74 results about "Nucleoside triphosphate synthesis" patented technology

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.

Methods to generate novel acyl-tRNA species deploy an orthogonal synthetase that accepts α-hydroxy acids, α-thio acids, N-formyl-L-α-amino acids, and / or α-carboxyl acid monomers (malonic acids) that are formally precursors to polyketide natural products.

The invention discloses a method for screening a non-natural nucleic acid aptamer of targeted thrombin based on streptavidin magnetic beads and application, and belongs to the technical field of biology. According to the method, an initial screening library is efficiently constructed through a primer extension technology, and a chemical modification SELEX technical process is simplified; a streptavidin magnetic bead separation technology is introduced, so that the screening specificity and efficiency are improved, the screening period is remarkably shortened, and the cost is reduced; by selecting non-natural nucleic acid modification sites and adopting a strategy of combining reverse screening and forward screening, high specificity and affinity of the aptamer are ensured, and limitation of an existing screening method is overcome; the screened non-natural nucleic acid aptamer shows high affinity and excellent stability, the aptamer with a specific structure and function is successfully obtained by optimizing the proportion of nucleoside triphosphate monomers and extension reaction conditions, and a wide application prospect is provided for the fields of drug development, biosensor preparation and the like. A new path is opened up for research and application of the nucleic acid aptamer.

The present invention is directed to terminal deoxynucleotidyltransferase (TdT) variants that (i) comprise an amino acid sequence that is at least a specified percent identical to an indicated SEQ ID NOs and have at least one substitution at Q455 or at least Q455 plus at least one further substitution at G186, S248, T331, Q390, K394 or H466 (where positions are with respect to SEQ ID NO 1 and functionally equivalent positions in indicated SEQ ID NOs), (ii) are capable of template-free extension of a polynucleotide, and (iii) exhibit enhanced stability or enhanced efficiency in incorporating 3′-0-blocked nucleoside triphosphates into a polynucleotide. The invention is also directed to the use of these TdT variants for synthesizing polynucleotides of any predetermined sequence.

The invention is directed to methods for synthesizing a plurality of oligonucleotides in the same reaction vessel, and in some embodiments, using the synthesized oligonucleotides in an oligonucleotide-based assay in such reaction vessel. In some embodiments, methods of the invention are implemented by steps of (a) providing a plurality of different initiators attached to one or more supports, each different initiator having a terminal nucleotide with a different 3-O-blocking group; (b) for each different initiator, synthesizing a polynucleotide by repeated cycles of template-free enzymatic additions of 3′-O-blocked nucleoside triphosphates, wherein the blocking group of the 3-O-blocked nucleoside triphosphate is removable under deblocking conditions orthogonal to the deblocking conditions for removing blocking groups of the other initiators; and (c) releasing the oligonucleotides from the polynucleotides and the one or more solid supports.

The present disclosure provides uses of type II and variant type I and type II inorganic pyrophosphatases under nonnatural conditions present in industrial applications. In another aspect, the present disclosure describes compositions and methods of using inorganic pyrophosphatases with nucleoside triphosphates and in coupled reactions with a second enzyme.

The invention discloses construction and activity detection methods of semisynthetic chassis cells for genetic material expansion based on vibrio natrieti. The VnDX / pET23b-PtNTT2 (66-575) cell has a relatively wide nucleoside triphosphate transport active substrate spectrum, and has recognition and transport activities on natural nucleoside triphosphate (dNTPs and NTPs), pentose modified nucleoside triphosphate (2 '-OMe-NTPs and 2'-F-NTPs), and nucleoside triphosphate (dTPT3TP, dNaMTP, dCNMOTP, TPT3TP and NaMTP) containing non-natural basic groups, and the VnDX / pET23b-PtNTT2 (66-575) cell has the recognition and transport activities on the natural nucleoside triphosphate (dNTPs and NTPs) and the pentose modified nucleoside triphosphate (2 '-OMe-NTPs and 2'-F-NTPs). By means of the transport characteristic of the nucleoside triphosphate transporter PtNTT2 (66-575) and by detecting the content of ATP transported out of cells within the same time, the relative strength of the transport activity of various nucleoside triphosphate of the PtNTT2 (66-575) is simply, conveniently and rapidly determined.

Cyclic GMP-AMP synthase (cGAS) enzymes have been engineered to produce polypeptides having increased cGAS activity in the cyclization of modified nucleoside triphosphates, including thiolated or fluorinated nucleoside triphosphates, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing cGAS enzymes, compositions comprising the cGAS enzymes, and methods of using the engineered cGAS enzymes are useful for the production of pharmaceutical compounds.

