185 results about "Deoxynupharidine" patented technology

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

The invention provides an AS01 derivative adjuvant, a preparation method thereof and application of the AS01 derivative adjuvant in preparation of vaccines or anti-tumor drugs, and belongs to the technical field of biological drugs or products. The AS01 derivative adjuvant is prepared from the following components in parts by mass: 0.5 to 1.0 part of cholesterol, 2.0 to 4.0 parts of dioleoyl lecithin and at least one of the following components in parts by mass: 0.1 to 0.2 part of monophosphoryl lipid A, 0.1 to 0.2 part of QS-21, 0.36 to 0.72 part of 3M-052, 0.4 to 0.8 part of muramyl dipeptide and 0.2 to 0.4 part of oligodeoxynucleotide. According to the AS01 derived adjuvant, a novel synergistic adjuvant system is developed by integrating various immunomodulators, so that the immunogenicity of a vaccine is enhanced. The HPV vaccine is prepared on the basis of the AS01 derived adjuvant, so that the immunogenicity can be enhanced, and good anti-tumor activity can be achieved. Therefore, the invention provides a new strategy for immunotherapy of HPV-related tumors.

The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful for template-independent polynucleotide synthesis, as well as compositions, methods of utilizing these engineered polypeptides, and polynucleotides encoding engineered terminal deoxynucleotidyl transferases.

The invention discloses a synthetic method of 2 '-fluoro-2'-deoxynucleosid-5 '-triphosphoric acid and application of the 2'-fluoro-2 '-deoxynucleosid-5'-triphosphoric acid. The synthesis method comprises the following steps: reacting in a system containing adenosine triphosphate, 2 '-fluoro-2'-deoxynucleoside, metal ions and deoxynucleoside kinase to obtain 2 '-fluoro-2'-deoxynucleoside-5 '-phosphoric acid; and reacting in a system containing polyphosphoric acid, 2 '-fluoro-2'-deoxynucleoside-5 '-phosphoric acid, metal ions and polyphosphate kinase to obtain 2'-fluoro-2 '-deoxynucleoside-5'-triphosphoric acid. The obtained 2 '-fluoro-2'-deoxynucleoside-5 '-triphosphoric acid can be used for preparing heterologous nucleic acid. The invention provides an economic and green new way for synthesis of 2 '-fluoro-2'-deoxynucleoside-5 '-triphosphate, solves the problems of complex steps, non-environment-friendly reagents and high cost in the existing chemical synthesis method, and provides a thought for preparation of nucleotide derivatives.

The invention belongs to the technical field of biological medicine, and particularly discloses a composite vaccine adjuvant as well as a preparation method and application thereof. The composite vaccine adjuvant provided by the invention is an oil-in-water emulsion, and simultaneously contains squalene, polysorbate 80, sorbitan trioleate, polyinosinic acid (Poly I: C) and CpG oligodeoxynucleotide (CpG-ODN). The sequence-optimized CpG-ODN and Poly I: C are used in a specific emulsion system in a combined mode, a remarkable synergistic immune enhancement effect can be generated, the induced humoral immunity and cellular immunity response level is remarkably higher than the sum of the effects when all the components are independently used, and the composite adjuvant is good in stability, high in immunostimulatory activity and suitable for clinical application. The immune effect can be remarkably improved by combined application with various vaccine antigens. The invention also provides a preparation method and application of the composite vaccine adjuvant.

The invention discloses an oligodeoxynucleotide for preventing and treating cognitive impairment in a perioperative period and application of the oligodeoxynucleotide. The oligodeoxynucleotide comprises an oligonucleotide unit as shown in SEQ ID NO: 1. The deoxyoligonucleotide drug disclosed by the invention is applied to in-vitro cultured neurons, so that the reduction of alpha-N-catenin expression of neurons caused by contact with anesthetic drugs and inflammatory factors can be smoothly blocked; the decoy medicine can also competitively block the combination of the FoxO3 and the ctnna2 enhancer, so that the transcription inhibition effect of the FoxO3 on the ctnna2 is blocked, and the cognitive impairment in the perioperative period is prevented and treated.

