54 results about "Capping enzyme" patented technology

The present invention relates to kits and methods for efficiently generating 5′ capped RNA having a modified cap nucleotide and for use of such modified-nucleotide-capped RNA molecules. The invention is used to obtain novel compositions of such modified-nucleotide-capped RNA molecules. In particular, the present invention provides kits and methods for capping RNA using a modified cap nucleotide and a capping enzyme system, such as poxvirus capping enzyme. The present invention finds use for in vitro production of 5′-capped RNA having a modified cap nucleotide and for in vitro or in vivo production of polypeptides by in vitro or in vivo translation of such modified-nucleotide-capped RNA for a variety of research, therapeutic, and commercial applications. The invention also provides methods and kits for capturing or isolating uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate, such as RNA synthesized in vitro or obtained from a biological source, including prokaryotic mRNA that is in a mixture with other prokaryotic and / or eukaryotic nucleic acids. The method for capturing modified-nucleotide-capped RNA also provides methods and kits for obtaining only type-specific or condition-specific modified-nucleotide-capped RNA by cap-dependent subtraction of that portion of the captured modified-nucleotide-capped RNA in cells of one type or condition that is the same as RNA in cells of another type or condition. The invention further provides methods and kits for using a capping enzyme system and modified cap nucleotides for labeling uncapped RNA comprising primary RNA transcripts or RNA having a 5′-diphosphate with detectable dye or enzyme moieties.

The present invention relates to enzymes involved in capping of fungal mRNAs, and molecules that inhibit such enzymes. In particular, the invention relates to the novel C. albicans capping enzyme genes ABD1 and CET1 and their encoded protein products, as well as derivatives and analogs thereof. The invention also relates to methods of using of fungal capping enzymes to screen for fungal inhibitors.

The invention provides methods to inhibit the replication of a flavivirus, methods of inhibiting the guanosine triphosphate (GTP)-binding and guanylyltransferase activity of a flavivirus RNA capping enzyme, and methods of treating a subject infected with a flavivirus. The methods can include contacting a flavivirus with an effective amount of a thioxothiazolidine compound described herein, or a derivative thereof, such as a compound of Formula (I).

The invention provides a method for preparing a recombinant eukaryotic mRNA molecule by using a prokaryotic transcription system. The method comprises the following steps: 1) expressing an enzyme system required for the transcription and capping process of the recombinant eukaryotic mRNA, and obtaining a prokaryotic in-vitro transcription-capping enzyme system; 2) preparing a linear transcriptiontemplate DNA; and 3) transcribing, capping and purifying the recombinant mRNA. The recombinant mRNA molecule prepared by the method provided by the invention has all mRNA characteristics required by eukaryotic organisms in the protein translation process, so that the recombinant mRNA molecule can be further identified and utilized by a eukaryotic translation system. The method provided by the invention overcomes the defects in the field of recombinant mRNA at present, can produce the recombinant eukaryotic mRNA with stable genetic characteristics and no carrier inhibition effect in a simple process and at low cost, and meets the large-scale industrial production demand.

The invention provides a method for detecting the activity of mRNA (messenger ribonucleic acid) 2 '-O-methyltransferase. The method specifically comprises the following steps: (S1) providing a single-stranded RNA molecule with 5 '-triphosphate at the 5'-terminal; (S2) the RNA molecules in the step (S1) are subjected to the following test reactions, the test reactions comprise reactions in a negative test system, a first test system and a second test system, the negative test system does not contain enzyme, and the first test system does not contain enzyme; the first test system comprises Cap0 capping enzyme; the second test system comprises Cap0 capping enzyme and mRNA (messenger ribonucleic acid) 2 '-O-methyltransferase; and (S3) detecting the formation conditions of the SAH in the negative test system, the first test system and the second test system in the step (S2), and comparing with a standard curve to obtain a quantitative result of the SAH. The method is high in safety, free of radioactive contamination, simple in step, low in experimental environment requirement and low in cost.

