324 results about "Cytidine" patented technology

The instant specification provides for evolved base editors which overcome deficiencies of those in art (including increased efficiency and / or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system. In particular, the instant specification provides for evolved cytidine base editors (e.g., based on APOBEC1, CDA, or AID cytidine deaminase domains) which overcome deficiencies of those in art (including increased efficiency and / or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system.

The invention relates to a metabolic marker for diagnosing diabetic secondary osteoporosis and application of the metabolic marker, and belongs to the technical field of biological medicine. The method comprises the following steps: firstly, screening out obviously different metabolites between osteoporosis (DOP) and osteoporosis-free groups (DM) in diabetic patients; and secondly, screening out significant difference metabolites between the primary osteoporosis patient and the healthy control group, and excluding the intersection of the two groups of difference metabolites to obtain metabolites which exclude the influence of the primary osteoporosis. Metabolic markers, namely cytosine nucleotide, ketoglutaric acid and triethylamine, are obtained through further screening, and when the three metabolic markers are independently used, AUCgt; the AUC during combined use is 0.99, so that the limitation that the sensitivity of the traditional bone mineral density detection on the osteoporosis recognition in the diabetic population is insufficient is effectively made up.

The invention discloses cytosine deaminase as well as a related biological material and application thereof, and belongs to the technical field of protein. The technical problem to be solved by the invention is to excavate natural cytidine deaminase without sequence preference and improve the base editing efficiency and range. The invention provides a 15th-143rd site protein of which the amino acid sequence is SEQ ID NO.2. The invention also provides a preparation method of the protein. The invention also provides application of the protein and related biological materials thereof in single base editing. According to the application, metagenome big data is utilized, comprehensive and systematic information mining analysis is carried out on novel cytidine deaminase, and a protein L85 which is highly similar to a known DddA protein structure but low in sequence similarity is selected from the novel cytidine deaminase for functional verification. The protein L85 has high catalytic activity as cytidine deaminase, is not limited by target sequence preference, can be widely applied to gene editing in vitro and in cells / tissues, and has good application potential and development value.

The invention discloses a genetically engineered bacterium for producing uridine diphosphate glucose as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. The following genes are overexpressed in the genetically engineered bacterium: a pyrimidine nucleoside operon gene, a carbamyl phosphate synthetase gene, an aspartate carbamyl transaminase gene, an orotic acid phosphoribose transferase gene, a phosphoribose pyrophosphate synthase gene, a uridylate kinase gene and a glucophosphate mutase gene. A uridine triphosphate glucose-1-phosphate uridine group transferase gene; the genetically engineered bacterium does not express the following genes: a purine nucleotide repressor protein gene, a deoxyribonucleoside triphosphate reductase gene, a nucleoside triphosphate pyrophosphohydrolase gene, a cytidine triphosphate synthase gene, a pyrimidine nucleotidase gene, a nucleotidase gene, an ornithine carbamoyltransferase gene, a UDPG dehydrogenase gene and a lacI repressor protein gene. The genetically engineered bacterium can be used for efficiently synthesizing UDPG by taking glucose as a substrate.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. The disclosure provides fusion proteins of nucleic acid programmable DNA binding proteins (napDNAbp), e.g., Cas9 or variants thereof, and nucleic acid editing proteins such as cytidine deaminase domains (e.g., novel cytidine deaminases generated by ancestral sequence reconstruction), and adenosine deaminases that deaminate adenine in DNA. Aspects of the disclosure relate to fusion proteins (e.g., base editors) that have improved expression and / or localize efficiently to the nucleus. In some embodiments, base editors are codon optimized for expression in mammalian cells. In some embodiments, base editors include multiple nuclear localization sequences (e.g., bipartite NLSs), e.g., at least two NLSs. In some embodiments, methods for targeted nucleic acid editing are provided.

