66 results about "Phosphodiester bond" patented technology

The invention discloses a conjugate of antisense oligonucleotide and polypeptide as well as a preparation method and application of the conjugate, belongs to the technical field of biological medicine, and provides the conjugate of antisense oligonucleotide and polypeptide, the structural general formula of the conjugate of antisense oligonucleotide and polypeptide is as shown in formula (I), O is antisense oligonucleotide, L is a connexon, and P is polypeptide; the antisense oligonucleotide is a nucleotide sequence obtained by modifying thiophosphate and 2 '-O-methyl on the basis of a nucleotide sequence as shown in SEQ ID NO.1 or a nucleotide sequence as shown in SEQ ID NO.2; and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.3. According to the conjugate of the antisense oligonucleotide and the polypeptide, a phosphodiester bond of RNA is catalyzed by the polypeptide P to break, the antisense oligonucleotide O is complementarily paired with a frame-shift element, the polypeptide P is brought to the vicinity of the frame-shift element for targeted cleavage, and a linker L is responsible for coupling, sequence-specific targeted degradation of pathogenic RNA and destruction of the expression condition of RNA in cells.

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 invention discloses a conjugate of an antisense oligonucleotide and a polypeptide, a preparation method thereof and an application thereof, belonging to the technical field of biomedicine. Provided is a conjugate of an antisense oligonucleotide and a polypeptide, the general structural formula of which is shown in formula (I), wherein O is an antisense oligonucleotide, L is a linker, and P is a polypeptide; the antisense oligonucleotide is a nucleotide sequence obtained by phosphorothioate and 2'-O-methyl modification based on the nucleotide sequence of SEQ ID NO.1 or the nucleotide sequence shown in SEQ ID NO.2; the amino acid sequence of the polypeptide is shown in SEQ ID NO.3. In the conjugate of the antisense oligonucleotide and the polypeptide of the present invention, the polypeptide P catalyzes the cleavage of the phosphodiester bond of RNA, the antisense oligonucleotide O is complementary paired with the frameshift element, brings the polypeptide P to the vicinity of the frameshift element, and performs targeted cleavage. The linker L is responsible for coupling, specifically targeting and degrading pathogenic RNA in a sequence-specific manner, and disrupting the expression of RNA in cells.

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.

Provided in the present invention are a preparation method for a non-natural RNA and a product. The preparation method for the non-natural RNA comprises: connecting the 3' end of a substrate donor with the 5' end of a substrate receptor by using an RNA ligase to form a phosphodiester bond, wherein the connection does not need a template chain, and the non-natural RNA is prepared. The substrate donor and / or the substrate receptor comprise one or more non-natural nucleotides. The 5' end of the substrate receptor is a phosphoric acid group; and the 3' end of the substrate donor is a hydroxyl group. The RNA ligase comprises any one or more enzymes in RNA ligase family Rnl1 and Rnl2. The solution solves the problem in the prior art of non-natural RNA being difficult to prepare using enzyme catalysis, and the method is suitable for the field of RNA synthesis.

Provided are a gRNA and a biosynthesis method therefor, suitable for the field of gRNA synthesis. The biosynthesis method comprises: connecting the 3' end and the 5' end of different substrates by using an RNA ligase to form a phosphodiester bond, so as to form a gRNA. The gRNA comprises natural gRNA or non-natural gRNA. The number of the substrates is greater than or equal to 2. The connection site of the substrate is located at any one or more positions in the stem-loop structure complementary region, the stem-loop structure annular region or a linker region between the stem-loop structures of the gRNA, and base complementary pairing can be carried out in a substrate or among substrates to form a secondary structure of the gRNA; wherein the RNA ligase comprises any one or more enzymes in the RNA ligase family 1, 2 or 3. The solution solves the problem in the prior art of low purity of synthesized gRNA.