The invention relates to a method for synthesizing deoxynucleoside triphosphate by an enzymic method, which comprises the following steps: by taking deoxynucleoside monophosphate dNMP as a raw material and taking ATP (adenosine triphosphate) and acetyl phosphoric acid as phosphoric acid donors, synthesizing dNTP (deoxynucleoside triphosphate) by using nucleotide kinase and acetokinase through a one-pot enzymic method. The one-pot enzyme method is used for preparing deoxynucleoside triphosphate, and the method has the advantages of being simple in operation, short in production period, low in production cost, high in yield, small in three-waste discharge, suitable for large-scale industrial production and the like.

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

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 discloses a method for improving the soluble expression of terminal deoxynucleotidyl transferase (TdT), and relates to the technical field of protein engineering and gene engineering, in particular to a method for improving the soluble expression of the TdT. TdT (TdT-B) with the BRCT structural domain removed serves as initial enzyme, hydrophilic amino acid replacement is conducted on hydrophobic amino acid on the surface of the TdT-B enzyme, and a TdT mutant is obtained. The mutant does not need any fusion tag and can be rapidly expressed under induction conditions of high temperatures such as 30 DEG C and 37 DEG C, the soluble protein expression level is remarkably improved, a large amount of soluble TdT is obtained in a short time, the soluble expression level of the TdT is remarkably improved, the fusion tag cutting step is not needed in subsequent downstream treatment, and the preparation method is simple and low in cost.

The invention relates to novel DNA polymerases of the polX family, and in particular Terminal deoxynucleotidyl Transferase (TdT) variants comprising specific mutations or substitutions, and to their uses.

The present invention relates to methods for producing a protected deoxyribonucleic acid (DNA) product with enhanced resistance to nuclease digestion. The methods comprise digesting a double-stranded DNA molecule with an endonuclease to generate a digested double-stranded DNA molecule comprising a first strand and a second strand, wherein the first strand has a 5' overhang; and extending the second strand 5' to 3' using a polymerase in the presence of one or more nuclease-resistant deoxynucleotide triphosphates to generate the protected DNA product, wherein the protected DNA product comprises an extended second strand comprising at least one nuclease-resistant nucleotide incorporated into the extended second strand by the polymerase using the 5' overhang of the first strand as template. Also provided are protected DNA products and uses thereof.

This invention discloses a method for the bioenzymatic synthesis of deoxynucleoside monophosphate. The invention provides a mutant protein of dNK, comprising mutating amino acid residues at positions 84, 88, 110, and 172 of the amino acid sequence shown in SEQ ID NO:1, while leaving other amino acid residues unchanged, to obtain a protein with deoxynucleoside kinase activity. This invention obtains a deoxynucleoside kinase mutant capable of stably and efficiently catalyzing the synthesis of 3'-O-NH2-deoxynucleoside monophosphate using 3'-O-NH2-deoxynucleoside as a substrate, providing a new approach and pathway for nucleotide production. The bioenzymatic synthesis of (deoxy)nucleoside monophosphate of this invention has advantages such as high conversion rate, mild reaction conditions, simple production process, environmental friendliness, and ease of large-scale production.

The invention discloses a method for synthesizing deoxynucleoside monophosphate by a biological enzyme method. The mutant protein of dNK provided by the invention is a protein with deoxynucleoside kinase activity, which is obtained by mutating amino acid residues at 84th, 88th, 110th and 172nd positions on an amino acid sequence as shown in SEQ ID NO: 1 and keeping amino acid residues at other positions unchanged. According to the present invention, the deoxynucleoside kinase mutant is obtained, 3 '-O-NH2-deoxynucleoside can be adopted as the substrate, the 3'-O-NH2-deoxynucleoside monophosphate can be stably and efficiently catalyzed and synthesized, and the new thought and the new approach are provided for nucleotide production. The (deoxy) nucleoside monophosphate synthesized by the biological enzyme method has the advantages of high conversion rate, mild reaction conditions, simple production process, environmental friendliness, easiness in large-scale production and the like.

