42 results about "Phosphodiester bond" patented technology

A number of T4 DNA ligase mutants exhibiting enhanced ligation activity in the presence of high salt concentrations compared to the wild-type ligase were engineered, characterized, and selected via gel electrophoresis of ligation products from a standard ligation assay. Ligase catalyzes the formation of phosphodiester bonds between the 5′ and 3′ ends of complementary cohesive ends or blunt ends of duplex DNA, a process that is vital to numerous molecular biology processes including cloning and sequencing.

A method for confirming the sequence of a nucleic acid is provided, comprising: (i) hydrolytic cleavage of predetermined phosphodiester bonds in the nucleic acid to generate a plurality of oligonucleotides; (ii) ionization of the plurality of oligonucleotides to provide a gas phase of ionized oligonucleotides, wherein a majority of the ionized oligonucleotides are singly charged; (iii) isolation of the ionized oligonucleotides using a plurality of mass-to-charge ratio (m / z) windows; (iv) detection of the isolated ionized oligonucleotides of each m / z window using a high-resolution mass spectrometer (HRMS) with a resolution of more than 30.000 at m / z 200, thereby obtaining a mass spectrum; (v) Confirming the sequence of the nucleic acid by comparing the mass spectrum obtained in step (iv) with a mass spectrum determined in silico after the same hydrolytic cleavage of the nucleic acid as in step (i), the confirmation comprising a comparison of the signal intensities of one or more mass envelopes resulting from the ionized oligonucleotides.

Provided herein is a particle made of a protein covalently bound to a prebiotic carbohydrate, thereby forming a conjugate, wherein at least two said conjugates are covalently crosslinked via their carbohydrate units (e.g. by a phospho-di-ester bond). The said particle may be used for selectively promoting probiotic bacteria growth in the colon and may be used to deliver additional probiotic growth factors, or other bioactives and drugs to the colon. Furthermore, provided herein are methods for preparing the particle, and for delivering a substance bound to, or entrapped within the particle, into the gastrointestinal tract of a subject in need thereof.

The invention belongs to the technical field of environmental functional materials and water pollution control, discloses a cerium monatomic nano-enzyme as well as a preparation method and application thereof, and effectively solves the problem that an existing advanced oxidation technology lacks specificity on ARGs degradation and the problems that a traditional nano-enzyme is non-selective in catalytic mechanism and undefined in active site structure. According to the cerium monatomic nano-enzyme, a metal organic framework UiO-67-N is used as a carrier, pore channels of the carrier are modified through a nitrogen-containing ligand 2, 2 '-bipyridine-5, 5'-dicarboxylic acid, anchoring sites of cerium monatomic are constructed, a monatomic active center with a Ce-N6 coordination structure is formed, and cerium stably exists in a material framework in an atomic-scale dispersion form. According to the method, efficient recognition and cutting of extracellular DNA phosphodiester bonds in a neutral water environment are achieved, ARGs structures are directly destroyed, horizontal transfer pathways of the ARGs structures are blocked, and an enzyme simulation catalysis tool is provided for precise blocking and controlling of the ARGs in a water body.

The present invention provides oligonucleotide and / or oligonucleotide analogue molecules which are antagonists of microRNAs, preferably of human microRNAs hsa-miR-23b-3p and hsa-miR-218-5p, which comprise a mixture of phosphorothioate and phosphodiester linkages, and which are conjugated to at least one oleic acid molecule. Inhibition of these microRNAs can increase the endogenous levels of the corresponding proteins MBNL1 and / or MBNL2. The present invention further provides compositions comprising said oligonucleotide and / or oligonucleotide analogue molecules and their use for the treatment and prevention of diabetes in a subject in need thereof.

The present invention relates to a chemically modified small interfering RNA (siRNA), and more particularly, to a chemically modified siRNA having enhanced nuclease degradation resistance and effectively exhibiting RNAi activity compared to an unmodified siRNA in which 15 bases are extended at the 5'terminal of a sense strand and the 15 bases are simultaneously subjected to phosphorothioate modification, i.e., a non-bridged oxygen atom of a phosphodiester bond is substituted with a sulfur atom. The invention provides a siRNA delivery tool, a method for promoting siRNA delivery to cause gene silencing and improving the efficiency of delivering siRNA to plant cells and application of the siRNA delivery tool, siRNA modified by thiophosphate can be effectively introduced into YFP (Yellow Fluorescent Protein) transformed arabidopsis thaliana leaves, and gene silencing of YFP is realized. The invention also can effectively silence endogenous genes of tobacco. Therefore, the thiophosphate modified siRNA can be used for silencing a certain gene of a plant to achieve the effect of regulating good growth of the plant or treating related plant diseases and insect pests, and has potential agricultural production value and wide application prospect.

