45 results about "Nucleotide synthesis" patented technology

Comprising a multivalent fusion protein and an exosome delivery carrier, and the content of the multivalent fusion protein is 70-90 [mu] g; wherein the multivalent fusion protein is a fusion protein formed by connecting a gB glycoprotein, a gC glycoprotein, a gD glycoprotein and a high-immunogenicity part of an important antigen segment selected from gE and gI; the exosome delivery carrier is a small vesicle extracted and purified from mesenchymal stem cells or HEK 293-F cells, the surface of the exosome delivery carrier is modified with a CD40L single-chain antibody, and the exosome delivery carrier is used for loading multivalent fusion protein; the porcine pseudorabies virus vaccine further comprises adjuvants, and the adjuvants comprise a TLR agonist, CpG oligonucleotide, synthetic liposome and a traditional aluminum salt adjuvant. The porcine pseudorabies virus vaccine also comprises a mucous membrane adsorbent, a penetration enhancer, a stabilizer and a preservative. The invention aims to construct a multivalent fusion protein complex containing a plurality of important antigen segments by using an advanced bioengineering technology, and provides a wider protection effect by combining an efficient exosome delivery platform and a method for enhancing immune response.

This invention provides an oligonucleotide synthesis catalyst and its preparation method, as well as a method for synthesizing oligonucleotides. The oligonucleotide synthesis catalyst comprises at least one of a tetrazolium, azole salt, pyridine salt, and imidazole salt compound as the main component, and a polar additive. The oligonucleotide synthesis catalyst of this invention solves the problems of current oligonucleotide synthesis processes, such as the difficulty in synthesizing complex oligonucleotide sequences (e.g., oligonucleotide chains that are too long, have high GC content, or have repetitive or continuous sequences that easily generate secondary structures), the difficulty in obtaining the target oligonucleotide fragment, or the high impurity content and poor quality of the target oligonucleotide fragment. When the oligonucleotide synthesis catalyst of this invention is applied to oligonucleotide synthesis, the success rate of synthesizing high-GC-content oligonucleotides can be increased, and the quality of oligonucleotides can be improved.

This invention provides a high-throughput method for constructing dual sgRNA libraries and its applications. The method utilizes high-throughput microarray synthesis technology to prepare a set of DNA fragments containing multiple dual sgRNA expression cassettes in a single step. After amplification, these fragments are assembled with a target vector containing a first promoter and a second gRNA backbone sequence in a first round of directed assembly to obtain a preliminary recombinant plasmid set. Then, linearized enzyme digestion and homologous recombination technology are used to insert a fragment containing a transcription termination sequence and a complete second promoter to complete the construction of the dual sgRNA expression unit. Finally, transformation and amplification yield the dual sgRNA plasmid library. This invention avoids the high error rate and high cost of long-chain oligonucleotide synthesis by utilizing microarray synthesis and simplifies the operation process through two rounds of directed assembly, significantly improving the throughput, fidelity, and efficiency of library construction. It is applicable to the construction of genome-wide dual sgRNA libraries, providing an efficient and reliable technical platform for high-throughput gene function screening, drug target discovery, and gene interaction research based on CRISPR.

The invention relates to the technical field of biological enzyme engineering, in particular to a mutant of nicotinamide adenine dinucleotide synthetase and application thereof. According to the nicotinamide adenine dinucleotide synthetase mutant, mutation of sites such as W11F, M12R, H37W, I40V, I40L, G43A, G43D, L46D, L46E, V55D, V55E, F60W, Y61S, L112I, L112K, L125E and L133T is carried out by taking NadR as a parent, so that the mutant can efficiently catalyze a low-cost substrate NR and ATP to generate NAD. Especially through mutation of the multiple mutation sites, the obtained mutant has better substrate binding capacity and catalytic efficiency and higher NAD conversion rate and enzyme activity (up to 12.49 U / mg), not only solves the technical problem of low activity of the existing NAD synthetase, but also remarkably reduces the production cost of NAD by using low-price NR instead of expensive NMN as a substrate, and has good application prospects. And a technical scheme with more economic benefits is provided for industrial production of NAD.

The present disclosure relates to methods and compounds for liquid phase oligonucleotide synthesis employing the use of small molecules with lipophilic groups. Methods for making an oligonucleotide by liquid phase oligonucleotide synthesis using the compounds described herein are also provided.

The purpose of the present disclosure is to provide an amide-crosslinked amidite monomer which contains a guanine base and can contribute to a reduction in the amount of the amidite monomer used during oligonucleotide synthesis. The present disclosure relates to a crystal of an amide-crosslinked compound represented by formula (1). (In the formula, P1 represents a protecting group for a hydroxyl group and P2 represents a protecting group for an amino group.