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 discloses a pharmaceutical nucleic acid synthesis process. In the synthesis process, the present invention temporarily protects unnecessary groups as much as possible. After a round of coupling reaction, the protecting groups on the previous round of groups are selectively removed to form a specific phosphodiester bond, thereby reducing side reactions as much as possible, improving the yield of the target product, and reducing the difficulty of purification. The present invention can achieve no transfer of the synthesis column during the aminolysis process by simply transforming the synthesis column, online aminolysis, elution of the oligonucleic acid product loaded on the surface of the solid phase support in the synthesis column, collection of the eluate containing the oligonucleic acid product and subsequent purification process. Since the synthesis column does not need to be offline during the whole process, the difficulty and complexity of the work are greatly reduced on the one hand, and the exhaust gas leakage during the disassembly and installation process is significantly reduced on the other hand.

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 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 thiophosphate linkages and phosphodieester 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 invention also provides compositions comprising said oligonucleotides and / or oligonucleotide analogue molecules and their use for the treatment and prevention of diabetes in a subject in need thereof.

The invention relates to a linearization method for NGS sequencing, a sequencing chip and a sequencing method. The linearization method comprises the following steps: introducing single ribonucleoside into a DNA primer, wherein the 2 '-OH position of the ribonucleoside is modified with a hydroxyl protecting group; during linearization, removing the hydroxyl protecting group by using a cutting reagent, and exposing 2 '-OH of ribonucleoside; a phosphodiester bond at the ribonucleoside position is cut off, so that one chain in the DNA primer is broken; the linear reaction formula is shown in the specification. According to the novel linearization method, one DNA chain can be cut and removed very quickly and efficiently, the single-stranded DNA template suitable for sequencing is obtained, and the novel linearization method has the advantages of low damage to DNA, capability of obviously improving the sequencing quality, good stability and capability of keeping stable sequencing quality in actual PE (Poly Ethylene) sequencing application. # imgabs0 #

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.

The present invention provides a preparation method of Patisiran. The Patisiran is a double-stranded RNA composed of a complementary sense strand and an 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 a sense strand, and the antisense strand substrate fragment can form an antisense strand; the sense strand substrate fragment and the antisense strand substrate fragment are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate fragment and the antisense strand substrate fragment are not connected to each other, forming a double-stranded nucleotide structure containing a nick; and using RNA ligase to connect the bases at both ends of the nick with a phosphodiester bond to form Patisiran. The method can solve the problem of low purity of Patisiran prepared in the prior art, and is applicable to the technical field of drug biosynthesis.

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 in the present invention is a preparation method for a siRNA for inhibiting PCSK9 gene expression. The siRNA in the preparation method is a double-stranded RNA consisting of a sense strand and an antisense strand which are complementarily paired. The preparation method comprises: mixing a sense strand substrate, an antisense strand substrate and an RNA ligase, using the RNA ligase for catalyzing the sense strand substrate and the antisense strand substrate to be connected by means of a phosphodiester bond, and obtaining a sense strand and an antisense strand, so as to obtain the siRNA. The sense strand substrate can form the sense strand, and the antisense strand substrate can form the antisense strand. The sense strand is a nucleic acid sequence as shown in SEQ ID NO: 45, and the antisense strand is a nucleic acid sequence as shown as SEQ ID NO: 46. The solution solves the problem in the prior art of low purity of the siRNA, and is applicable in the field of medicine biosynthesis.

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.

The invention provides a probe, a freeze-drying reagent, a lateral flow chromatography test strip, a kit and a method for detecting RNase activity, and relates to the technical field of biology. According to the present invention, based on the specific antigen-antibody reaction and the immunochromatography technology, the RNA probe with the 5'end labeled with digoxin and the 3 'end labeled with biotin is selected as the substrate, and under the condition that the sample to be detected contains RNase and is mixed with the substrate, the RNase can specifically recognize and cut the phosphodiester bond on the RNA probe so as to fragment the RNA; then, the test strip is inserted into the reacted reagent, if the sample does not contain RNase, the RNA probe is not subjected to enzyme digestion and can be fixed on the quality control line and combined with streptavidin, the quality control line shows a red strip, and the detection line does not develop color; if RNase exists in the sample, the RNA probe is fixed on the detection line after being subjected to enzyme digestion and is combined with the secondary antibody, and a red strip appears on the detection line.

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.