22 results about "Nucleoside triphosphate synthesis" patented technology

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.

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.

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 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.

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 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.

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 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.

The invention provides an application of a nucleoside triphosphate hydrolase superfamily protein BnaGET3B in regulation and control of the oil content of rape seeds, a homozygous mutant material with a BnaGET3B function deletion is obtained by mutating the gene in wild type rape, and it is found that the gene BnaGET3B positively regulates and controls the oil content of the rape seeds. Therefore, a new rape germplasm with significantly improved oil content of the seeds can be obtained by overexpressing the gene in the rape, and low-oil-content rape can also be obtained by mutating the gene, so that important genetic resources are provided for molecular mechanism and regulatory network research of rape grease synthesis. The biological function of the BnaGET3B gene in regulating the oil content of the rape seeds is disclosed for the first time, a brand new target and genetic resources and materials with direct application value are provided for rape breeding, and the application prospect is wide.

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 non-natural base DNA aptamer targeting bacillus cereus 5 / B / 6 metal beta-lactamase and a screening method of the non-natural base DNA aptamer. The screening method comprises the following steps: respectively taking dNaMTP and dTPT3TP, or dCNMOTP and dTPT3TP as non-natural base nucleoside triphosphate substrates, and amplifying a DNA library containing non-natural bases by utilizing OneTaq DNA polymerase; the method comprises the following steps: carrying out PCR (Polymerase Chain Reaction) reaction on bacillus cereus 5 / B / 6 metal beta-lactamase, degrading 5 '-end phosphorylated DNA chains in a PCR product by virtue of lamda exonuclease to obtain two ssDNA libraries, and screening non-natural base DNA aptamers targeting bacillus cereus 5 / B / 6 metal beta-lactamase on the basis of the libraries to obtain three 70 nt non-natural base DNA aptamers. A biological membrane layer interference analysis result shows that the three non-natural base DNA aptamers have good binding affinity to bacillus cereus 5 / B / 6 metal beta-lactamase.

The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful in template-independent polynucleotide synthesis using a nucleoside triphosphate-3′-O-removable blocking group (NTP-3′-O-RBG), as well as compositions, methods of utilizing these engineered polypeptides, and polynucleotides encoding the engineered terminal deoxynucleotidyl transferases.

The present invention is directed to methods and compositions for template-free enzymatic synthesis of a polyribonucleotide of a predetermined sequence from 3′-O-reversibly blocked nucleoside triphosphates using poly(A) and poly(U) polymerases.