The present invention provides a method for preparing an siRNA for treating α1-antitrypsin deficiency. The siRNA is Fazirsiran. Fazirsiran is a double-stranded RNA composed of a complementarily paired sense strand and antisense strand. The preparation method comprises: mixing sense strand substrates, antisense strand substrates, and an RNA ligase, and using the RNA ligase to catalyze the linkage between the sense strand substrates and between the antisense strand substrates by means of phosphodiester bonds, to obtain a sense strand and an antisense strand, thereby obtaining Fazirsiran, wherein the sense strand substrates can form the sense strand, and the antisense strand substrates can form the antisense strand. Compared with the preparation of Fazirsiran by means of chemical synthesis, the product obtained by the preparation method of the present application has higher purity and fewer impurities, and the reaction conditions are mild, facilitating industrial large-scale production.

The invention provides oligonucleotide and / or oligonucleotide analogue molecules that are antagonists of a microRNA, preferably antagonists of human microRNAs hsa-miR-23b-3p and hsa-miR-218-5p, that comprise a mixture of phosphorothioate and phosphodiester linkages, and that are conjugated to at least one oleic acid molecule. Inhibiting these microRNAs allows to increase the endogenous levels of the corresponding proteins MBNL1 and / or MBNL2. The present invention further provides compositions comprising said oligonucleotides and / or oligonucleotide analogue molecules and their uses for the treatment and prevention of DM in a subject in need thereof.

The present application relates to a double-stranded DNA molecule comprising a first double-stranded DNA molecule (1) connected to a second double-stranded DNA molecule (2) by at least one covalent bond which is not a phosphodiester, phosphorothioate, phosphoramidate or phosphorodiamidate bond, preferably by a tether, said tether preferably being a double-stranded DNA molecule.

This invention discloses a circular RNA agent targeting PCSK9 and its applications, relating to the field of biomedical technology. The circular RNA agent is a single-stranded closed-circular oligonucleotide containing an antisense sequence complementary to the transcription of the PCSK9 gene. It lacks free 5' and 3' ends, and does not contain overhangs, hairpin structures, or double-stranded regions. The molecules are linked by 3'-5' phosphodiester bonds. This circular RNA agent can be modified with GalNAc or prepared into pharmaceutical compositions for the treatment of diseases or conditions related to abnormal PCSK9 gene expression or activity. The circular RNA agent provided by this invention exhibits high stability, a long half-life, low immunogenicity, low modification toxicity, and high target gene inhibitory activity. It can significantly prolong the dosing interval, improve patient compliance, and provide a safer, longer-acting drug option for lipid-lowering therapy.

The present application relates to the technical field of biological medicine, and in particular to a synthesis method and application of circular shRNA with gene knockdown function. The present application provides a synthesis method of circular shRNA with gene knockdown function. The ends of linear shRNA are connected by 2', 5'-phosphodiester bond to form circular shRNA, which can improve the stability of delivery in the environment of exonuclease. Moreover, after entering the cell, the 2', 5'-phosphodiester bond can be cleaved by DBR1 (Debranching RNA Lariats 1), so as to obtain shRNA, which can play the gene knockdown function of shRNA and play the role of inhibiting gene expression. The present application provides a new direction for developing a new nucleic acid drug for knocking down gene expression, and has a broad application prospect.

Provided is a method for preparing siRNA for inhibiting FXI gene expression. The siRNA is Fitusiran, and the Fitusiran is a double-stranded RNA composed of a sense strand and an antisense strand that complement and are paired with each other. The preparation method comprises: mixing sense strand substrates, antisense strand substrates and an RNA ligase, wherein the sense strand substrates can constitute the sense strand, the antisense strand substrates can constitute the antisense strand, the sense strand substrates and the antisense strand substrates are connected via hydrogen bonds formed by means of complementary base pairing, and no linkage is formed between head and tail bases of the sense strand substrates and between head and tail bases of the antisense strand substrates, thereby forming a nicked double-stranded nucleotide structure; and ligating the bases at both ends of the nick via a phosphodiester bond using the RNA ligase to form the Fitusiran. The preparation method can solve the problem of Fitusiran prepared by means of the prior art having low purity.

