36 results about "Ribonuclease" patented technology

The present application relates to a deoxyribonuclease mutant and its application in preparing PDRN. The amino acid sequence of the deoxyribonuclease mutant is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2. The present application also provides the application of the deoxyribonuclease mutant and / or the encoding gene of the deoxyribonuclease mutant in preparing PDRN, and the specific application method. The present application is based on the deoxyribonuclease expressed by the DNASE1 gene of bovine pancreas tissue, and the amino acids at positions 69, 132 and 215 are selected for iterative saturation mutation. Compared with the wild-type enzyme, the enzyme activity of the deoxyribonuclease mutant A69H-S132R-Q215C is increased by 2.15 times, reaching 4.1x10 6 U / mL, and the DNA extraction rate of salmon PDRN prepared by using the mutant enzyme prepared by the present application is 15.2%, the DNA content is 99.1%, and the molecular weight is 1.05 million Daltons, which is significantly improved compared with the wild-type enzyme, and the PDRN in this molecular weight range has more significant biological activity.

The present disclosure is generally directed to anchoring bridge nucleic acid probes in a polymer matrix, which are configured to hybridize to cellular nucleic acid, embedding in a polymer matrix, clearing cellular components including the cellular nucleic acid, contacting the anchored bridge probes with primary probes that hybridize with the bridge probes and imaging. The disclosure describes methods for imaging cellular RNA in an RNase insensitive method.

This invention relates to the field of pharmaceutical technology, and discloses a stress particle targeted degrader, its preparation method, and its applications. The compounds provided by this invention include ternary complexes composed of stress particle protein, a linker group, and an E3 ubiquitin ligand, or ternary complexes composed of stress particle protein, a linker group, and a ribonuclease L ligand. Compounds with different ligands exhibit good targeting and degradation effects on stress particles and can be used as stress particle targeted degraders in the preparation of antitumor drugs and anti-neurodegenerative disease drugs, showing promising application prospects in treating stress particle-induced tumor drug resistance and neurodegenerative diseases.

The application discloses a single cell whole genome amplification sequencing method. The application comprises the following steps: 1) separating a single cell, adding a single cell lysis solution to sufficiently lyse the single cell and digest proteins combined on the genomic DNA; 2) using a Tn5 transposome with a specific adapter sequence to fragment the DNA, while adding adapters to both ends of the fragments; 3) after end repair, using a ribonuclease to cut modified ribonucleotide residues on the adapter sequence; 4) using the adapter sequence as a primer to linearly amplify the fragmented DNA by using a single primer PCR method; and 5) adding sequencing adapters to both ends of the DNA fragments by using a chain extension and PCR method to obtain a final sequencing library. The method disclosed by the application can amplify single cell whole genome DNA with high coverage, uniformity and fidelity, and simultaneously and accurately detect chromosomal copy number variation CNV and single nucleotide mutation SNV on a single cell genome.

The invention relates to the technical field of biotechnology, enzyme engineering and biological pharmacy, in particular to a ribonuclease purification production method. According to the method, a deep sea antifreeze polypeptide and rare earth nanocrystal composite bionic system is constructed, quorum sensing signal molecules and a pulsed electric field are introduced in a fermentation stage for two-factor regulation, and a light-operated micro-fluidic chip selective extraction technology is combined, so that efficient separation of target enzyme is realized. A double-ligand magnetic microsphere chromatographic column is innovatively adopted for gradient elution, and the enzyme activity retention rate and the long-term storage stability are remarkably improved through thermotropic liquid crystal capsule stabilization and radio frequency assisted freeze drying processes. According to the invention, the destructiveness of the traditional chromatography technology to enzyme conformation is broken through, the problem of batch fluctuation caused by multi-step purification is solved, a ribonuclease product with high purity, high activity and environmental response characteristic is provided for the field of biological medicine, and the ribonuclease is especially suitable for industrial preparation of gene therapy products and virus vectors.

This disclosure provides several ribonuclease R variants and their preparation methods. Compared to the parent enzyme, the variants described in this disclosure exhibit increased protease activity per unit and show promising prospects for industrial applications. Furthermore, this disclosure also provides the application of ribonuclease R variants in linear RNA degradation.

The invention relates to gene therapy, and more specifically, to AAV gene therapy vectors containing a novel chimeric deoxyribonuclease (DNase) protein transgene and methods of treating ailments such as cancer and neurodegeneration.