Aspects of the disclosure relate to methods and systems for solid phase oligonucleotide synthesis (SPOS). In some embodiments, the methods described by the disclosure comprise systems and methods related to washing after deblocking during solid phase oligonucleotide synthesis. In some embodiments, used deblocking solution is combined with a used washing solution and distilled to provide a distillate wash solution that can be used in a later washing step. In some embodiments, wash solutions are heated or pyridine is added for use during countercurrent washing procedures to increase efficiency. In some embodiments, the methods are combined.

A process of liquid-phase oligonucleotide synthesis (LPOS) is provided which uses the photolabile protecting group 2-(2-nitrophenyl)propyloxycarbonyl (NPPOC). The process may be used for synthesizing an NPPOC-protected oligonucleotide or salt thereof or a precursor thereto. Also provided is the use of NPPOC in liquid phase oligonucleotide synthesis, as well as an NPPOC-protected oligonucleotide or salt thereof which is in solution.

Click-labeled uridine bases, nucleosides, and phosphoramidites are provided, including improved methods of synthesis, oligonucleotides comprising the click-labeled nucleosides, methods of synthesizing spin-labeled oligonucleotides using click-labeled nucleotides, and spin-labeled oligonucleotides comprising click-labeled nucleosides.

Described herein are aptamers for temperature-dependent reversible inhibition of thermostable polymerase activity in order to improve sensitivity and specificity of various reactions and assays involving hot start polynucleotide synthesis. Methods for use of the aptamers and related compositions and kits are also provided.

A reactor system is disclosed. The reactor system includes a vessel configured to contain a solid support, the vessel including: a vessel wall defining a reaction chamber, the reaction chamber having a first end and a second end opposite the first end; a piston operatively arranged at the first end and configured to translate within the reaction chamber; a force measuring device coupled to the piston and configured to measure a load on the piston; a piston driver coupled to the piston; and a processor operably coupled to the force measuring device and the piston driver, the processor configured to: measure a load on the piston using the force measuring device, and adjust a position of the piston using the piston driver based on measuring the load on the piston.

Embodiments for the synthesis of sensitive oligonucleotides as well as insensitive oligonucleotides are provided. Sulfur-based groups are used for the protection of exo-amino groups of nucleobases, phosphate groups and 2′-OH groups, and as cleavable linker for linking oligonucleotides to a support. Oligonucleotide syntheses are achieved under typical conditions using phosphoramidite chemistry with important modifications. To prevent replacing sulfur-based protecting groups by acyl groups via cap-exchange, special capping agents are used. To retain hydrophobic tag to assist RP HPLC purification, special phosphoramidites are used in the last synthetic cycle. With the sulfur-based groups for protection and linking, oligonucleotide deprotection and cleavage are achieved via oxidation followed by beta-elimination under mild conditions. Therefore, besides for insensitive oligonucleotide synthesis, the embodiments of the invention are capable for the synthesis of oligonucleotide analogs containing sensitive functional groups that cannot survive the harsh conditions used in prior art oligonucleotide synthesis technologies.

The application provides a promoter capable of improving the transcription level of a formylglycine-generating enzyme gene and application, the promoter is obtained by mutating the sequence shown in SEQ ID NO:1, and five promoters capable of enhancing the transcription level of a formylglycine-generating enzyme gene and capable of improving the expression of an alpha-amylase amyE gene from Bacillus subtilis subsp. subtilis str. 168 in coryneform bacteria are obtained.

The present disclosure relates to aptamers for temperature-dependent reversible inhibition of thermostable polymerase activity in order to improve sensitivity and specificity of various reactions and assays involving hot start polynucleotide synthesis. Methods for use of the aptamers and related compositions and kits are also provided.

The present application relates to the technical field of biological enzyme engineering, and particularly relates to a nicotinamide adenine dinucleotide synthetase mutant and application thereof. The nicotinamide adenine dinucleotide synthetase mutant is obtained by mutating W11F, M12R, H37W, I40V, I40L, G43A, G43D, L46D, L46E, V55D, V55E, F60W, Y61S, L112I, L112K, L125E, L133T and other sites of NadR as a parent, so that the mutant can efficiently catalyze low-cost substrates NR and ATP to generate NAD. In particular, by using the above-mentioned multi-mutation point mutation, the mutant has better substrate binding capacity and catalytic efficiency, higher NAD conversion rate and enzyme activity (up to 12.49 U / mg), not only solves the technical problem of low activity of the existing NAD synthetase, but also significantly reduces the production cost of NAD by using the low-priced NR to replace the expensive NMN as the substrate, and provides a more economically beneficial technical scheme for the industrial production of NAD.

The present invention provides compounds useful as selectively soluble supports (or tags) for oligonucleotide and peptide synthesis that can be removed in a high yield under basic conditions and can afford a resulting product at a high purity in an organic synthesis reaction such as peptide synthesis, oligonucleotide synthesis, and the like. The protecting reagent is a selectively soluble compound of Formula (I) or Formula (II), which is derived from 5-hydroxyisophthalic acid.