This application discloses a linker and its preparation method. The linker includes pharmacophore P, pharmacophore Q, and deoxynucleotides and / or deoxynucleotide derivatives connecting pharmacophore P and pharmacophore Q. Pharmacophore P is obtained by removing the protecting group from group B, and pharmacophore Q is obtained by removing the protecting group from group X. Group B is obtained by chemically modifying the benzylquinolone carboxylic acid pharmacophore, and group X is obtained by chemically modifying the zenomeprazole pharmacophore. The linker is prepared by linking group B and group X with deoxynucleotides and / or deoxynucleotide derivatives to form the linker, including solid-phase synthesis technology. The method of this application can automatically synthesize molecular probes or bivalent drugs containing two pharmacophores, thereby replacing the traditional stepwise linking method, enabling efficient construction of screening libraries, and allowing control of the spatial distance and spatial orientation of the pharmacophores by adjusting the number and base arrangement of deoxynucleotides.

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-terminal deoxynucleotidyl transferase (TdT) antibody and application thereof. The amino acid sequences of CDR1-3 on a light chain variable region of the antibody are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of CDR1-3 on a heavy chain variable region of the antibody are respectively shown as SEQ ID NO.8-10. The antibody disclosed by the invention can specifically recognize TdT protein naturally expressed in cells and tissues, has the advantages of high specificity, high affinity, high sensitivity and the like when being used for immunoblotting, immunohistochemistry and other immunodetection systems, is beneficial to reducing false positive and false negative rates in pathological diagnosis, improves the accuracy of immunodiagnosis, and has a good application prospect. The method has wide application potential in the field of immunological detection of living biological samples. In addition, the antibody disclosed by the invention has cross-species reaction characteristics on homologous TdT proteins of human, mice and rats, so that the application scene of the antibody is greatly expanded.

Disclosed herein is a composition comprising a lipid nanoparticle and a double-stranded oligodeoxynucleotide (dsODN) encapsulated in the lipid nanoparticle. According to the embodiments of the present disclosure, the dsODN comprises two strands complementary to each other, in which the first strand comprises the nucleotide sequence of SEQ ID NO: 1. Also disclosed herein are methods of treating cancers by using the present composition.

In one aspect, a method of treating an NK cell to induce a gene edit. The method may include contacting an NK cell with a transfection composition including poly-L-glutamic acid (y- PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition including: a DNA-PK inhibitor, and an HD AC inhibitor; electroporating the NK cell; and contacting the NK cell with a recovery composition including DNase I. In another aspect, a method of preparing an engineered NK cell comprising a fluorescent reporter at an endogenous locus. In another aspect, a method of treating an NK cell to introduce an exogenous nucleic acid.

The invention relates to a preparation method of a CpG ODN nano immune complex, which comprises the following steps: (1) uniformly mixing a protamine solution with a certain concentration and a CpG motif-containing oligodeoxynucleotide CpG ODN aqueous solution with a certain concentration according to a certain ratio, and carrying out electrostatic compounding assembly at room temperature to form a suspension; and (2) applying energy to the suspension to disperse / homogenize uniformly to form the suspension of the nano-composite. The nano-composite prepared by the method disclosed by the invention is controllable in particle size and controllable in surface charge. The nano particles formed under the preparation condition of an optimized proportion are uniformly dispersed, good in stability, good in resolubility after freezing, and good in long-term storage stability. The preparation method provided by the invention is simple, does not involve toxic and harmful chemical substances, and is suitable for amplification. Compared with the prior art, the preparation method provided by the invention can obviously improve the effective concentration of the effective active substance CpG ODN in the nano immune complex. The invention also relates to the nano immune complex prepared by the method and application thereof. Cell and animal experiments prove that the nano immune complex prepared by the method has good immunostimulatory activity, can stimulate the maturation of related immune cells and induce the secretion of related cytokines, can be independently used as an immunomodulator, and can also be used as an immunologic adjuvant to be combined with various antigens to be used as a vaccine preparation as a whole. The nano immune complex has good application prospects in the fields of inducing and activating immune cells to mature, releasing cytokines, resisting allergic diseases, preventing and treating microbial infectious diseases, vaccine adjuvants, resisting tumor immunotherapy and resisting Alzheimer's disease.