The invention discloses an Escherichia coli mutant strain which is classified and named as Escherichia coli, the strain number is CR-TK01, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.35554. The invention further discloses a preparation method of the Escherichia coli mutant strain. The Escherichia coli CR-TK01 with high yield of cytidine provided by the invention has the characteristics of high yield and stable high-yield character, glucose is used as a carbon source, the Escherichia coli CR-TK01 is used for fermentation production of cytidine under a certain fermentation condition, and the yield of cytidine is as high as 97.23 g / L; the strain is not degraded after passage for more than 20 generations, the performance of high-yield cytidine is maintained in the passage fermentation process, and the yield of cytidine in passage fermentation is improved. The method for producing cytidine through fermentation is simple in process, high in yield and suitable for industrialization.

The invention discloses a compound derivative represented by a formula I or an isotope labeled compound thereof, or an optical isomer, a geometric isomer, a tautomer or an isomer mixture thereof, or a pharmaceutically acceptable salt thereof. According to the application of a double protection strategy, diversified carrier fragments are introduced to 5-hydroxyl and hydroxylamine in N4-hydroxycytidine molecules, the N4-hydroxycytidine prodrug is invented, the affinity of lung cell membranes (phospholipid membranes) is improved, and the physicochemical properties of the drug are made to meet the requirements of the physiological environment of lung tissue. Therefore, the compound disclosed by the invention has a wide application prospect in the aspect of broad-spectrum respiratory virus resistance. # imgabs0 #

Described herein are methods, compositions, and kits for removing false positive uracils due to the deamination of unmethylated cytosines in assays using engineered cytosine deaminases to deaminate methylated cytosines. The methods, compositions, and kits utilize a dual enzymatic process. After cytidine deaminase treatment, the DNA is first incubated with Uracil DNA Glycosylase (UDG), which generates abasic sites where dC to dU conversions have occurred. The DNA is then incubated with a high-fidelity polymerase supplemented with a deoxycytidyl transferase, such as Rev1, to repair the lesion with the installation of a cytidine.

The present disclosure relates to recombinant microorganisms, such as bacteria, e.g., E. coli, engineered to have improved flux and / or yield of the DXP pathway. Cells can be engineered to comprise nucleotide sequences comprising dxs polypeptides (EC 2.2.1.7); dxr polypeptides (EC 1.1.1.267); ispD polypeptides (EC 2.7.7.60); ispE polypeptides (EC 2.7.1.148); ispF polypeptides (EC 4.6.1.12); ispG polypeptides (EC 1.17.7.1 or EC 1.17.7.3); ispH polypeptides (EC 1.17.7.4); and idi polypeptides (EC 5.3.3.2). Cells can be further engineered to express redox polypeptides, e.g., ferredoxins, flavodoxins, and / or flavodoxin / ferredoxin--NADP reductases (EC 1.19.1.1 or EC 1.18.1.2), e.g., to increase the activity of the ispG and / or ispH polypeptides. Some or all of the nucleotide sequences can be heterologous to the cells. The recombinant microorganisms can be used in methods of isoprenoid production.

The invention relates to the field of anti-tumor drugs, and discloses an anti-diffuse large B-cell lymphoma pharmaceutical composition, a preparation method and application thereof, the composition comprises azacitidine and Sanilisoma, the composition can improve the accumulation amount of VDUP1 protein in a cell nucleus through the combination of the methylation effect of DNMT3b on VDUP1 protein and the inhibition of XPO1 activity by Sanilisoma, and the anti-diffuse large B-cell lymphoma pharmaceutical composition can be used for preparing anti-diffuse large B-cell lymphoma. The chemotherapy effect is remarkably improved, the sensitivity of the DLBCL to the Sanisole is remarkably improved, and particularly, the pharmaceutical composition has a remarkable curative effect on R / R DLBCL patients.