The present disclosure relates to aptamers for temperature-dependent reversible inhibition of thermostable polymerase activity in order to improve sensitivity and specificity of various reactions and assays involving hot start polynucleotide synthesis. Methods for use of the aptamers and related compositions and kits are also provided.

The invention discloses a preparation method and application of a TLR9 targeted CpG oligonucleotide adjuvant, and belongs to the technical field of vaccine adjuvants, and the preparation method comprises the following steps: sequentially adding a phosphoramidite monomer solution, a Unylinker350 carrier, a deprotection reagent, a coupling activator, a sulfo reagent and a capping reagent into an oligonucleotide synthesizer; automatically synthesizing an oligonucleotide fragment in an oligonucleotide synthesizer by adopting a phosphoramidite method to obtain a reaction solution containing the oligonucleotide fragment; carrying out ammonolysis reaction to obtain an ammonolysis crude product; purifying the ammonolysis crude product by using an ion exchange method, collecting components with the purity of more than or equal to 90%, and merging and ultra-filtering; according to the method, the synthesis process conditions are optimized, so that the purity of the finally synthesized CpG oligonucleotide is more than 95%, and the yield is more than 70%.

The invention is suitable for the technical field of nucleotide synthesis, and provides a high-efficiency green synthesis method of 3 '-amino nucleotide, which is green and environment-friendly, and efficiently adapts to industrial production. Pure water is used as a unique solvent, so that no harmful waste is discharged; 5% of the Pd / C catalyst can be recycled, the low-concentration TEAB eluent can be recycled, and the green chemistry concept is met. The Pd / C catalyst has the advantages of high selectivity, few side reactions (impurities are less than or equal to 5%), mild reaction conditions (normal-pressure hydrogen and 50 DEG C), time consumption of only 2 hours and raw material conversion rate of more than or equal to 99%. DEAE resin and low-concentration gradient elution purification are adopted, the HPLC purity of the product is larger than or equal to 95% (part is larger than or equal to 98%), and the molar yield is 46-85% and is superior to that of an existing method; only two-step core operation is adopted, post-treatment is simple and convenient, corrosion to equipment is small, the method can adapt to 3 '-azido nucleotide raw materials with various substituent groups, and the preparation requirements of different drug intermediates are met.

The present invention relates to the use of nucleotide synthesis pathway inhibitors and macropinocytosis inhibitors for the treatment of tumors. The present disclosure provides the use of nucleotide synthetic pathway inhibitors and macropinocytosis inhibitors in the preparation of medicaments or kits for the treatment of tumors, where the nucleotide synthetic pathway inhibitors may include pyrimidine and / or purine synthetic pathway inhibitors such as orotic dehydrogenase (DHODH) inhibitors and phosphoribose pyrophosphate synthetase 1 (PRPS) inhibitors, the macropinocytosis inhibitor can comprise a macropinocytosis substrate uptake inhibitor, a PAK1 inhibitor, an EGFR signal pathway inhibitor, an ErbB signal pathway inhibitor, an NF-KB signal pathway inhibitor, an EREG inhibitor, a p65 inhibitor and an AREG inhibitor. The invention discloses a new way for activating macropinocytosis by using intracellular signals, provides a new insight for a regulation mechanism in tumor cells, and provides a new method for tumor treatment.

The invention discloses a high-freshness fermented protein marinade and a preparation method thereof, and relates to the technical field of fermentation engineering. The method comprises the following steps: preparing a mushroom enzymolysis extracting solution, preparing a bacillus subtilis induced fermentation solution and a saccharomyces cerevisiae induced fermentation solution, and carrying out mixed fermentation. According to the invention, the efficient proteolysis capability of bacillus subtilis and the flavor nucleotide synthesis capability of saccharomyces cerevisiae are organically combined for the first time. The bacillus subtilis secretes high-activity neutral protease under the induction of a substrate, and mushroom protein is deeply hydrolyzed into small-molecule umami peptide; metabolizing the saccharomyces cerevisiae under the induction of the mushroom extract to generate 5 '; -nucleotides (GMP, IMP, etc.) and alcohol ester flavor substances. Peptide-nucleotide molecule level synergy is achieved in synergy fermentation of the two components, the palatable taste is mellow and mellow, the aftertaste is lasting, and the problems that in the prior art, the single enzymolysis flavor is thin, and physical compounding synergy is insufficient are solved.