Provided herein is a method for efficiently capping RNA in vitro. In some embodiments the capping reaction may be done at high temperature using Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reactions may comprise a capping enzyme from a large virus of amoeba, e.g., Faustovirus, mimivirus or moumou virus, or a variant thereof. Compositions and kits for practicing the method are also provided.

The present invention relates to an RNA capping enzyme comprising or consisting of an amino acid sequence having high identity with any of the sequences of SEQ ID NO: 1-28, which can be expressed in vitro, in human cells, in mouse cells or in yeast cells, and which has the activities of RNA 5 '-triphosphatase (RT), guanylate transferase (GT) and RNA guine-N7 methyltransferase (RNMT) at the same time. The invention also relates to a mutant obtained by carrying out substitution, addition and / or deletion of amino acid on any sequence in SEQ ID NO: 1-28, a nucleotide sequence for coding the capping enzyme and / or the mutant thereof, and an expression construct or a host cell containing the nucleotide sequence. The invention also relates to a composition comprising the capping enzyme as well as a method and a reaction system for capping RNA by using the capping enzyme, a mutant or a composition thereof.

Provided herein is a method for efficiently capping RNA in vitro. In some embodiments the capping reaction may be done at high temperature using Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reactions may comprise a capping enzyme from a large virus of amoeba, e.g., Faustovirus, mimivirus or moumouvirus, or a variant thereof. Compositions and kits for practicing the method are also provided.

The invention relates to the field of molecular biology, and particularly relates to a chimeric enzyme, application thereof in-vitro one-step reaction synthesis of Cap0mRNA and a method. The method comprises the steps that the chimeric enzyme and an in-vitro transcription template are mixed and then subjected to a co-reaction, and a capped and tailed mRNA product is obtained through a one-step reaction, wherein the chimeric enzyme comprises an RNA polymerase structural domain and a capping enzyme catalytic structural domain, and the chimeric enzyme has the dual functions of in-vitro transcription and capping through chimeric design. The method for in-vitro one-step reaction synthesis of the mRNA is developed, and the synthesis efficiency and the mRNA yield are improved.

A method for capping a polynucleotide, comprising: S1, providing a composition, which at least comprises: an uncapped polynucleotide, a capping enzyme, and a cap structure analog, the capping enzyme comprising a polynucleotide ligase polypeptide; and S2, forming a capped polynucleotide on the basis of the composition of S1.

The invention relates to a vaccinia virus capping enzyme mutant, a recombinant vector, a recombinant engineering bacterium and application of the vaccinia virus capping enzyme mutant. The vaccinia virus capping enzyme mutant comprises: (a) a polypeptide formed by deleting, replacing or adding one or more amino acids to an amino acid sequence as shown in SEQ ID No.2; or (b) a polypeptide having at least 90% of homology with the polypeptide composed of the amino acid sequence as shown in SEQ ID No.2. The half-life period of the vaccinia virus capping enzyme mutant at 45 DEG C is 35 min or above and is higher than that of a wild type vaccinia virus capping enzyme mutant, and when mRNA is capped at a high temperature, the vaccinia virus capping enzyme mutant still shows excellent catalytic activity and can play an important role in the fields of clinical medicine, influenza vaccines, tumor vaccines, biological pharmacy and the like.

Provided is a method for producing an artificial recombinant virus of the family Reoviridae, the method comprising the steps of:(1) introducing a FAST protein expression vector and / or a capping enzyme expression vector into host cells;(2) introducing a vector containing expression cassettes for individual RNA genome segments of a virus or introducing a set of single-stranded RNA transcripts from the expression cassettes into host cells; and(3) culturing the host cells.The method of the present invention allows more efficient production of an artificial recombinant virus of the family Reoviridae as compared with conventional methods and allows artificial recombinant rotavirus production without using a helper virus.