A method for amplifying a DNA target region is provided. The method includes: linearly amplifying a fragmented DNA including the target region by a specific primer to provide a linear amplification product, where 3′ end of the specific primer is modified by a duality functional group, a phosphodiester bond of a part of nucleotide backbone at the 3′ end of the specific primer is modified by phosphorothioate, and the duality functional group is used to prevent the 3′ end of the specific primer from ligation with other oligonucleotides and can be removed by a specific enzyme to perform a linear amplification of the specific primer. The method for amplifying a DNA target region effectively reduces a non-specific amplification during the linear amplification by introducing a phosphorothioate modification into the specific primer.

Provided in the present invention is a method for preparing cemdisran, wherein cemdisran is an siRNA composed of a sense strand and an antisense strand by means of complementary pairing. The preparation method comprises mixing sense strand substrates, antisense strand substrates and an RNA ligase, and using the RNA ligase to catalyze the ligation between the sense strand substrates and between the antisense strand substrates via phosphodiester bonds, so as to obtain the sense strand and the antisense strand, and thereby obtaining cemdisran. The sense strand substrates can form the sense strand, and the antisense strand substrates can form the antisense strand. Compared with the preparation of cemdisiran by means of chemical synthesis, a product obtained by means of the preparation method of the present application has a relatively high purity and few impurities. Moreover, the reaction conditions are mild, thus facilitating the realization of industrial scaled-up production.

Provided is a method for preparing givosiran. Givosiran is a double-stranded siRNA composed of a sense strand and an antisense strand by means of complementary pairing. The preparation method therefor comprises mixing a sense strand substrate, an antisense strand substrate and an RNA ligase, wherein the sense strand substrate can form the sense strand, the antisense strand substrate can form the antisense strand, the sense strand substrate and the antisense strand substrate are linked via a hydrogen bond formed by means of base complementary pairing, and the terminal bases of the sense strand substrate and antisense strand substrate are not linked to each other, thereby forming a double-stranded nucleotide structure containing a nick. The RNA ligase is used to link the bases at both ends of the nick via a phosphodiester bond to form givosiran. The preparation method can solve the problem of the relatively low purity of givosiran in the preparation in the prior art.

Provided are methods for non-enzymatically synthesizing nucleic acids. The methods include submerging a first portion of the outer surface of a cylinder in a non-enzymatic nucleic acid synthesis reaction mixture. The reaction mixture has a pH of 4 or less and includes an organizing matrix reagent and monophosphate nucleotides. The methods further include rotating the cylinder about its axis of radial symmetry so that the first portion of the outer surface of the cylinder is no longer submerged in the reaction mixture, thereby providing a thin film of the reaction mixture on the first portion of the outer surface of the cylinder. The methods further include heating and drying the thin film to form phosphodiester bonds between the monophosphate nucleotides of the thin film. Also provided are devices that find use, e.g., in practicing the methods of the present disclosure.

The invention provides for an oligonucleotide lipid group conjugate, wherein the oligonucleotide comprises at least two alpha anomeric bicyclo-DNA residues connected by a phosphodiester bond, and wherein the lipid group is attached to the oligonucleotide via a linker. The invention also provides for methods of modulating gene expression using an oligonucleotide lipid group conjugate.

The present disclosure relates to a delivery platform for payload-specific and efficient directing of stable RNAi agents to hepatocytes in a subject. The delivery platform disclosed herein includes a metabolically stable N-acetylgalactosamine (NAG or GalNAc) targeting ligand conjugated to one or more oligonucleotides by a metabolically stable bond that is more stable than a phosphodiester bond to facilitate delivery of oligonucleotide-based payloads to cells, including hepatocytes. Also described are pharmaceutical compositions comprising the metabolically stable RNAi agent conjugate delivery platform, as well as methods for treating various diseases and conditions in which a therapeutic payload is required to be delivered to hepatocytes.

Provided in the present application is a method for preparing an siRNA for treating non-arteritic anterior ischemic optic neuropathy. The siRNA is cosdosiran, which is a double-stranded RNA composed of a complementarily paired sense strand and antisense strand. The preparation method comprises mixing sense strand substrates, antisense strand substrates and an RNA ligase, and using the RNA ligase to catalyze the ligation between the sense strand substrates and between the antisense strand substrates via phosphodiester bonds, so as to obtain the sense strand and the antisense strand, and thereby obtaining cosdosiran. The sense strand substrates can form the sense strand, and the antisense strand substrates can form the antisense strand. Compared with the preparation of cosdosiran by means of chemical synthesis, a product obtained by means of the preparation method of the present application has a relatively high purity and few impurities. Moreover, the reaction conditions are mild, thus facilitating the realization of industrial scaled-up production.