The invention discloses a preparation method and application of beta-nicotinamide mononucleotide, and belongs to the technical field of nucleotide synthesis. Tetraacetyl ribose and ethyl nicotinate are used as raw materials, a catalyst is added for a condensation reaction, the catalyst comprises a component A and a component B, the component A is mercaptosilane modified mesoporous silica loaded trimethylsilyl trifluoromethanesulfonate, and the component B is nitrogen-doped carbon nanotube loaded stannous chloride. And after the condensation reaction, carrying out ammonolysis deacetylation reaction, phosphorylation reaction and post-treatment to obtain the beta-nicotinamide mononucleotide. According to the catalyst provided by the invention, the component A and the component B are two catalytic components loaded by different carriers, so that the catalyst is favorably filtered and recycled, the catalyst residue is reduced, double activation of a glycosyl donor and a nucleophilic reagent can be realized by adjusting the ratio of the component A to the component B and through a synergistic effect, the forward proceeding of a reaction is promoted, and the catalyst has high selectivity and is suitable for industrial production. The method is suitable for the application field of cosmetics.

The invention is applicable to the technical field of biomedicine, provides a preparation method of a multifunctional composite nano-enzyme with triple cooperative regulation, and constructs an intelligent nano-enzyme system AgAu C-L based on a triple environment self-adaptive regulation mechanism. According to the system, the catalytic reaction temperature is accurately optimized through the photothermal effect of an AgAu nanocage, meanwhile, the local pH is actively reduced to the optimal activity range of CeO2 in combination with the probiotic metabolism characteristic of lactobacillus reuteri, and the charge transfer efficiency is enhanced in cooperation with an AgAu-coated CeO2 heterojunction interface electron transfer optimization strategy; under the combined action of the three, the catalytic efficiency of the CeO2 nano-enzyme in a physiologically neutral / weakly alkaline environment, especially the peroxidase-like activity of the CeO2 nano-enzyme is remarkably improved. The intelligent system not only can efficiently and physically remove a periodontal pathogenic bacterium biological membrane, but also can disintegrate a microorganism collaborative network by accurately interfering key paths such as pathogenic bacterium nucleotide synthesis and arginine metabolism, and finally remodeling of oral cavity micro-ecological balance is achieved.

Provided is a method for enzymatic synthesis of a 3'-O-phosphoric acid-modified nucleic acid, the method comprising using a phosphokinase to catalyze a nucleic acid monomer having a length of 1 nt or an oligonucleotide chain having a length of ≥ 2 nt, so as to obtain a 3'-O-phosphoric acid-modified nucleic acid monomer or oligonucleotide chain. The method can fill the technical blank that 3'-O-phosphoric acid-modified nucleic acids are difficult to synthesize via enzyme catalysis in the prior art, and the method is suitable for the field of synthesis of non-natural nucleotides.

The invention relates to DNA-based digital information storage. Provided herein are compositions, devices, systems, and methods for generating and using biomolecule-based information for storage. Further provided are devices comprising addressable electrodes that control polynucleotide synthesis (deprotection, extension or cleavage, etc.). The compositions, devices, systems, and methods described herein provide improved storage, density, and retrieval of biomolecule-based information.

Functionalized solid supports are useful in the synthesis of oligonucleotides. The functionalized solid supports contain an extended linker to a terminal functional group or a first nucleotide or nucleoside moiety. The extended linker permits oligonucleotide synthesis to take place at a greater distance from the solid support with greater efficiency.

The present disclosure pertains to methods of synthesizing a polynucleotide by associating a polynucleotide scaffold with a plurality of adaptor oligonucleotides and a plurality of fragment oligonucleotides to form a mixture. Each of the adaptor oligonucleotides includes subsequences that become hybridized with the polynucleotide scaffold and other subsequences that become hybridized with subsequences of the fragment oligonucleotides to form adaptor oligonucleotide: fragment oligonucleotide complexes. A ligase is then added to the mixture to ligate the adjacent fragment oligonucleotides to one another and form the polynucleotide. In some embodiments, the methods of the present disclosure also include a step of purifying the formed polynucleotides.

The present disclosure relates to methods and compounds for liquid phase oligonucleotide synthesis employing the use of small molecules with lipophilic groups. Methods for making an oligonucleotide by liquid phase oligonucleotide synthesis using the compounds described herein are also provided.

To provide an efficient solid phase oligonucleotide synthesis (SPOS) method.SOLUTION: A method of adding an oligonucleotide to a solid support in a bed reactor, the method comprising: removing a protecting group from a 5' - position of an oligonucleotide bound to the solid support; and adding an activated amidite solution to the bed reactor, wherein: Wherein the activated amidite solution comprises an amidite and flows up and down or is fluidized by nitrogen bubbling or other agitation in the bed reactor and reacts at the 5 ' position of the oligonucleotide, wherein the phosphite linkage found in the amidite comprises a P atom in oxidation state III, and then converting the P atom from oxidation state III to oxidation state V.SELECTED DRAWING: None