48 results about "Nucleoside triphosphate" patented technology

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 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 disclosure relates to the production of highly pure N,N-diisopropylphosphoramidite compounds, such as 6-bromohexyl 2-cyanoethyl N,N-diisopropylphosphoramidite, and highly pure starting material used therein, such as 6-bromohexan-1-ol. The resulting N,N- diisopropylphosphoramidite compounds are useful, for example, for the improved preparation of expandable nucleotide triphosphates (XNTPs) for use in sequencing by expansion.

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 invention relates to a new diastereoselective synthesis method for highly modified nucleoside triphosphates with two conjugable groups. The method works in liquid phase with conventional laboratory equipment as used in any chemical laboratory or production. For this, a new chiral P(V) phosphorylation reagent has been designed that allows highly efficient diastereoselective synthesis under mild conditions. Overall, a highly efficient, diastereoselective, high quality, cost-effective synthetic method of modified nucleoside triphosphates has been developed.

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 present disclosure provides enzymatic methods for the production of 3'-phosphatenucleoside-5'-triphosphate (NQP). The present disclosure further provides enzymatic methods for the production of nucleotide-5'-triphosphates. In some embodiments, a method of producing a nucleotide triphosphate with a phosphate group at the 3' position of the sugar moiety (NQP), the method comprising at least: contacting a nucleoside diphosphate (NDP) with a nucleoside diphosphate kinase, a 3' -O-kinase, and a phosphate donor under reaction conditions such that a NTP with a phosphate group on the 3' position of the sugar moiety (NQP) is produced.

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.

The invention discloses an efficient cell-free protein synthesis buffer solution as well as a preparation method and application thereof, and belongs to the field of bioengineering and synthetic biology. The efficient cell-free protein synthesis buffer solution comprises 20-50 mM of HEPES, a cationic substance, an energy regeneration component, 9.5-38 mM of amino acid, an auxiliary factor, a stabilizer and 0.01%-0.03% / v of a bacteriostatic agent, the cationic substances are 8-15 mM of Mg < 2 + >, 60-150 mM of K < + > and 1-10 mM of NH4 < + >; the energy regeneration system comprises 1 to 4 mM of nucleoside triphosphate, 30 to 50 mM of phosphocreatine and 0.2 to 0.6 U / [mu] L of creatine kinase; the stabilizer comprises 1 to 3 mM of dithiothreitol; 1% to 3% w / v of PEG 8000; and a molecular chaperone GroEL / GroES with a concentration of 0.02 to 0.08 mg / mL. The activity is high after long-time normal-temperature preservation; the buffer solution is high in compatibility, low in reagent cost and suitable for industrial production.

Among other things, the present invention provides a conjugate comprising a polymerase and a nucleoside triphosphate, wherein the polymerase and the nucleoside triphosphate are covalently linked via a linker comprising a cleavable bond. Also provided is a set of such conjugates, wherein the conjugates correspond to G, A, T (or U), and C. Also provided is a method for synthesizing a nucleic acid of a defined sequence. The conjugates can also be used in sequencing applications.

The application provides a cytosine nucleoside producing strain and a construction method and application thereof, and the strain is constructed by using CRIPSR / Cas9 gene editing technology E. coli The cdd gene and cmk gene are knocked out on the UR12 genome, the nucleoside triphosphate reductase gene nrdD is weakened, and the nucleoside triphosphate pyrophosphatase gene nudG, the nucleoside diphosphate kinase gene ndk and the uridine acid kinase gene pyrH carrying a mutation point are overexpressed (D93A) The cytosine triphosphate synthase gene pyrG carrying a mutation point is introduced into the Corynebacterium glutamicum heterologously and in multiple copies (D160E、E162A、E168K，cgl) The bifunctional nucleotidase gene of Saccharomyces cerevisiae PHM8 (sce) The obtained strain has good genetic stability, high fermentation yield and can stably produce cytosine nucleoside.

The application discloses a non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metal beta-lactamase and a screening method thereof. The screening method uses dNaMTP and dTPT3TP, or dCNMOTP and dTPT3TP as non-natural base nucleoside triphosphate substrates, uses OneTaq DNA polymerase to amplify a DNA library containing non-natural bases, and uses lamda exonuclease to degrade 5' end phosphorylated DNA chains in PCR products, so as to obtain two ssDNA libraries. The non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metal beta-lactamase is screened based on the libraries, and three non-natural base DNA aptamers with a length of 70 nt are obtained. The results of a biological membrane layer interference analysis show that the three non-natural base DNA aptamers have good binding affinity to Bacillus cereus 5 / B / 6 metal beta-lactamase.

The invention relates to a diastereomer of nucleoside triphosphates suitable for use in sequencing by expansion. The diastereomer provides a better acceptance and incorporation by a DNA polymerase and better performance in sequencing by expansion workflows. The invention also relates to sequencing methods using the diastereomer of the nucleoside triphosphates.

The present invention relates to a novel enzymatic method for producing nucleoside triphosphates (NTP), in particular modified NTPs, using polyphosphate kinase 2. The NTPs generated by the methods of the invention may be used in methods of producing polynucleotides, including oligonucleotides, for use in therapy.

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.

The present invention addresses the problem of providing a novel method for synthesizing a deoxynucleoside triphosphate or nucleoside triphosphate.　This method for synthesizing a deoxynucleoside triphosphate (dNTP) from a deoxyribonucleoside or synthesizing a ribonucleoside triphosphate (NTP) from a ribonucleoside is characterized by: adding, to a reaction vessel, (i) a deoxyribonucleoside or ribonucleoside as a starting material, (ii) a kinase capable of producing a deoxyribonucleoside monophosphate from the deoxyribonucleoside or a ribonucleoside monophosphate from the ribonucleoside, a kinase capable of producing a deoxyribonucleoside diphosphate from the deoxyribonucleoside monophosphate or a ribonucleoside diphosphate from the ribonucleoside monophosphate, and a pyruvate kinase as enzymes, and (iii) a nucleoside triphosphate or deoxynucleoside triphosphate and a phosphoenolpyruvic acid (PEP) as phosphoric acid donors, thereby adjusting a reaction solution; and reacting the same in one pot.

Described herein are methods and compositions that provide efficient primer annealing and denaturation during nucleic acid amplification. The basic method uses destabilized nucleoside triphosphate (dNTP) in the amplification reaction mixture. The incorporation of such dNTP into the amplicon produced during amplification reduces the Tm of the amplicon, promoting denaturation / strand separation of the next round of primer annealing.

The invention is directed to systems and methods of recording digital information using nucleic acids. A DNA template strand has alpha and beta segments, each with a set of three nucleotides differing from each other. When a DNA polymerase contacts the template in the presence of a first solution of three nucleotides corresponding to an alpha segment, a new strand is extended according to available base pairing. The extension halts when the polymerase encounters a beta strand, as the first solution lacks a base-pairing nucleotide triphosphate. Similarly, when the polymerase is provided with a second solution of three nucleotides corresponding to the beta segment, the new strand continues extension according to available base pairing. As first and second solutions are alternated, chemically-altered nucleotide triphosphates are introduced, providing mutations in certain segments of the new strand. When sequenced, mutation segments are assigned a '1' and non-mutation segments are assigned a 'O'.