2013 results about "Double strand" patented technology

The invention belongs to the technical field of microorganisms, and particularly relates to recombinant escherichia coli containing a PDRN sequence and a preparation method thereof, and the preparation method comprises the following steps: S1, preparing a PDRN solution; s2, complementing the double strands of the PDRN into blunt ends; s3, constructing a recombinant plasmid; s4, carrying out mixed culture on a product obtained in the step S3 and escherichia coli DH5alpha; s5, screening the recombinants; s6, carrying out PCR (Polymerase Chain Reaction) amplification, and recording a strain from a fragment with the length of more than 200bp; s7, preparing a seed solution by adopting the strain prepared in the step S6; s8, strain fermentation; and S9, centrifugally collecting thalli. The method comprises the following steps: constructing a recombinant plasmid containing a PDRN fragment, introducing the recombinant plasmid into Escherichia coli, subsequently culturing the recombinant Escherichia coli to copy DNA in the Escherichia coli, extracting a purified exogenous gene fragment to obtain a strain containing a single fragment PDRN sequence, and getting rid of dependence on raw materials during subsequent extraction.

A modification template for improving the inhibitory effect of a double-stranded oligonucleotide on a target gene, a combination thereof and the use thereof. The double-stranded oligonucleotide is an siRNA and contains a sense strand and an antisense strand, and each nucleotide of the sense strand and antisense strand is a modified nucleotide. The double-stranded oligonucleotide is modified with a special modification template, and has a significantly improved inhibition rate on a target gene compared with a basic sequence.

The invention generally relates to sequencing library preparation methods. In certain embodiments, two or more template nucleic acids are joined together by a linking molecule, such as a PEG derivative. Identical copies of a nucleic acid fragment or both strands of a duplex fragment may be linked together. The linked nucleic acids are amplified, creating linked amplicons. Emulsion PCR with linked primers creates linked template nucleic acids for seeding sequencing clusters and errors can be readily identified by their presence on only one of the linked fragments.

The invention generally relates to sequencing library preparation methods. In certain embodiments, two or more template nucleic acids are joined together by a linking molecule, such as a PEG derivative. Identical copies of a nucleic acid fragment or both strands of a duplex fragment may be linked together. The linked nucleic acids are amplified, creating linked amplicons. Emulsion PCR with linked primers creates linked template nucleic acids for seeding sequencing clusters and errors can be readily identified by their presence on only one of the linked fragments.

The present invention provides a method and a kit for detecting a target nucleic acid to be detected by using a melting curve. The method includes designing a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, performing PCR amplification in a PCR amplification system containing the first primer, the second primer, the detection probe, the sample to be detected, a nicking enzyme and a DNA polymerase, generating a reporter primer that is complementary to the detection probe to form a double-stranded product, obtaining the melting curve of the double-stranded product, and the melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected. The method of the present invention is a non-target-dependent melting curve method, and the melting point (T m value) of each double-stranded body can be calculated in advance, solving the problems of melting curve peak shift and easy misjudgment caused by the easy mutation of the target nucleic acid sequence to be detected.

The present invention provides a method and a kit for detecting a target nucleic acid to be detected through a melting curve. The method includes designing a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, performing PCR amplification in a PCR amplification system containing the first primer, the second primer, the detection probe, the sample to be detected, a restriction endonuclease and a DNA polymerase, generating a reporter primer that is complementary to and pairs with the detection probe to form a double-stranded product, obtaining the melting curve of the double-stranded product, and the melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected. The method of the present invention is a non-target-dependent melting curve method, and the melting point (T m value) of each double-stranded body can be calculated in advance, solving the problems of melting curve peak shift and easy misjudgment caused by easy mutation of the target nucleic acid sequence to be detected.

Provided are a double-stranded oligonucleotide agent and uses. The double-stranded oligonucleotide agent comprises a specific cleavage site for an endonuclease, wherein the cleaved double-stranded oligonucleotide agent is capable of inhibiting expression of the target gene via RNA interference (RNAi).

The specification provides programmable base editors that are capable of introducing a nucleotide change and / or which could alter or modify the nucleotide sequence at a target site in mitochondrial DNA (mtDNA) with high specificity and efficiency. Moreover, the disclosure provides fusion proteins and compositions comprising a programmable DNA binding protein (e.g., a mitoTALE, a mitoZFP, or a CRISPR / Casp) and double-stranded DNA deaminase that is capable of being delivered to the mitochondria and carrying out precise installation of nucleotide changes in the mtDNA. The fusion proteins and compositions are not limited for use with mtDNA, but also may be used for base editing of any double-stranded target DNA.