Compounds and pharmaceutical compositions useful to treat cancer (e.g., lymphoma), neurodegenerative diseases, and autoimmune disorders include (1) a first nucleic acid sequence capable of binding to a transcription factor, for example, a signal transducer and activator of transcription (STAT) factor, such as STAT3, (2) a second nucleic acid sequence capable of binding a Toll-like receptor protein, for example, a CpG oligodeoxynucleotide, and (3) a ubiquitin ligase binding compound capable of binding a ubiquitin ligase protein, for example, a compound that is capable of binding a cereblon protein, such as lenalidomide, pomalidomide, or thalidomide.

The invention relates to a high-flux enzymatic DNA synthesis method based on framework nucleic acid. Specifically, the invention provides a TdT-framework nucleic acid compound which can accurately regulate and control the position and distance of terminal deoxynucleotidyl transferase (TdT) so as to achieve the purpose of high-flux enzymatic DNA synthesis. The invention also provides a method for preparing the TdT-framework nucleic acid compound, and a method for synthesizing DNA (Deoxyribonucleic Acid) by using the TdT-framework nucleic acid compound. When the TdT-framework nucleic acid compound is used for synthesizing DNA, high-flux enzymatic DNA synthesis can be realized, and the TdT-framework nucleic acid compound has important significance for developing a high-flux DNA synthesis technology with mild conditions.

Multiple polynucleotides having different, arbitrary sequences are synthesized on the surface of an array by spatial control of polymerase activity. The polymerase is a template-independent polymerase such as terminal deoxynucleotidyl transferase (TdT). Spatial control of polymerase activity is implemented by localized changes in redox-pH conditions. A single species of nucleotide is added and incorporated on growing polynucleotide strands at locations on the array where the polymerase is active. A washing step removes the polymerase and free nucleotides. This process may be repeated multiple times changing both the location of polymerase activity and the species of nucleotide thereby synthesizing different polynucleotides in parallel on the surface of the array. Polymerase activity may be regulated by removing a blocking group attached to a His-tag sequence on the polymerase, a change in pH, or release of encapsulated inhibitors.

The application discloses CpG-containing oligodeoxynucleotide (ODN) with the function of regulating the body's immune capacity and application thereof. The oligodeoxynucleotide in the application is different from the design concept of traditional A-type CpG ODN in sequence and structure, embodies the complex drug-making rule and overcomes the problem of traditional A-type CPG in drug-making. The oligodeoxynucleotide has excellent activity of regulating the body's immune function in vitro and in vivo, can stimulate the production of I, II and III type interferons, regulates the Th1 type immune response, and inhibits the Th2 type and Th17 type inflammatory response. Therefore, the oligodeoxynucleotide can prevent and treat allergic diseases caused by Th2 type immune response, including allergic rhinitis, atopic dermatitis, asthma, chronic obstructive pulmonary disease, eosinophil and Th17 related diseases, can prevent and treat the infection and spread of respiratory or non-respiratory pathogenic microorganisms including viruses, bacteria, fungi and parasites, can improve the immune level of the elderly and immunodeficient population, can convert "cold tumor" into "hot tumor", improve the effect of cancer immunotherapy, can prevent and treat central nervous system diseases related to immune dysfunction such as Alzheimer's disease, and can be applied to other diseases related to immune dysfunction.

The application relates to the technical field of biological medicine, and particularly discloses CpG oligodeoxynucleotides and application thereof. The CpG oligodeoxynucleotide has a sequence as shown in any one of SEQ ID NO. 3-6, 9, and preferably has a sequence as shown in SEQ ID NO. 3-6. The CpG oligodeoxynucleotide can effectively activate the immune system of a human body or an animal, has the functions of preventing and treating pathogenic microorganism infection in a broad spectrum, has the function of improving the immunogenicity of a common vaccine, and can be applied as a human or animal vaccine adjuvant.