The invention discloses a method for efficiently eliminating a primer dimer during construction of an amplicon library and a construction method of the amplicon library, and belongs to the technical field of biology. The method for efficiently eliminating the primer dimer comprises the following steps: carrying out multiple PCR (Polymerase Chain Reaction) by using a multiple primer modified by a 3'end phosphodiester bond sulfo group to obtain a multiple PCR reaction product; and digesting the multiplex PCR reaction product by using exonuclease, degrading and eliminating the primer dimer, and simultaneously removing primer sequences on the two sides of the amplification product to obtain the amplification product without the primer dimer. The invention discloses a construction method of an amplicon library, which comprises the following steps of: eliminating a primer dimer by adopting the method, performing purification, terminal modification, linker sequence connection, linker purification and linker PCR reaction on an amplification product, and finally constructing the amplicon library. The method disclosed by the invention not only can efficiently eliminate the primer dimer, but also is low in cost and suitable for popularization and application.

The present disclosure relates to delivery platforms that specifically and efficiently direct stabilized RNAi agent payloads to hepatocytes in a subject, in vivo. The delivery platforms disclosed herein include metabolically stabilized N-Acetylgalactosamine (NAG or GalNAc) targeting ligands conjugated to one or more oligonucleotides through a metabolically stabilized linkage that is more stable than a phosphodiester linkage, to facilitate the delivery of the oligonucleotide-based payloads to cells, including to hepatocytes. Pharmaceutical compositions that include the metabolically stabilized RNAi agent conjugate delivery platform are also described, as well as methods of use for the treatment of various diseases and disorders where delivery of a therapeutic payload to a hepatocyte is desirable.

The present invention relates to a method for decapping a 5'-dinucleotide-capped nucleic acid molecule, wherein the dinucleotide is linked to the nucleic acid molecule via a phosphodiester bond and the dinucleotide comprises a diphosphate, triphosphate or tetraphosphate linkage between the two nucleotides of the dinucleotide, and wherein the method comprises contacting the 5'-dinucleotide- capped nucleic acid molecule with (I) an enzyme being capable of cleaving the diphosphate, triphosphate or tetraphosphate linkage, wherein the enzyme (a) comprises or consists of the amino acid sequence of SEQ ID NO: 21, (b) comprises or consists of an amino acid sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical to SEQ ID NO: 21, (c) comprises or consists of the amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 17, or (d) comprises or consists of an amino acid sequence being encoded by a nucleotide sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical to SEQ ID NO: 17; (II) a nucleic acid molecule, preferably a vector, encoding in expressible form the enzyme of (I); and / or (III) a host cell comprising the nucleic acid molecule, preferably the vector of (II).

Provided is a method for preparing patisiran. Patisiran is a double-stranded RNA composed of a complementarily paired sense strand and antisense strand. The preparation method comprises mixing a sense strand substrate fragment, an antisense strand substrate fragment and an RNA ligase, wherein the sense strand substrate fragment can form the sense strand, the antisense strand substrate fragment can form the antisense strand, the sense strand substrate fragment and the antisense strand substrate fragment are linked via a hydrogen bond formed by means of base complementary pairing, and the terminal bases of the sense strand substrate fragment and antisense strand substrate fragment are not linked to each other, thereby forming a double-stranded nucleotide structure containing a nick. The RNA ligase is used to link the bases at both ends of the nick via a phosphodiester bond to form patisiran. The preparation method can solve the problem of the relatively low purity of patisiran in the preparation in the prior art.

The invention belongs to the technical field of molecular biology, and particularly relates to template conversion oligonucleotide, a kit and application of the template conversion oligonucleotide. In the first aspect of the invention, a template conversion oligonucleotide is provided, the template conversion oligonucleotide comprises a template conversion region at the 3'end, the template conversion region comprises a plurality of nucleotides, the nucleotides comprise sugars and basic groups, and the basic groups are guanine; wherein in the plurality of nucleotides, the 3 '-hydroxyl site of the glycosyl of the nucleotide at the 3'-terminal is provided with a modification for preventing the 3 '-hydroxyl from forming a phosphodiester bond. TSO of conventional reverse transcription is specifically modified, then conventional reverse transcription processes of single cells, space-time omics and the like are carried out, additional enrichment operation is not needed, and the operation process is simple. Not only is the data utilization rate of single molecule sequencing improved, but also the sensitivity of target gene capture is improved, and background interference is reduced.