The invention belongs to the field of biological medicines, and particularly relates to a modified double-stranded oligonucleotide, and a conjugate and a composition thereof. The double-stranded oligonucleotide provided by the invention effectively enhances the activity of the double-stranded oligonucleotide for inhibiting the expression of the target gene by performing specific modification on the nucleotide at a specific site, can efficiently inhibit the expression of the target gene in both in-vitro and in-vivo experiments, and surprisingly, the double-stranded oligonucleotide provided by the invention has a good application prospect. The modification on the double-stranded oligonucleotide can be generally used for siRNA designed for various different target genes, and the inhibition rate of the siRNA on the target genes can be effectively improved.

Systems and methods for sequencing polynucleotides using nanopores are disclosed. In some embodiments, a polynucleotide including a single-stranded region and a double-stranded region, in which the single-stranded region is disposed through a nanopore. The polynucleotide can be moved relative to the nanopore by electric forces while one or more structural locks keep the polynucleotide close to the nanopore. A characteristic signal based on nanopore ionic current blockade and associated with the regions of the polynucleotide at or near the nanopore recognition zone is measured and used to infer the nucleobase sequence of the polynucleotide. In some examples, the double-stranded region is extended by a polymerase, and the polymerase is removed from the polynucleotide. In some examples, signals measured under different applied voltages provide nonredundant information regarding the polynucleotide sequence.

The present invention belongs to the technical field of bioengineering. Provided are an IscB mutein and the use thereof. Compared to the wild-type IscB protein, the IscB mutein comprises arginine at at least one of the following amino acid positions: position 84, position 96, position 102, position 111, position 159, position 368 and position 386; preferably, the IscB mutein further comprises arginine at position 401 and position 456. The relatively strong interaction between the IscB mutein and a double-stranded DNA target improves the editing activity for mammalian cell lines, which can greatly enhance the application potential of the IscB mutein as a new-generation underlying tool for gene editing, and promote the use of the IscB mutein in the aspects of precision medicine, animal model construction and crop breeding.

The invention discloses a modification template for improving the target gene inhibition effect of double-stranded oligonucleotide and a combination and application of the modification template, the double-stranded oligonucleotide is siRNA and contains a positive-sense strand and an antisense strand, and each nucleotide of the positive-sense strand and the antisense strand is a modified nucleotide. The double-stranded oligonucleotide is modified by a special modification template, and compared with a basic sequence, the inhibition rate of the double-stranded oligonucleotide to a target gene is remarkably improved.

Relating to the technical field of gene therapy, provided is an siRNA molecule for inhibiting PCSK9 expression, and use thereof. Particularly, an siRNA molecule for inhibiting PCSK9 expression (comprising a sense strand and an anti-sense strand that are complementary to form a double-stranded region) and a pharmaceutical composition comprising the same are disclosed. The siRNA molecule has a high silencing efficiency and a long-lasting silencing effect for PCSK9 gene, and is useful in the manufacture of products for inhibiting PCSK9 protein expression, products for reducing the concentration of low-density lipoprotein or low-density lipoprotein cholesterol in serum; and products for relieving the symptoms of PCSK9 gene-mediated diseases.

Provided are an INHBE gene-targeted double-stranded oligonucleotide, a conjugate, and a composition, which relate to the technical field of nucleic acid drugs. The double-stranded oligonucleotide comprises a sense strand and an antisense strand. The antisense strand is complementary or substantially complementary to the sense strand; the sense strand comprises a nucleotide sequence identical or substantially identical to at least 15 consecutive nucleotides in a sequence of SEQ ID NO: 309, and said substantially identical means that there is no more than three nucleotide differences between the sense strand and the at least 15 consecutive nucleotides in the sequence of SEQ ID NO: 309. The double-stranded oligonucleotide can effectively inhibit the INHBE gene and treat patients suffering from metabolic diseases or metabolic syndrome and related diseases such as diabetes, hypertension and cardiovascular diseases.

The present disclosure is directed to controlling pest infestation by inhibiting one or more biological functions in an invertebrate pest. The disclosure discloses methods and compositions for use in controlling pest infestation by feeding one or more different recombinant double stranded RNA molecules to the pest in order to achieve a reduction in pest infestation through suppression of gene expression. The disclosure also discloses methods and compositions for targeted genome editing in the pest in order to achieve a reduction in pest infestation through disruption of protein activity. The disclosure is also directed to methods for making transgenic plants that express the double stranded RNA molecules and targeted genome editing constructs for use in protecting plants from pest infestation.

A nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a nucleotide sequence comprising a cardiovascular disease associated gene to be silenced and wherein said cardiovascular gene is proprotein convertase subtilisin kexin 9 (PCSK9) there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ end of said sense strand; and ii) a second nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a different cardiovascular disease associated gene to be silenced wherein said cardiovascular gene is lipoprotein (a) (Lp(a)) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ end of said sense strand wherein the single stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single stranded deoxyribonucleic acid (DNA) molecule set forth in i) above and anneals by complementary base pairing to form a double stranded DNA linker that links the first and second double stranded inhibitory ribonucleic acid (RNA) molecules.