A method of sequencing a nucleic acid characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one oligonucleotide used probe by reacting, in the presence of a polymerase, at least one of the single nucleoside triphosphates with a corresponding biological probe comprising (a) a first single-stranded oligonucleotide including an exonuclease blocking-site, a restriction endonuclease recognition-site located on the 5' side of the blocking-site and including a single nucleotide capture-site for capturing the single nucleoside triphosphate, and at least one fluorophore region located on the 5' side of the recognition-site arranged so as to render the fluorophore(s) quenched and (b) a second and optionally a third single-stranded oligonucleotide each separate from the first oligonucleotide and capable of hybridising to complementary regions on the first oligonucleotide flanking the 3' and 5' sides of the capture-site; (3) cleaving the first oligonucleotide strand of the used probe at the recognition-site with a restriction endonuclease to create a first oligonucleotide component bearing the fluorophores; (4) digesting the first oligonucleotide component with an enzyme having 3'-5' exonucleolytic activity to yield fluorophores in a detectable state and (5) detecting the fluorophores released in step (4). A corresponding method for use with enzymes having 5'-3' exonucleolytic activity as well as new biological probes and a kit derived therefrom is also provided.

The invention discloses base-modified mRNA (messenger Ribonucleic Acid) and application thereof in a cell-free protein synthesis system, and belongs to the technical field of nucleic acid modification. Natural nucleoside triphosphate is completely replaced by different types of basic group modified nucleoside triphosphate, mRNA with different chemical modifications is obtained through an in-vitro transcription method and purification, and the mRNA is put into an in-vitro cell-free protein synthesis system for reaction. A reaction element recombination system-PURE system based on prokaryote Escherichia coli purified protein and an eukaryote CFPS system taking rabbit reticulated red lysate as a material are mainly selected to detect the corresponding translation efficiency of mRNA modified by different bases in two cell-free protein synthesis systems respectively. Different regulatory activities may be shown in different cell-free protein synthesis systems when different modified nucleotides are doped into mRNA, base modified triphosphoric acid capable of improving the translation efficiency of an in-vitro protein synthesis system is screened out, and the stability of mRNA in the cell-free protein synthesis system and the corresponding protein expression level are improved.

A method for DNA synthesis using protected nucleosides is disclosed. The nucleosides may be nucleoside triphosphates or nucleoside phosphoramidites with nucleobases attached to electrochemically-cleavable linkers. Removal of a protecting group by application of a voltage in solution triggers a cyclization reaction that cleaves the electrochemically-cleavable linkers. The electrochemically-cleavable linkers may include an amide linkage and an amide that forms a lactam or an ester linkage and a protected alcohol that forms a lactone when the protecting group is removed. The voltage used to cleave the electrochemically-cleavable linkers may be generated by activation of individual electrodes on a microelectrode array. The microelectrode array can be a substrate for solid-phase synthesis of oligonucleotides. Activation of specific electrodes removes the protecting groups at those electrodes and thus enables spatially-controlled extension of the oligonucleotides. Protected nucleosides linked to protecting groups by electrochemically-cleavable linkers are also disclosed.

The invention discloses a luciferase mutant capable of efficiently utilizing non-natural nucleoside triphosphate. In the mutant, histidine (H) at the 244 site is mutated into tryptophan (W) on the basis of a starting enzyme Luc146-1H2 (K357E) at which amino acid No.357 is mutated from lysine (K) to glutamic acid (E). Compared with an evolutionary starting enzyme, the mutant Luc-WE not only can more efficiently utilize ATP (adenosine triphosphate) as an energy donor, but also can more efficiently utilize non-natural nucleoside triphosphate as an energy donor to catalyze the oxidation of D-fluorescein, so that a luminescence value higher than that of the evolutionary starting enzyme is generated; for different nucleoside triphosphate energy donors, the luminescence value is 2-40 times that of an evolution starting enzyme. Under the reaction condition of lower nucleoside triphosphate concentration, the luciferase mutant Luc-WE shows higher sensitivity, and has wider application range and practical value.

This application relates to mutant polymerases. This application discloses mutant phage-type RNA polymerases, such as a mutant T7, SP6, and T3 RNA polymerase, may use 2′-modified nucleoside triphosphates or deoxynucleotide triphosphates as substrates. Methods for producing nucleic acid molecules using these mutant polymerases are also disclosed.