The invention discloses application of N4-hydroxycytidine and a derivative thereof in preparation of a medicine for treating cat abdominal infection. The test result shows that the N4-hydroxycytidine and the derivative thereof have a very small toxic effect on normal cat cells, have an obvious inhibiting effect on cat coronavirus (FCoV), and can obviously reduce the virus titer of the virus in the cells. Therefore, the N4-hydroxycytidine and the derivative thereof can be used for preparing a feline coronavirus inhibitor for treating feline infectious peritonitis, and are good in curative effect and high in safety.

The invention discloses escherichia coli recombinant engineering bacteria for producing citicoline as well as a construction method and application of the escherichia coli recombinant engineering bacteria, and belongs to the technical field of biology. A genome cytidine deaminase coding gene cdd is knocked out on the basis of Escherichia coli K-12 MG1655, a temperature control expression vector PBV220 is used for expressing a SpnCCT-SpnCKI-pRpLpyrG mutant gene cluster, the expression of choline transporter protein BetT is enhanced, the gene locus of Escherichia coli ptsH-ptsI-crr is destroyed, meanwhile, a glucose permease gene glf and a glucokinase gene glk from Zymomonas mobilis are introduced, and the mutant gene cluster of the Escherichia coli K-12 MG1655 is obtained. The escherichia coli recombinant engineering bacterium ZMCB02NCL capable of efficiently biosynthesizing citicoline is obtained, and the preservation number of the escherichia coli recombinant engineering bacterium ZMCB02NCL is CCTCC NO: M 2025483.

The invention discloses a method for improving the survival rate of probiotics based on cell membrane regulation and control. The method comprises the following steps: adding a lipid component in a probiotic culture process and / or adding the lipid component as a protective agent after probiotic culture, the lipid component added in the probiotic culture process comprises cytidine diphosphate diacylglycerol and / or stearic acid; the lipid component added as the protective agent after culture comprises cardiolipin and / or oleic acid. The method breaks through the research category of a traditional optimized freeze drying process (such as a protective agent and parameters), and key lipid molecules directly related to resistance are screened out through a lipidomics means and establishment of a screening method of stress resistance related lipid molecules; through a dual mode of supplementing key lipid components and precursor substances thereof, the tolerance of the strain is improved from the metabolic level, and the freeze-drying resistance and the survival rate of the strain are remarkably improved.

The invention relates to an RNAN4-acetylcytidine modification site prediction method and an RNAN4-acetylcytidine modification site prediction system based on an RNAErnie pre-training model. The method comprises the following steps: collecting an RNA sequence data set, inputting the RNA sequence data set into an RNAErnie pre-training model, capturing a context dependency relationship, and extracting global features; performing feature coding through an RNAErnie pre-training model in combination with six traditional feature coding methods; and performing feature dimension reduction in the deep neural network model, inputting the feature dimension reduction result into a soft voting integration model, and integrating prediction results of different classifiers to obtain an RNAN4-acetylcytidine modification site prediction result. Multistage masking is carried out through an RNAErnie pre-training model, and details and physical and chemical attributes of the sequence can be captured in combination with six traditional feature coding modes; feature dimension reduction is automatically performed by using a deep neural network, so that the calculation complexity is reduced and key information is reserved; and the dimensionality-reduced features are input into a soft voting integrated model, and a final prediction result is obtained by integrating a plurality of classifiers, so that the prediction accuracy and robustness are remarkably improved.

The invention provides inter alia methods of treating cancer comprising administering to a subject a cytidine triphosphate synthase 1 (CTPS1) inhibitor and a B-cell lymphoma 2 (BCL2) inhibitor.

The present disclosure generally relates to an evolved cytidine deaminase derived from a cytidine deaminase, and a method of editing DNA using the same. In some aspects, the present disclosure describes directed evolution of a TadA-derived adenosine deaminase (TadA-CD) for cytidine deamination. In some embodiments, the TadA-CD comprises a plurality of mutations as compared to the parent TadA variant. In some embodiments, the TadA-CD is fused to a programmable DNA binding protein. Other aspects of the present disclosure generally relate to a cytosine base editor (CBE) comprising a programmable DNA binding protein and a TadA-CD. In some embodiments, the disclosed cytosine base editor has an improved conversion efficiency and a reduced off-target editing frequency compared to a naturally occurring CBE. Polynucleotides, vectors, and kits for generating and delivering CBE are also provided. Also provided are cells containing such vectors and CBE. In addition, methods of treatment comprising administration of CBE are also provided.