The invention relates to the technical field of biological medicine, in particular to DNase I loaded lipidosome and application thereof. The invention provides a bionic long-acting deoxyribonuclease I (DNase I) liposome with free DNA (cfDNA) responsiveness and a dual-targeting function, the liposome is subjected to layered fusion modification through an erythrocyte membrane and a neutrophile granulocyte membrane, a release window is accurately regulated and controlled by utilizing a 4ME-PC proportion, and a liposome drug carrier for cfDNA targeted combination and accurate delivery of an inflammation part is realized. The polypeptide can be used for cfDNA-related inflammatory diseases, and has an important application prospect.

The invention discloses a ribosome group or selective ribosome group detection and analysis method based on an immune principle, and relates to the field of molecular biology and genomics. Comprising the following steps: sample preparation and cross-linking: treating cells by using a translation inhibitor to suspend ribosome translation, and then cross-linking RNA-protein in the cells by using a membrane permeable cross-linking agent; then cracking the cells, digesting RNA of a cracking product by using ribonuclease, and retaining ribosome and mRNA fragments protected by the ribosome; immune enrichment: carrying out specific immune enrichment on the digested sample by using an antibody combined with a carrier, and eluting to obtain a ribosome-mRNA compound; then decrosslinking and removing protein, extracting mRNA fragments and carrying out phosphorylation treatment so as to add joints, build a library and carry out sequencing; and finally, library construction, sequencing and bioinformatics analysis are carried out. The method has the advantages of high-specificity enrichment, accurate positioning of translation sites, wide application range and accurate sequence information.

The present disclosure provides, among other things, methods for removing linear ribonucleic acid molecules from a sample by contacting the sample with ribonuclease R (RNase R) from a thermophilic organism, for example, Geobacillus stearothermophilus, Fervidobacterium pennivorans or Thermus thermophilus expressed and purified in E.coli. Also provided herein are methods of producing circular RNA and removing uncircularized linear RNA molecules, linear RNA byproducts, or linear RNA side products using an RNase R expressed or purified from a thermophilic organism. The present disclosure also provides methods of purifying RNase R from a thermophilic organism in E. coll, wherein the method comprises heat treatment at high temperatures, and compositions and kits comprising the same.

The invention relates to the technical field of gene engineering, in particular to a fusion protein expression cassette for rice multi-gene editing and application of the fusion protein expression cassette. The expression of the fusion protein for multiple gene editing of rice provided by the invention comprises an endonuclease Csy4, a DNA (Deoxyribose Nucleic Acid) endonuclease nickase Cas9, an M-MLV reverse transcriptase and a Cas12a nuclease; in the guide editing fusion protein frame, the ribonuclease Csy4 is located at the N end of the guide editing fusion protein frame, the DNA incision enzyme nickase Cas9 is located between the ribonuclease Csy4 and the M-MLV reverse transcriptase, the M-MLV reverse transcriptase is located between the DNA incision enzyme nickase Cas9 and the Cas12a nuclease, and the Cas12a nuclease is located at the C end of the fusion protein frame. The fusion protein expression cassette not only can ensure the editing efficiency, but also can improve the editing accuracy.

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes that comprise a ribonucleotide position that is susceptible to endoribonuclease (e.g., RNase H) cleavage in the presence of target nucleic acid molecules. Probes of the embodiments may also comprise non-natural nucleotide linked to a reporter and / or quenching moiety.

The purpose of the present invention is to provide an immunogenic composition containing a new HBV polypeptide or polynucleotides encoding the polypeptide. The immunogenic composition contains at least one polypeptide selected from among polypeptides a) through c) or polynucleotides encoding the polypeptide, and can be used for treating or preventing HBV infection. a) an HBV surface protein polypeptide including a specific amino acid substitution; b) an HBV polymerase polypeptide including an inactivated reverse transcriptase domain and an inactivated ribonuclease H domain, and including a specific amino acid substitution; and c) an HBV core protein polypeptide including a specific amino acid sequence.