The invention discloses a diagnosis and treatment integrated nano antibody for targeting shrimp iridovirus deoxyuracil nucleoside triphosphate hydrolase as well as a preparation method and application of the diagnosis and treatment integrated nano antibody. According to the invention, a nano-antibody library is constructed on the basis of a squalus leukoderma new antigen receptor variable fragment VNAR, a nano-antibody 4A12-coated VNAR targeting shrimp iridovirus deoxyuracil nucleoside triphosphate hydrolase dUTPase protein is screened from the nano-antibody library, the nucleotide sequence of the nano-antibody 4A12-coated VNAR is shown as SEQ ID No.5, and the amino acid sequence of the nano-antibody 4A12-coated VNAR is shown as SEQ ID No.6. The invention further discloses a preparation method of the nano-antibody 4A12-coated VNAR. The molecular weight of the nano antibody 4A12atVNAR is much smaller than that of a conventional full-length antibody, and the nano antibody 4A12atVNAR has better tissue permeability and capability of being combined with an epitope with a steric hindrance effect, can be massively expressed in a prokaryotic expression system, and is short in period and low in production cost. The nano antibody 4A12-coated VNAR can be used for detection, diagnosis and treatment of shrimp iridovirus.

The invention relates to a nucleoside triphosphate tetrabutylammonium salt and a preparation method thereof. The nucleoside triphosphate tetrabutylammonium salt is prepared by reaction with nucleoside triphosphate and tetrabutylammonium hydroxide as raw materials. The invention prepares the nucleoside triphosphate tetrabutylammonium salt by using simple and readily available raw materials, has low requirements on process equipment, and can prepare milligram-level products.

The invention discloses a high-yield cell-free protein synthesis system based on a cell crude extract and application of the high-yield cell-free protein synthesis system. The system comprises an escherichia coli extract, a DNA template, a buffer solution, an amino acid mixture, a nucleoside triphosphate mixture, an ATP regeneration system and antibiotics, the antibiotics are selected from compounds having inhibitory activity on metabolic processes except ribosome functions in prokaryotic microorganisms, including but not limited to one or more combinations of interfering DNA replication, DNA transcription, cell wall synthesis, energy metabolism, lipid synthesis or other non-translational related metabolic pathways. According to the method disclosed by the invention, the escherichia coli crude extract is used as a bacterial chassis, and non-essential metabolic pathways in a CFPS system are selectively inhibited by introducing different types of antibiotics, so that the yield of protein synthesis is increased.

Disclosed are methods and compositions for glycosylation of a sugar acceptor, comprising the steps of: (i) converting an exogenous nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP); (ii) converting the NDP to NDP-sugar, comprising contacting at least one Leloir glycosyltransferase (GT) with the NDP and an exogenous sugar donor (SD) in a reaction medium; and (iii) converting a sugar acceptor (SA) to a glycosylated SA, comprising contacting the at least one GT with the NDP-sugar and the SA in the reaction medium.

The present invention relates to a novel and efficient method for the sialylation of a glycoside, the method comprising mixing said glycoside with sialic acid, cytidine monophosphate, a nucleoside triphosphate and one or more cell-free extracts of a microorganism, said microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein said one or more cell-free extracts comprise: at least one polypeptide having cytidine monophosphate kinase activity, at least one polypeptide having N / -acylneuraminate citydyltransferase activity, and at least one polypeptide having sialyltransferase activity, thereby sialylating said glycoside.

Provided herein, inter alia, are methods for improving the growth of cells (such as probiotic cells or recombinant cells) by culturing the cells in a liquid media comprising one or more enzymes that hydrolyze at least one nucleoside triphosphate present in the media. Cells cultured in accordance with these methods exhibit improved growth compared to cells cultured in media that does not comprise one or more enzymes that hydrolyze at least one nucleoside triphosphate.

The invention relates to engineered terminal deoxynucleotidyl transferase (TdT) enzymes or the homologous amino acid sequence of Polμ, Polβ, Polλ, and Polθ of any species or the homologous amino acid sequence of X family polymerases of any species and their use in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of new terminal deoxynucleotidyl transferases and 3′-blocked nucleoside triphosphates in a method of template independent nucleic acid synthesis.

A method of RNA synthesis via a fluid flow device. The method may involve: introducing a plurality of reactants into a first fluid flow module via a plurality of inlet ports, the plurality of reactants including: at least one nucleoside triphosphate (NTP), a reaction buffer, and DNA, a DNA-based compound, or a DNA-based mixture; allowing at least some of the reactants to react within a reaction channel or well within a first module of the flow system; retaining the DNA at the first reactor module or recirculating the NDA at the first reactor module and allowing a reaction product flow of the reactants to the first fluid filtration module; and filtering the reaction product in the first filtering module.