Provided herein is a method for efficiently capping RNA in vitro. In some embodiments the capping reaction may be done at high temperature using Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reactions may comprise a capping enzyme from a large virus of amoeba, e.g., Faustovirus, mimivirus or moumouvirus, or a variant thereof. Compositions and kits for practicing the method are also provided.

The invention provides methods to inhibit the replication of a flavivirus, methods of inhibiting the guanosine triphosphate (GTP)-binding and guanylyltransferase activity of a flavivirus RNA capping enzyme, and methods of treating a subject infected with a flavivirus. The methods can include contacting a flavivirus with an effective amount of a thioxothiazolidine compound described herein, or a derivative thereof, such as a compound of Formula (I).

The invention provides a small-amount preparation method of self-replicating RNA (Ribonucleic Acid), which comprises the following steps: (1) preparing an IVT reaction template, (2) carrying out IVT reaction, (3) digesting a DNA (Deoxyribonucleic Acid) template, (4) purifying an IVT sample and (5) carrying out capping reaction: adding a capping buffer solution, GTP (Glutathione Triphosphate), SAM (Soluble Amplified Matrix), an RNA enzyme inhibitor, capping enzyme, 2-O-methoxytransferase and denatured RNA into a reaction container, uniformly mixing, and then incubating; and (6) purification: purifying the sample after the capping reaction. According to the method, the in-vitro transcription and capping process of saRNA is optimized, the method is suitable for small-amount preparation of self-replicating RNA, the operation is simple, the self-replicating RNA with high integrity and normal expression can be obtained, and the system can be amplified.

The invention provides a method for in-vitro detection of the activity of vaccinia virus capping enzyme. Specifically, the method comprises the following steps: A, transcribing target gene RNA by using T7 RNA polymerase; b, carrying out capping reaction on the RNA in the step A by using 3 'biotin-GTP and a vaccinia virus capping enzyme; c, carrying out capping RNA adsorption reaction on the RNA in the step B by using streptavidin magnetic beads; and D, detecting the difference of the CT values in the steps B and C by using an RT-qPCR method so as to judge the activity of the vaccinia virus capping enzyme in the step B. The method disclosed by the invention is good in repeatability, the measured capped mRNA yield and non-capped mRNA yield are obviously different, and the capped enzyme activity of the vaccinia virus can be successfully and quantitatively detected.

Provided is a method for producing an artificial recombinant virus of the family Reoviridae, the method comprising the steps of:(1) introducing a FAST protein expression vector and / or a capping enzyme expression vector into host cells;(2) introducing a vector containing expression cassettes for individual RNA genome segments of a virus or introducing a set of single-stranded RNA transcripts from the expression cassettes into host cells; and(3) culturing the host cells.The method of the present invention allows more efficient production of an artificial recombinant virus of the family Reoviridae as compared with conventional methods and allows artificial recombinant rotavirus production without using a helper virus.

To provide a method for efficiently capping RNA in vitro.SOLUTION: In some embodiments, the capping reaction can be done at high temperature using Vaccinia capping enzyme or variants thereof. In other embodiments, the capping reaction may comprise capping enzymes from amoebic large viruses, such as Faust, Mimivirus, or Moumouvirus, or variants thereof. Compositions and kits for practicing the methods are also provided.SELECTED DRAWING: None

Provided herein is a method for efficiently capping RNA in vitro. In some embodiments the capping reaction may be done at high temperature using Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reactions may comprise a capping enzyme from a large virus of amoeba, e.g., Faustovirus, mimivirus or moumouvirus, or a variant thereof. Compositions and kits for practicing the method are also provided.

The present invention provides a composition that includes (i) an RNA-capping enzyme derived from Faustovirus S17, (ii) uncapped target RNA, (iii) guanosine triphosphate (GTP), (iv) a methyl group donor, and (v) a buffer. The present invention also provides a target RNA capping method that includes a step for incubating a composition that includes (i) an RNA-capping enzyme derived from Faustovirus S17, (ii) uncapped target RNA, (iii) guanosine triphosphate (GTP), (iv) a methyl group donor, and (v) a buffer.