The present invention belongs to the technical field of genetic engineering. Provided are a single-base editor, a deaminase used therein, and the use thereof. The technical problems to be solved are to identify a naturally occurring cytosine deaminase without sequence preference, construct a base editor, and improve the efficiency and scope of base editing. In order to solve the technical problems above, a cytosine base editor is provided. The cytosine base editor is a fusion protein, wherein the fusion protein is a protein containing a cytidine deaminase, a Cas protein and a uracil-DNA glycosylase inhibitor, and the cytidine deaminase is a protein having an amino acid sequence of positions 28-164 of SEQ ID NO. 2. Further provided is the use of the fusion protein above and a biomaterial related thereto in plant single-base editing. The single-base editor can improve the efficiency of cytosine base editing and accurately mediate the base mutation of a target, and is widely applicable in the cells of maize and even other plants.

The present disclosure relates to methods for increasing cell-mediated immunity against varicella zoster virus (VZV) in a human subject in need thereof by administration of an immunogenic composition comprising effective amounts of a VZV glycoprotein E antigen and an oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif. The immunogenic compositions are suitable for prevention of herpes zoster and / or postherpetic neuralgia.

The invention belongs to the technical field of bioengineering, and particularly relates to a phosphorylcholine cytidyltransferase mutant and application thereof. On the basis of phosphorylcholine cytidyltransferase MsCCT001, any one or more of N194D, K91E, G98S, S148A and C156V are mutated respectively, a series of phosphorylcholine cytidyltransferase mutants are obtained through screening, and the obtained mutants have the advantages that in the reaction of catalyzing phosphorylcholine and cytidine triphosphate to generate citicoline, the yield of the obtained mutants is increased, and the yield of the obtained mutants is increased. The catalytic efficiency of the compound is 1.90-2.41 times that of initial phosphorylcholine cytidyltransferase, and a biological enzyme preparation with higher catalytic capacity is provided for preparing citicoline by a biological conversion method.

The invention discloses a single-base editor, deaminase used by the single-base editor and application of the single-base editor, and belongs to the technical field of genetic engineering. The technical problems to be solved in the invention are as follows: natural cytosine deaminase without sequence preference is mined, a base editor is constructed, and the base editing efficiency and range are improved. In order to solve the technical problem, the application provides a cytosine base editor, the cytosine base editor is a fusion protein, the fusion protein is a protein containing cytidine deaminase, a Cas protein and a uracil glycosylase inhibitor, and the cytidine deaminase is a protein at the 28th-164th site of an amino acid sequence SEQ ID NO.2. The invention also provides application of the fusion protein and related biological materials thereof in plant single base editing. The single-base editor provided by the invention can improve the efficiency of cytosine base editing, can accurately mediate the base mutation of a target site, and can be widely applied to corn and even other plant cells.

The invention belongs to the field of synthetic biology and metabolic engineering, and particularly relates to high-yield sialyl acyl-N-tetrasaccharide a escherichia coli as well as a construction method and application thereof. According to the method, recombinant escherichia coli is taken as an initial strain, and a beta1, 3-galactosyl transferase gene wbgO from escherichia coli O55: H7 is integrated into a chromosome in a multi-copy form, so that a lactyl-N-tetrasaccharide efficient synthesis chassis strain without plasmid dependence is constructed. And overexpression neuC, neuB, neuA and Nm3ST are introduced through plasmids, so that the supply of glycosyl donors is improved. The expression intensity of uridine diphosphate-N-acetylglucosamine synthesis pathway genes glmM and glmUS is regulated through ribosome binding site engineering, and a cytidine triphosphate regeneration module is introduced, so that the yield of sialyloyl-N-tetrasaccharide a is increased. And finally, the maximum titers of the sialyloyl-N-tetrasaccharide a respectively reach 2.238 g / L and 8.178 g / L by shaking a flask and culturing in batches. The invention provides an efficient technical scheme for industrial production of complex breast milk oligosaccharide.