The invention relates to a novel liposome polydopamine nanoparticle and application thereof in treating renal cell carcinoma. The preparation method of the nanoparticles comprises the following steps: adding liposome into si-circPVT1 dissolved in ribonuclease-free water, and carrying out vortex mixing to form a si-circPVT1 liposome suspension; dispersing the dopamine PDA modified liposome into a Tris-HCl solution, then adding dopamine hydrochloride, and stirring to obtain dopamine PDA modified liposome; then dispersing in a streptavidin solution, and fully oscillating at 4 DEG C under a dark condition to obtain a compound; and mixing the complex with a biotin-labeled MUC12 antibody solution for incubation, and washing the obtained suspension with a PBS (Phosphate Buffer Solution) to obtain the novel liposome polydopamine nanoparticles marked as SCLPM-NPs. The product prepared by the invention realizes targeted delivery of kidney cell cancer cell lines, and the SCLPM-NPs can effectively target tumor tissue parts and inhibit the growth of kidney tumors.

The invention relates to the technical field of molecular biology, in particular to a structural probe and application thereof. The invention provides a nucleic acid probe which is single-stranded oligonucleotide, and the single-stranded oligonucleotide has a stem-loop structure; the number of the stem rings is larger than or equal to 2. The detection method provided by the invention is high in sensitivity, has ultrahigh sensitivity higher than that of a common single-chain fluorescent probe as an RNase reaction substrate, and can detect RNase A as low as 0.1 pg / ml. The probe disclosed by the invention has broad spectrum and has detection capability on various ribonuclease.

Compositions and methods relating to ApoA-1 fusion polypeptides are disclosed. The fusion polypeptides include a first polypeptide segment corresponding to an ApoA-1 polypeptide or ApoA-1 mimetic, and may also include a dimerizing domain such as, e.g., an Fc region, which is typically linked carboxyl-terminal to the first polypeptide segment via a flexible linker. In some embodiments, the fusion polypeptide further includes a second polypeptide segment located carboxyl-terminal to the first polypeptide segment and which confers a second biological activity (e.g., an RNase, paraoxonase, platelet-activating factor acetylhydrolase, cholesterol ester transfer protein, lecithin-cholesterol acyltransferase, polypeptide that specifically binds to proprotein convertase subtilisin / kexin type 9, or polypeptide that specifically binds to amyloid beta). Also disclosed are dimeric proteins comprising first and second ApoA-1 fusion polypeptides as disclosed herein. The fusion polypeptides and dimeric proteins are useful in methods for therapy.

The present disclosure provides a plurality of RNase R variants and a preparation method thereof, wherein the expression amount of the variant RNase R is significantly increased relative to the parent enzyme, and the enzyme activity of the variant RNase R can be increased by 3 times, and the variant RNase R has a good industrial application prospect. In addition, the present disclosure also provides the application of the RNase R variant in the degradation of linear RNA and the preparation of circRNA.

The invention provides an integrated DNA interference excluding in-situ hybridization probe and an in-situ hybridization method for detecting hepatitis B virus cccDNA, the in-situ hybridization probe is designed based on a Basescope detection method, and comprises an HBV cccDNA probe specifically targeting an integrated breakpoint of HBV negative chain DNA, an HBV DNA probe targeting an HBV negative chain, an HBV cccDNA probe targeting an HBV negative chain, an HBV cccDNA probe targeting an HBV negative chain, an HBV cccDNA probe targeting an HBV negative chain, and an HBV cccDNA probe targeting an HBV negative chain. The HBV RNA probe with the targeted HBV positive strand gap structure and the matched ribonuclease of the HBV RNA probe can be used for detecting cccDNA and integrating HBV DNA after being treated, and the targeting sequences of the HBV cccDNA, the HBV DNA and the HBV RNA probe are respectively shown as SEQ ID No.1 to SEQ ID No.3. The in-situ hybridization probe and the in-situ hybridization method can be applied to screening medicines for preventing or treating hepatitis B virus infection and researching the life cycle of the hepatitis B virus, so that the understanding on the characteristics of the hepatitis B virus is promoted, and the evaluation on a treatment strategy is optimized.

The application provides a mutant ribonuclease R (RNase R) and application thereof. The application provides 10 mutant ribonucleases R by creatively selecting and screening the site mutation of Escherichia coli ribonuclease R, and mutant T495E, A246E, K457F, A246F, D281F, D122L with significantly improved enzyme activity relative to the parent enzyme are obtained. The mutant T495E has the most significant enzyme activity improvement, and the relative enzyme activity is improved by 37.48%, and the enzyme activity of other mutants is respectively improved by 24.74%, 21.74%, 10.04%, 10.34% and 6.75%. The application also provides a recombinant Escherichia coli with high expression of ribonuclease R, which is used for realizing efficient production of RNase R mutants and has a good industrial application prospect. The mutant ribonuclease R with improved enzyme activity provided by the application is suitable for industrial large-scale preparation of mRNA, and is particularly suitable for application in the field related to degradation of linear RNA or production of circRNA.