The application relates to the field of organic membrane separation, and specifically discloses a method for replacing deoxyribonucleotide triphosphate amine salt with sodium salt by adopting nanofiltration roll type membrane. The method specifically comprises the following steps: a concentration step, concentrating deoxyribonucleotide triphosphate amine salt fraction after high-pressure liquid phase purification; a replacement step, adding 1.5mol / L-6mol / L sodium (potassium) carbonate or sodium (potassium) hydrogen carbonate 0.5V-2V salt solution to the deoxyribonucleotide triphosphate amine salt solution for low-temperature replacement; and a desalination step, introducing carbon dioxide into the system in the above step, and washing with water until sodium ions cannot be detected in the filtered liquid, so as to obtain a deoxyribonucleotide triphosphate sodium salt solution. The preparation method can replace the organic amine salt with sodium salt, and 100% replacement of the organic amine salt can be realized.

A pharmaceutical composition comprising: i) a herpes gE protein, an active fragment of the protein, a variant of the protein, or a mixture of at least two of them; ii) an immunostimulatory composition comprising a saponin and a CpG oligodeoxynucleotide, or consisting of an adjuvant comprising a saponin and a CpG oligodeoxynucleotide. Use of the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a varicella-zoster virus infection and / or a varicella-zoster virus mediated disease. The pharmaceutical composition achieves an unexpected technical effect and can mediate a stronger immune response.

The invention relates to the field of DNA sequencing, in particular to a short-chain ssDNA sequencing library construction method, a sequencing method and application. The construction method of the sequencing library of the short-chain ssDNA comprises the following steps: a step of adding a polynucleotide tail: taking the short-chain ssDNA as a template, carrying out enzyme reaction by utilizing terminal deoxynucleotidyl transferase, and adding the polynucleotide tail at the 3'terminal of the short-chain ssDNA to obtain tailed short-chain ssDNA; carrying out PCR (Polymerase Chain Reaction) amplification on the short-chain ssDNA; a tail end repairing step; a linker connection step: taking the product obtained in the terminal repairing step as a template, carrying out a reaction by using ligase to obtain a template with a linker, and purifying the template with the linker; and a PCR enrichment and purification step of dsDNA: designing a second primer by taking a purified product obtained in the step of connecting the joints as a template, and carrying out PCR enrichment and purification to obtain the sequencing library. According to the short-chain ssDNA sequencing library construction method, the defects in the prior art are overcome, the short-chain ssDNA sequencing library construction method is not limited by ssDNA in a fixed state any more, and library construction of special ssDNA is achieved.

Methods, devices, and compositions are provided for inkjet assisted synthesis of a plurality of polynucleotides at a reaction site on a substrate using a template-free polymerase (e.g., a terminal deoxynucleotidyl transferase (TdT)). Compositions include printable stable formulations of synthetic reagents for inkjet delivery, including, but not limited to, template-free polymerases, divalent cations, and nucleotides.

The application discloses a TdT signal amplification PAM-independent Cas12a lateral chromatography nucleic acid detection method and application. The method comprises the following steps: performing a first reaction on a first reaction system comprising a to-be-detected nucleic acid, a recombinase polymerase amplification reaction component, a recombinase polymerase amplification primer pair and an exonuclease, so as to obtain a single-stranded target nucleic acid; performing a second reaction on a second reaction system comprising the single-stranded target nucleic acid, a CRISPR / Cas12a reaction component, a reporter probe, a terminal deoxynucleotidyl transferase and a deoxynucleotide triphosphate substrate, so as to obtain an extension product; and contacting and hybridizing the extension product with a gold nanoparticle-labeled detection probe in a lateral chromatography process, and when a visual detection signal is obtained, it is judged that the to-be-detected nucleic acid is a target nucleic acid. The method of the application can significantly improve the detection sensitivity and signal contrast while getting rid of the PAM restriction, the detection process does not need complex instruments, the result can be judged by naked eyes, and the method is suitable for on-site rapid visual detection of pathogenic microorganism nucleic acid.