A method for preparing pseudouridine through whole-cell catalysis of recombinant Escherichia coli. The recombinant Escherichia coli is obtained by overexpressing pseudouridine-5-phosphoglycosidase, ribokinase, ribonucleoside hydrolase, cytosine deaminase and nucleoside permease, and not expressing pseudouracil transport protein in Escherichia coli E.coHMG1655; and whole-cell catalytic preparation of pseudouridine taking uridine or cytidine as a substrate is realized by using the recombinant Escherichia coli. A novel pseudouridine enzyme catalytic synthesis route is designed, and in optimal reaction conditions, the maximum yield of pseudouridine reaches 27.24 g / L, the conversion rate reaches 90.8%, the production efficiency is 1.135 g·h-1·L-1, and the pseudouridine yield and the production efficiency are high.

The invention relates to a T7RNA polymerase-guided bacillus subtilis cytosine base editor and application thereof, and belongs to the technical field of gene editing. A T7RNAP guided cytosine base editor is developed in bacillus subtilis, four kinds of cytidine deaminase from different sources are selected to be fused with T7RNAP, the cytidine deaminase is fused at the N end of the T7RNAP, and a fusion body with the highest mutation activity is obtained through screening; the cytosine base editor is used for evolving a global transcriptional regulatory factor CodY, in-situ evolution of genome protein is achieved, and the expression level of bovine-derived beta-lactoglobulin (beta-Lg) is improved. According to the cytosine base editor, the operable range of the cytosine base editor on a genome is widened, multi-site base (C-T) editing can be realized, and an editing window is large.

The application discloses cytosine deaminase and related biological materials and applications thereof, and belongs to the technical field of proteins. The technical problem to be solved by the application is to mine natural sequence-unpreferred cytosine deaminase, and to improve base editing efficiency and range. The application provides a protein with amino acid sequence of SEQ ID NO. 2 at positions 15-143. The application also provides application of the above protein and related biological materials thereof in single base editing. The application uses macro-genome big data, performs comprehensive and systematic bioinformatics mining analysis on novel cytosine deaminase, and selects a protein L85 highly similar to known DddA protein structure but with low sequence similarity for function verification. The protein L85 has high catalytic activity as cytosine deaminase, and has no target sequence preference restriction, and can be widely applied to in-vitro and in-cell / tissue gene editing, and has good application potential and development value.

This invention provides a simple synthesis method for Fe-Ag / CMP nanopolymers with catalase-like activity and their application in hydrogen peroxide detection, belonging to the field of bioanalytical technology. The method of this invention includes the following steps: (1) preparing a 4-hydroxyethylpiperazine thiosulfonic acid (HEPES) standard buffer solution; (2) adding cytidine monophosphate (CMP) to the solution obtained in step (1) and stirring until the CMP is fully dissolved. Then, under vigorous stirring, ferric nitrate aqueous solution and silver nitrate aqueous solution are added sequentially, at which point the colorless solution turns pale yellow; (3) transferring the mixed solution obtained in step (2) to a centrifuge tube, centrifuging at 12000 rpm for 8 min to collect the precipitate, washing it three times with distilled water, and finally adding 4 mL of distilled water to obtain a 1 mg / mL Fe-Ag / CMP suspension. This invention utilizes CMP as a biological ligand and Ag... + Fe 3+ Simultaneous coordination, and the reducing amino groups on CMP can directly transfer Ag... + In situ reduction to silver nanoparticles (AgNPs) led to the one-step synthesis of a bimetallic Fe-Ag / CMP nanopolymer with hydrogen peroxide-like nanozyme activity, enabling the detection of hydrogen peroxide.