The present invention relates to a method for decapping a 5'-dinucleotide-capped nucleic acid molecule, wherein the dinucleotide is linked to the nucleic acid molecule via a phosphodiester bond and the dinucleotide comprises a diphosphate, triphosphate or tetraphosphate linkage between the two nucleotides of the dinucleotide, and wherein the method comprises contacting the 5'-dinucleotide- capped nucleic acid molecule with (I) an enzyme being capable of cleaving the diphosphate, triphosphate or tetraphosphate linkage, wherein the enzyme (a) comprises or consists of the amino acid sequence of SEQ ID NO: 21, (b) comprises or consists of an amino acid sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical to SEQ ID NO: 21, (c) comprises or consists of the amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 17, or (d) comprises or consists of an amino acid sequence being encoded by a nucleotide sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical to SEQ ID NO: 17; (II) a nucleic acid molecule, preferably a vector, encoding in expressible form the enzyme of (I); and / or (III) a host cell comprising the nucleic acid molecule, preferably the vector of (II).

The invention provides a synthesis method of a single-chain aptamer and application of the single-chain aptamer in synthesis of a C5 complement inhibitor, and relates to the technical field of aptamer synthesis, the synthesis method comprises the following steps: cutting off the single-chain aptamer into two fragments, pairing the two fragments through complementary bases carried by the two fragments to form a double-chain secondary structure with a single gap, and under the enzymatic action, synthesizing the C5 complement inhibitor by using the double-chain secondary structure to obtain the C5 complement inhibitor. A phosphodiester bond is formed at a notch of the double-chain secondary structure to obtain a single-chain aptamer, and the base length ratio of the two fragments is (14-26): (25-13). The self-complementary nucleic acid fragment of the to-be-synthesized single-stranded aptamer is used as a substrate, specific connection is realized under the action of ligase, an exogenous template chain is not needed, and the final purity and yield of the single-stranded aptamer are improved. The technical problems that in the prior art, enzymatic connection of single-stranded oligonucleotides needs to depend on an exogenous template, so that an experimental system is complex, mismatching is likely to happen, and connection position errors are likely to happen are solved.

Provided is a method for preparing Vutrisiran. Vutrisiran is a double-stranded RNA composed of a complementarily paired sense strand and antisense strand. The preparation method therefor comprises: mixing sense strand substrate fragments, antisense strand substrate fragments, and an RNA ligase, wherein the sense strand substrate fragments can form the sense strand, and the antisense strand substrate fragments can form the antisense strand. The sense strand substrate fragments and the antisense strand substrate fragments are linked by means of hydrogen bonds formed by base complementation, and terminal bases of the sense strand substrate fragments and the antisense strand substrate fragments are not linked to each other, to form a double-stranded nucleotide structure containing a nick. The bases at two ends of the nick are linked by means of a phosphodiester bond by using the RNA ligase to form Vutrisiran. The preparation method can solve the problem in the prior art that the purity of prepared Vutrisiran is relatively low.

A method for confirming a sequence of a nucleic acid is provided, the method comprising: (i) hydrolytic cleavage of pre-determined phosphodiester bonds in the nucleic acid to produce a plurality of oligonucleotides; (ii) ionizing the plurality of oligonucleotides to provide ionized oligonucleotides in the gas phase, wherein a majority of the ionized oligonucleotides are singly charged; (iii) separating the ionized oligonucleotides using a plurality of mass-to-charge (m / z) windows; (iv) detecting the separated ionized oligonucleotides of each m / z window using a high resolution mass spectrometer (HRMS) having a resolution greater than 30,000 at m / z 200, thereby obtaining a mass spectrum; (v) confirming the sequence of the nucleic acid by comparing the mass spectrum obtained in step (iv) to a mass spectrum determined by computer simulation after the same hydrolytic cleavage of the nucleic acid in step (i), wherein the confirming comprises comparing signal intensities of one or more mass envelopes produced by the ionized oligonucleotides.

The present application relates to the field of nucleic acid detection, and particularly relates to a novel nucleic acid detection method based on CRISPR-Cas12. By introducing chemical modification (such as locked nucleic acid modification), the present application constructs a method for site-specific cleavage of specific phosphodiester bonds in single-stranded nucleic acid by Cas protein containing RuvC domain and a method for detecting target DNA in a sample to be tested, so as to realize site-specific cleavage and trigger signal cascade amplification, and then realize rapid and high-sensitivity detection of nucleic acid.