Disclosed herein are improved broad-spectrum nanozymes for targeting RNA. The disclosed nanozymes are synthesized using recombinant ribonuclease with site-specific cysteine-substituted mutations that can be covalently functionalized with a length-tunable multithiol tether and then loaded onto gold particles through multiple gold-sulfur bonds, or inorganic particles with specific multiple ligand-to-particle-surface bonds. The disclosed nanozymes are also densely loaded with protective DNA oligonucleotides. In some embodiments, the disclosed nanozyme are core-free hollow forms. The removal of the inorganic nanoparticle cores from nanozymes can effectively eliminate the potential long-term toxicity induced by the core, and also creates a cavity for loading and delivery of small molecule drugs.

The present invention relates to a process for obtaining a yeast protein comprising the following steps: a) providing a yeast cream; b) exposing the yeast cream to heat plasmolysis at a temperature of 70 to 95°C for 30 seconds to 4 hours, preferably 1 minute to 3 hours, more preferably 40 minutes to 2 hours; b') separating the insoluble fraction and the soluble fraction; c) subjecting the insoluble fraction to the activity of at least one ribonuclease and one glucanase, sequentially or simultaneously, at a temperature of 40 to 65°C, preferably 60°C, for 8 to 24 hours, preferably 18 hours; d) separating the insoluble fraction from the soluble fraction; wherein the insoluble fraction collected in step d) has no taste, a nucleotide content of less than 3% and a true protein content of at least 72%. Step b') is optional. In this case, the whole composition obtained after heat plasmolysis of the yeast cream is subjected to the enzymatic activity.

The application discloses application of a high-efficiency and heat-stable ribozyme SiRe_0902 in specific recognition of a GG site. The amino acid sequence of the ribozyme SiRe_0902 is shown as SEQ ID NO. 2, and the nucleotide sequence is shown as SEQ ID NO. 1. The ribozyme SiRe_0902 has very high and specific hydrolysis activity on RNA, and has no non-specific cutting on DNA. In addition, the enzyme is derived from a thermophilic archaea, and is detected to have good heat stability and very good RNA cutting activity under conditions of 37 DEG C and 65 DEG C. The enzyme is detected to be capable of specifically recognizing and cutting a GG sequence, and also has high cutting activity on a GG sequence in a secondary structure region. Therefore, the ribozyme SiRe_0902 is a new heat-stable ribozyme, and has very high RNA hydrolysis activity under normal temperature and high temperature conditions, and meets the needs of clinical, production and molecular cloning fields.

This application belongs to the field of bioanalytical detection technology, specifically disclosing a detection system and method for live Salmonella based on deoxyribonuclease activation. The detection system includes a Sub probe, a deoxyribonuclease probe, a hairpin probe, hyperbranched dendritic nanomolecules, a crRNA / Cas12a binary complex, a fluorescent probe, and magnetic beads. The hyperbranched dendritic nanomolecules include substrate A, substrate B, and a trigger strand B probe. Substrate A consists of an A-F strand probe, an A-Q strand probe, and a helper strand A probe, while substrate B consists of a B-F strand probe, a B-Q strand probe, and a helper strand B probe. This detection system specifically recognizes ribonuclease H2 released only by the metabolism of live Salmonella and utilizes it to activate the Sub-Dz substrate, ensuring from the source that only live bacteria can trigger the subsequent reaction. This effectively solves the problem of interference from dead bacteria, achieving highly sensitive, highly specific, and rapid detection of live Salmonella.

This invention provides a mutant ribonuclease R (RNase R) and its applications. Through creative selection and screening of mutations at the RNase R site in *E. coli*, this invention provides 10 mutant RNase R variants, obtaining mutants T495E, A246E, K457F, A246F, D281F, and D122L with significantly increased enzyme activity relative to the parental strain. Among these, mutant T495E shows the most significant increase in enzyme activity, with a relative increase of 37.48%. The other mutants show increases of 24.74%, 21.74%, 10.04%, 10.34%, and 6.75%, respectively. This invention also provides recombinant *E. coli* strains that highly express RNase R, enabling efficient production of RNase R mutants with promising industrial application prospects. The RNase R mutants with increased enzyme activity provided by this invention are suitable for large-scale industrial production of mRNA, particularly in the degradation of linear RNA or the production of circRNA.