Aspects of the disclosure relate to mRNAs comprising a relatively low abundance of cytidine: adenosine (CA) dinucleotides that benefit from increased stability relative to mRNAs containing more CpA dinucleotides. The disclosure also relates to methods of modifying an mRNA sequence to improve stability. In some aspects, the disclosure relates to mRNAs comprising modified mRNA sequences with relatively reduced numbers of CpA dinucleotides, and compositions comprising mRNAs with relatively reduced numbers of CpA dinucleotides.

This invention discloses a method for directed evolution of antibiotic-resistant Escherichia coli strains based on cytidine deaminase, belonging to the field of microbial directed evolution and genetic engineering technology. This method uses E. coli as the host, introducing a recombinant plasmid expressing an optimized double-stranded cytidine deaminase mutant. Utilizing the low toxicity and high mutagenicity of this mutant, continuous passage evolution is carried out under gradient concentrations of aminoglycoside antibiotics (kanamycin and streptomycin). Combined with whole-genome sequencing, molecular docking, and reverse genetics verification, the A145T missense mutation in the wcaE gene is identified as the core functional site. This invention overcomes the shortcomings of traditional spontaneous and chemical mutagenesis, which suffer from low efficiency and significant strain damage. The mutation type is controllable, the evolutionary cycle is short, and the obtained engineered strains can tolerate up to 300 mg / L kanamycin while exhibiting streptomycin cross-resistance, and the genetic stability of the tolerance trait is strong. This method is simple, highly reproducible, and suitable for industrial breeding of stress-resistant E. coli, and can be widely applied in antibiotic fermentation, industrial microbial culture, and other scenarios.

This disclosure relates to certain N4-hydroxycytidine derivatives, pharmaceutical compositions, and methods related thereto. In certain embodiments, the disclosure relates to the treatment or prophylaxis of viral infections, such as Eastern, Western, and Venezuelan Equine Encephalitis (EEE, WEE and VEE, respectively), Chikungunya fever (CHIK), Ebola, Influenza, RSV, and Zika virus infection with the disclosed compounds.

Provided are a new CRISPR-Cas12b-based base editor and the use thereof in gene expression and / or protein evolution. The base editor uses a fusion protein comprising a deaminase, a Cas protein mutant dBhCas12b and a uracil glycosylase inhibitor domain UGI, wherein the deaminase is located at the N-terminus of dBhCas12b, and comprises cytidine deaminase CDA as shown in SEQ ID NO. 1 or adenosine base editor ABE8e as shown in SEQ ID NO. 2, and the uracil glycosylase inhibitor domain UGI is located at the C-terminus of dBhCas12b, and has an amino acid sequence as shown in SEQ ID NO. 4. Compared with an original sequence as shown in SEQ ID NO. 3, dBhCas12b has mutations comprising the following: aspartic acid at position 574, glutamic acid at position 828 and aspartic acid at position 952 are mutated into alanine A.

The invention belongs to the technical field of genetic engineering and microbial engineering, and particularly relates to a method for improving the production level of cytidine and application. According to the recombinant escherichia coli, a glucose-6-phosphate dehydrogenase gene zwf and a 6-phosphogluconate dehydrogenase gene gnd are subjected to overexpression. Specifically, the fusion expression of a glucose-6-phosphate dehydrogenase gene zwf and a 6-phosphogluconate dehydrogenase gene gnd is carried out, and the fusion expression is carried out by adopting Linkers with different properties. During application, the recombinant escherichia coli can be used for producing the cytidine by fermentation, so that the production level of the cytidine is improved. According to the invention, recombinant Escherichia coli which is over-expressed and fused with a glucose-6-phosphate dehydrogenase gene zwf and a 6-phosphogluconate dehydrogenase gene gnd is constructed, and the recombinant Escherichia coli can be used for efficiently fermenting and producing cytidine.