The invention discloses a terminal transferase mutant, a recombinant protein, a nucleic acid molecule and an oligonucleotide single base extension method, and relates to the technical field of oligonucleotide synthesis. The terminal transferase mutant has at least 80% of identity with an amino acid sequence as shown in SEQ ID NO: 1, and has amino acid mutation at at least one of the following positions: R323, W427 and R438. Compared with wild-type terminal transferase, the terminal transferase mutant has the advantages that after amino acid in a catalytic structural domain of the terminal transferase mutant is changed, the catalytic capability of the terminal transferase mutant on nucleoside triphosphate with a 3 '-O-blocking group can be remarkably improved, so that the nucleoside triphosphate with the 3'-O-blocking group is efficiently coupled to a single-stranded oligonucleotide chain. On the basis, the invention further develops an oligonucleotide single base extension method. The method has the technical advantages that the humidity does not need to be controlled, the usage amount of chemical reagents is small, the product purity is high, and few chemical synthesis byproducts are generated.

This invention provides a method for producing adenosylcobalamin. First, cyanocobalamin, cobalt chloride, adenine, nucleoside triphosphate, and water are mixed and added to a reaction vessel to prepare an adenosylcobalamin mixture. High-purity nitrogen and a NaBH4-NaOH aqueous solution are added during the reaction. Then, the adenosylcobalamin filtrate is purified by passing it through a purification column. The purified solution is then subjected to adsorption and elution on a resin column, and the eluent is crystallized to obtain pure adenosylcobalamin. This purification process effectively removes impurities introduced during the synthesis of adenosylcobalamin, with total impurities ≤0.5%, ensuring the safety and controllability of product quality.

The utility model discloses a disposable reaction bag for in-vitro transcription reaction, which comprises a bag body, the whole bag body is of a disc-shaped structure and is provided with thickening layers of which the upper side and the lower side are overlapped, an annular connecting part is connected to the side between the thickening layers on the two sides, a round hole is formed in the center of the thickening layer on the upper side, and a disc is fixed at the position of the round hole. A liquid injection hole is formed in the position, corresponding to the round hole, of the disc and used for external communication through a hose. The disc is used for positioning and installing the hose, the hose is used for injecting solutions such as a DNA template, nucleoside triphosphate and RNA polymerase, the solutions are prepared into the concentration according to the proportion, and the solutions are injected into the bag body through the liquid injection hole after being prepared according to the proportion. The round hole can be sealed by the disc, and meanwhile, the disc is of a rigid structure and can support the hose to a certain extent for shaping. And then putting the bag body into a constant-temperature oscillator for oscillation cultivation.

The present invention relates to a novel and efficient method for sialylation of a glycoside comprising mixing said glycoside with sialic acid, cytidine monophosphate, nucleoside triphosphate and one or more cell-free extracts of a microorganism, the present invention relates to a microorganism comprising one or more endogenous polypeptides having inorganic diphosphatase activity and one or more endogenous polypeptides having phosphotransferase activity, and wherein the one or more cell-free extracts comprise: at least one polypeptide having cytidine monophosphate kinase activity, at least one polypeptide having N-acyl neuraminic acid cytidyltransferase activity, and at least one polypeptide having sialyltransferase activity, thereby sialylating the glycoside.

This invention claims processes that append a single nucleotide having a 3′-ONH2 moiety to the 3′-ends of an oligonucleotide primer using 3′-deoxynucleoside triphosphates that have, instead of a 3′-OH moiety, a 3′-ONH2 moiety, where the nucleotides contain both standard and non-standard nucleobases, and where as a key claim limitation, substantially no hydroxylamine is present in the solutions used in the claimed processes.

The application provides a preparation method and application of a coumarin derivative labeled nucleoside triphosphate, and the preparation method comprises the following steps: (1) mixing compound AA-dUTP, compound Boc-6-Ahx-Osu and an alkali to react in the dark to obtain compound 1; (2) mixing compound 1 and an acid solution, and then mixing with an alkali solution to obtain compound 2; (3) mixing compound 2 and compound DEAC-NHS to react in the dark to obtain compound DEAC-12-dUTP, namely the coumarin derivative labeled nucleoside triphosphate. The preparation method provided by the application can efficiently synthesize the target product, and has the advantage of high synthesis yield.

The present disclosure relates to the technical field of DNA fragmentation for technologies such as next generation sequencing (NGS) or any other technologies where fragmentation of DNA to a certain uniform size is required. In particular, the present disclosure relates to a variant of a Nucleoside Triphosphate (NTP)-dependent restriction enzyme (McrBC variants selected from McrBL68YC, McrBL68FC, and combinations of certain ratios thereof) for nucleic acid fragmentation for methods that require fragmentation of DNA.

The present disclosure generally relates to methods for the enzymatic synthesis of nucleotide triphosphates. In particular, the methods include a one-pot preparation of a modified nucleotide triphosphate starting from 3´-vinyl blocked nucleoside monophosphate.