Compositions and methods relating to ApoA-1 fusion polypeptides are disclosed. The fusion polypeptides include a first polypeptide segment corresponding to an ApoA-1 polypeptide or ApoA-1 mimetic, and may also include a dimerizing domain such as, e.g., an Fc region, which is typically linked carboxyl-terminal to the first polypeptide segment via a flexible linker. In some embodiments, the fusion polypeptide further includes a second polypeptide segment located carboxyl-terminal to the first polypeptide segment and which confers a second biological activity (e.g., an RNase, paraoxonase, platelet-activating factor acetylhydrolase, cholesterol ester transfer protein, lecithin-cholesterol acyltransferase, polypeptide that specifically binds to proprotein convertase subtilisin / kexin type 9, or polypeptide that specifically binds to amyloid beta). Also disclosed are dimeric proteins comprising first and second ApoA-1 fusion polypeptides as disclosed herein. The fusion polypeptides and dimeric proteins are useful in methods for therapy.

The invention belongs to the technical field of biological analysis and detection, and particularly discloses a live salmonella detection system and method based on deoxyribozyme activation, the detection system comprises a Sub probe, a deoxyribozyme probe, a hairpin chain probe, a hyperbranched dendritic nano-molecule, a crRNA / Cas12a binary compound, a fluorescent probe and magnetic beads, the hyperbranched dendritic nano-molecule comprises a substrate A, a substrate B and a trigger chain B probe, the substrate A is composed of an A-F chain probe, an A-Q chain probe and an auxiliary chain A probe, and the substrate B is composed of a B-F chain probe, a B-Q chain probe and an auxiliary chain B probe. According to the detection system, ribonuclease H2 released only by metabolism of living salmonella is specifically recognized, and is utilized to activate a Sub-Dz substrate, so that the subsequent reaction can be triggered only by viable bacteria from the source, the problem of dead bacteria interference is effectively solved, and high-sensitivity and high-specificity rapid detection of the viable salmonella is realized.

Provided herein are modular programmable synthetic receptors comprising a ligand binding domain, a kinase domain, and an RNase domain. Also provided herein are engineered synthetic RNAs recognized by synthetic receptors provided herein, as well as cells comprising the synthetic receptors and engineered synthetic RNAs, and methods of making and using the same.

The invention relates to gene therapy, and more specifically, to AAV gene therapy vectors containing a novel chimeric deoxyribonuclease (DNase) protein transgene and methods of treating ailments such as cancer and neurodegeneration.

This invention discloses a universal method for preparing nuclear suspensions suitable for flow cytometry of Rosa species, belonging to the field of plant biotechnology. The method uses WPB dissociation buffer and follows a standardized procedure to process Rosa leaves in different storage states through sample dissociation, filtration, staining, and incubation steps: leaf tissue is dissociated using WPB dissociation buffer pre-cooled to 4°C, allowed to stand on ice, filtered through a 40µm cell filter, and brought to volume. Propidium iodide and ribonuclease A are added for staining and incubation before flow cytometry analysis. This invention is directly applicable to Rosa leaves in three storage states: fresh, silica gel dried, and -80°C frozen. It is simple to operate, highly versatile, and the prepared nuclear suspension yields high-quality peak histograms with low coefficient of variation when analyzed by flow cytometry, providing an efficient and standardized technical solution for large-scale ploidy analysis and genome size determination of Rosa species.

The application discloses a virus storage solution and a preparation method thereof, and belongs to the technical field of virus storage. The virus storage solution prepared by the application can rapidly inactivate and lyse collected viruses, and free RNA in solution can be prevented from being degraded by ribonuclease under the action of the storage solution, so that stable storage of a sample containing virus nucleic acid is realized. The virus storage solution of the application can protect a virus RNA sample, reduce the decomposition effect of RNAase on virus RNA, improve the sensitivity of nucleic acid detection, and is particularly suitable for collection of epidemic virus samples with strong infectivity.