-cell infection dose (TCID) of FMDV. 50 It can significantly reduce the replication ability of foot-and-mouth disease virus and has the effect of antagonizing foot-and-mouth disease virus infection; and there are currently no clear clinical studies or drug instructions mentioning that there is a direct physical / chemical incompatibility between the two, so it can be used to prepare drugs against foot-and-mouth disease virus and has good application prospects.

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.

This invention discloses a method and kit for detecting RNA using a three-stage amplification melting curve based on isothermal self-circulation. The method includes designing a first primer, a second primer, and a circular amplification probe targeting the target nucleic acid sequence. The first primer consists of an RNA polymerase promoter sequence and a target nucleic acid-specific binding sequence, sequentially from the 5' end to the 3' end. The circular amplification probe is a circular DNA segment whose sequence consists of three parts: the first part completely contains a continuous sequence composed of the tag sequence and the nicking enzyme-specific recognition sequence from the second primer; the second part is a self-cleaving DNase sequence. In the technical solution used in this invention, a cascaded nucleic acid amplification system is constructed, achieving exponential signal amplification of the target nucleic acid through the synergistic effect of rolling circle amplification and RNA polymerase-mediated transcriptional amplification.

Disclosed are methods for detecting a target molecule in a test sample using a cell-free protein synthesis (CFPS) reaction. The methods may be utilized for detecting target molecules which may include small molecules and / or metabolites of small molecules. The methods employ one or more transcription templates that encode and conditionally express one or more exogenous RNA polymerases in the presence of the target molecule. The expressed RNA polymerases in turn induce expression of one or more reporter molecules from transcription templates comprising promoters for the RNA polymerases, thereby amplifying an output signal that is generated in the presence of a detected target molecule.

Disclosed are recombinant vectors comprising one or more polynucleotides encoding at least one modified ribonucleic acid (RNA) dependent RNA polymerase (RdRp), as well as pharmaceutical compositions and methods of their use.

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.

This disclosure provides a class of RNA polymerase variants and their uses. These variants exhibit improved catalytic activity compared to wild-type T7 RNA polymerase, increasing the capping rate of mRNA products during in vitro transcription (co-transcriptional capping). Furthermore, this disclosure provides methods for preparing RNA using these variants. Using this method to prepare RNA molecules, a higher amount of capped mRNA can be obtained.