41 results about "Nucleoside triphosphate synthesis" patented technology

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 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.

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.

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 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.

This document provides a composition comprising an enzyme that releases pyrophosphate from a substrate and a dye of Formula 1. A method for detecting pyrophosphate is also provided. A kit is also provided comprising a polymerase that releases pyrophosphate via the hydrolysis of nucleoside triphosphates during nucleic acid replication, a divalent manganese salt, and a dye. This composition, method, and kit provide a way to detect and / or quantify the products of substrates or enzyme reactions associated with pyrophosphate release (such as nucleic acid amplification reactions and other ATP-hydrolyzing reactions) by means of a noticeable color change without materially affecting the sensitivity and / or specificity of the reaction.

The present invention relates to a method of synthesizing a polynucleotide, wherein the method comprises the steps of (a) performing a cycle comprising the steps of (i) contacting a template- independent polymerase with an initiator nucleic acid and a 3'-O-protected nucleoside triphosphate, so that the initiator nucleic acid is elongated by the template-independent polymerase activity by incorporation of the 3' -O-protected nucleoside triphosphate, to form a 3'-O-protected elongated initiator nucleic acid, (ii) deprotecting the 3'-O-protected elongated initiator nucleic acid to form an elongated nucleic acid having a free 3'- hydroxyl, (b) repeating cycles of step (a) to further elongate the initiator nucleic acid, until the polynucleotide is synthesized, wherein the template-independent polymerase is anchored to a solid support during cycles of step (a). The invention further relates to polymerases, kits, solid supports, devices for use in the invention and related uses.

The invention discloses a transporter mutant capable of efficiently transporting non-natural nucleoside triphosphate. The molecular structure of a truncated body PtNTT2 (66-575) of a nucleoside triphosphate transporter PtNTT2 is analyzed, the PtNTT2 (66-575) is taken as an evolution starting point, key amino acid residues: 271th and 474th amino acids are selected, site-specific saturated mutation libraries are respectively constructed, and screening is carried out aiming at the transport activities of different non-natural nucleoside triphosphate, so that the site-specific saturated mutation libraries can be used for screening the transport activities of different non-natural nucleoside triphosphate. The mutant capable of efficiently transferring non-natural nucleoside triphosphate (dCNTMOTP, dNaMTP and NaMTP) is obtained by adopting the method provided by the invention. The mutant disclosed by the invention can be used for high-efficiency cell transmembrane delivery of various non-natural nucleoside triphosphates, the substrate range of nucleoside triphosphate transporter is expanded, and a foundation is laid for construction of semi-synthetic life bodies for efficiently expanding genetic letters.

The application belongs to the field of biological medicine engineering, and relates to a preparation method of a cyclic dinucleotide, comprising the following steps: synthesizing the cyclic dinucleotide in a reaction system containing a dinucleotide cyclase and nucleoside triphosphates, centrifuging a reaction product, and obtaining supernatant; and purifying the cyclic dinucleotide from the supernatant by using a macroporous adsorption resin, wherein gradient elution is performed by using a 5-100 mM triethylamine-acetic acid solution as phase A and a mixed solution containing 70-80% phase A and 20-30% methanol as phase B. The macroporous adsorption resin is used to separate and purify the cyclic dinucleotide, the operation is relatively simple, the separation and purification time can be significantly shortened, the separation and purification speed is improved, and the obtained product has high purity.

The invention discloses a kit and a method for detecting bean weevil based on enzyme-mediated double-amplification nucleic acid amplification. The system comprises a specific sequence of the acanthobruchus manihot, a detection primer of the specific sequence, a probe for detecting the specific sequence, an E nucleic acid amplification group enzyme system (recombinase, single-stranded binding protein, polymerase and ATP energy regeneration enzyme), a signal amplification group enzyme system (positioning enzyme and cutting enzyme), an activating enzyme NTP (nucleoside triphosphate), a buffer solution, an RNA primer, a DNA upstream primer, a DNA downstream primer and RNase-free Water. The invention discloses a system composed of a freeze-drying protective agent and pre-subpackaged micro-freeze-dried balls prepared from the system. The kit containing the system provided by the invention can realize the detection of DNA with the concentration of 0.001 ng / mu L, can quickly and qualitatively detect the bean weevil, and is convenient to operate and simple in steps.

One or more enzymes are used to repair damage in synthetic DNA molecules that encode digital information. The enzymes are included in a repair mixture containing one or more of DNA polymerase, DNA ligase, T4 Endonuclease, Endonuclease IV, Endonuclease VIII, and uracil glycosylase. The repair mixture may also contain one or more of a buffering solution, oxidized nicotinamide adenine dinucleotide (NAD+), and deoxyribose nucleoside triphosphates (dNTPs). The synthetic DNA molecules are incubated with the repair mixture for approximately four hours. Use of the repair solution allows recovery of the digital information from damaged DNA molecules.