38 results about "Nucleotide synthesis" patented technology

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.

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 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.

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.

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

The present invention provides compounds useful as selectively soluble supports (or tags) for oligonucleotide 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), which is derived from a tricarboxylic acid.

Fluorophores are used during the synthesis of oligonucleotides to achieve real-time quality control of the synthesis process. Fluorescence may indicate successful addition of individual nucleotides to a growing oligonucleotide strand or removal of a blocking group. The oligonucleotides may be created by enzymatic synthesis using terminal deoxynucleotidyl transferase (TdT). The synthesis is performed on an addressable array so that oligonucleotides with different sequences are created in parallel on different regions of the array. The oligonucleotide sequences are predetermined and the locations of synthesis on the array are controlled. Observed fluorescence is compared to expected locations of fluorescence as determined by the oligonucleotide sequences and the arrangement on the array. Thus, the fidelity of oligonucleotide synthesis is checked as synthesis proceeds. If a variation is found, a mitigating action is taken such as repeating addition of a species of nucleotide or repeating a deblocking step.

Enzymatic polynucleotide synthesis with a template-independent polymerase is used to create multiple polynucleotides having different, arbitrary sequences on the surface of an array. The array provides a spatially-addressable substrate for solid-phase synthesis. Blocking groups are attached to the 3′ ends of polynucleotides on the array. Prior to polynucleotide extension, the blocking groups are removed at a selected location on the array. In an implementation, the blocking groups are acyl groups removed with a negative voltage created at an electrode. The array is then incubated with the polymerase and a single species of nucleotide. Nucleotides are incorporated onto the 3′ ends of the polynucleotides without blocking groups. Washing removes the polymerase and free nucleotides. To create polynucleotides with different sequences at different locations on the array, the location where the blocking groups are removed and the species of nucleotide may be changed during repeated cycles of synthesis.

Methods of synthesizing oligonucleotides via new intermediates on a cleavable support having an azidomethyl moiety are disclosed. The method comprises multiple reaction cycles, each of which comprises sequential coupling a nucleoside or oligonucleotide subunit on a cleavable support having an azidomethyl moiety and a nucleoside phosphoramidite or an oligonucleotide phosphoramidite, capping, oxidation / thiolation and deblocking; followed by orthogonal cleavage of the azidomethyl support while keeping all other protecting groups intact. The method can be used in combination with a support moiety for either solid phase or liquid phase oligo synthesis. The soluble support facilitates homogeneous reactions and efficient separations by simple precipitation. The methods also provide novel intermediates useful in the synthesis of oligonucleotide conjugates.

The present invention provides a protecting reagent that can be removed in a high yield even under acidic 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), which is derived from citric acid.

The present invention provides a protecting reagent that can be removed in a high yield even under acidic 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), which is derived from a triamide.