The invention provides a method for detecting the activity of capping enzyme of vaccinia virus in vitro. Specifically, the method includes: A. using T7 RNA polymerase to transcribe the target gene RNA; B. using 3'biotin-GTP and vaccinia virus capping enzyme to cap the RNA in step A; C. using streptavidin Avidin magnetic beads perform capping RNA adsorption reaction on the RNA in step B; D. Use RT-qPCR method to detect the difference in CT value in steps B and C, so as to determine the activity of vaccinia virus capping enzyme in step B. The method of the invention has good reproducibility, and the measured yield of capped and uncapped mRNA has significant difference, and can successfully quantitatively detect the capping enzyme activity of vaccinia virus.

The invention discloses a high-efficiency expression and purification method of vaccinia virus mRNA (messenger Ribonucleic Acid) Cap 2 oxygen methyltransferase, which comprises the following steps: based on an escherichia coli expression system, adding a tag and a TEV (Tetraethyl Virus) restriction enzyme cutting site to an optimized vaccinia virus mRNA Cap 2 oxygen methyltransferase gene sequence, connecting to a multiple cloning site of a pGST (Glutathione Sequence Transferase)-parall2 expression vector, and transforming into escherichia coli BL21 for expression; finally, the vaccinia virus mRNA Cap 2 oxygen methyltransferase is obtained through GS4B affinity chromatography, Heparin affinity chromatography and gel filtration chromatography purification, the invention provides a more economical and practical technical route for synthesizing the vaccinia virus mRNA Cap 2 oxygen methyltransferase, and a technical foundation is laid for large-scale production of vaccinia virus capping enzyme.

An RNA capping method including a step of reacting a compound (1) represented by the following general formula (1) with an RNA in the presence of a Vaccinia virus capping enzyme (Rb1 represents an oxo group, an alkoxy group, or a halogen atom; Rb2 is either absent or represents an alkyl group; Rb3 represents an amino group or a hydrogen atom; Rb4 is either absent or represents a hydrogen atom or an alkyl group; Rr1 represents a hydroxy group, an alkoxy group having 1 to 3 carbon atoms, an amino group, an azide group, —OR1C≡CH, or —R2R3; Rr2 represents a hydrogen atom, a hydroxy group, a halogen atom, an alkoxy group, an amino group, an azide group,—OR1C≡CH, or —R2R3; and A1 represents an oxygen atom or a sulfur atom).

Disclosed herein is an engineered fusion enzyme composed of a prokaryotic phage RNA polymerase (T7 RNAP) and a viral capping enzyme (NP868R) for the generation of RNA transcripts containing a 5′ 7-methylguanylate cap.

The invention relates to an enzymatic RNA capping method. Provided herein are methods for efficiently capping RNA in vitro. In some embodiments, a capping reaction may be performed at elevated temperatures using a vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reaction may include a capping enzyme from a megavirus from amoeba, such as a Faustovirus, a Bacteroides virus, or a momuvirus, or a variant thereof. Compositions and kits for practicing the methods are also provided.

Provided herein are methods for effectively capping RNA in vitro. In some embodiments, a capping reaction can be performed at high temperature using a vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reaction can include a capping enzyme from an amoeba giant virus, such as a Faustovirus, a mimivirus, or a moumouvirus, or a variant thereof. Compositions and kits for practicing the methods are also provided.

Provided herein is a method for efficiently capping RNA in vitro. In some embodiments the capping reaction may be done at high temperature using Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reactions may comprise a capping enzyme from a large virus of amoeba, e.g., Faustovirus, mimivirus or moumouvirus, or a variant thereof. Compositions and kits for practicing the method are also provided.

The present disclosure relates, according to some embodiments, to compositions, methods, and / or kits for producing vaccinia capping enzyme. For example, active, heterodimers of vaccinia capping enzyme may be produced as fusions comprising D1 and D12 subunits. Vaccinia capping enzyme fusion proteins may further comprise a linker.