The present disclosure provides, in part, double stranded compounds, including oligonucleotides such as siRNA, that comprise one or more independent occurrences of a modified nucleotide components of Formula (I), Formula (II), Formula (III), or Formula (IV) as described herein, as well as their methods of use in inhibiting gene expression and the treatment of various disorders.

The present disclosure relates to RNAi agents, e.g., double stranded RNAi agents such as siRNAs, able to Inhibin Subunit Beta E (INHBE) gene expression. Also disclosed are pharmaceutical compositions that include INHBE RNAi agents and methods of use thereof. The INHBE RNAi agents disclosed herein may be conjugated to targeting ligands to facilitate the delivery to cells, including to hepatocytes. Delivery of the INHBE RNAi agents in vivo provides for inhibition of INHBE gene expression. The RNAi agents can be used in methods of treatment of diseases, disorders, or symptoms mediated in part by INHBE gene expression, such as obesity, diabetes, liver inflammation, dyslipidemia, or metabolic disease.

The present invention relates to CRISPR / Cas based biosensing materials, assays, and methods. Specifically, the present technology relates to CRISPR / Cas-based ultra-sensitive detection methods for nucleic acid assays using specific molecular constructs, including constructs referred to as nucleic acid mediators, comprising single-stranded and double-stranded nucleic acid sequences of a cyclic conformation, and palindromic oligonucleotides. The materials and methods according to the invention can also be used to enhance the sensitivity of existing bioassays.

The invention provides a tumor exosome (TDEs) fluorescence detection method based on tandem hybrid chain reaction (HCR) and selective coordination, and aims to combine an aptamer, a tandem enzyme-free amplification technology and selective coordination of Cu < 2 + > to analyze a clinical lung cancer blood sample. The programmed cell death ligand 1 (PD-L1) biomarker overexpressed on the surface of the TDEs is selected as a target for detecting the exosome. The HCR can easily generate a large amount of double-stranded DNA, so that the HCR can be used as a means for signal amplification. The high affinity of the aptamer to the protein will affect the tandem HCR reaction and then modulate the HCR product. Cu < 2 + > and a double-stranded DNA product are utilized to form a compound, and a fluorescence signal of calcein can be accurately adjusted, so that accurate fluorescence quantification is performed on the concentration of TDEs.

The invention discloses a method for detecting the concentration of cyclosporine in blood through fluorescence and electrochemical double signals, and belongs to the technical field of biochemical analysis and medical detection. Aiming at the technical problems of insufficient sensitivity and high false positive rate of traditional single-signal detection of blood concentration of cyclosporine, the method comprises the following steps: activating carboxyl microspheres by a coupling agent, fixing double-stranded DNA (deoxyribonucleic acid) formed by a cyclosporine aptamer and an initiator chain, inducing the initiator to release by using a cyclosporine sample, realizing strand displacement through an A2-PEI composite system, and detecting the blood concentration of cyclosporine by using an A < 2 >-PEI composite system. DNA modified cadmium telluride quantum dots are respectively adopted to detect fluorescence signals, eATRP electrochemical signal amplification is carried out to detect electrochemical signals, concentration values obtained by the two signals are verified, and an average value is taken as a result. The method can accurately capture low-concentration cyclosporine signals in blood, eliminates interference of a single method, is mainly used for accurately monitoring the blood concentration of cyclosporine, and provides a reliable detection means for medication safety and curative effect control of cyclosporine in clinical scenes such as organ transplantation.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The disclosure provides, for example, a double stranded DNA (dsDNA) molecule comprising chemically modified nucleotides. In some embodiments, the dsDNA molecule comprises an upstream DNA end form which is a closed end, a downstream DNA end form which is a closed end, and a double stranded region comprising a sense strand and an antisense strand, in which the sense strand comprises one or more chemically modified nucleobases, and the antisense strand is free of chemically modified nucleobases.

The present invention relates to methods and compositions for editing a polynucleotide, e.g, a polynucleotide comprising a SNP associated with a disease or disorder.

The present disclosure provides using a double-stranded oligonucleotide compound as a novel therapy to improve the patient reported outcomes and quality of life of patients with hemophilia A or hemophilia B.

The present disclosure relates to isolated nucleic acid molecules comprising at least two double-stranded inhibitory ribonucleic acid (RNA) molecules adapted to silence the same or different genes by RNA interference to enhance silence, thereby modulating gene expression.

Aspects of the disclosure relate to methods for treating disorders such as cutaneous squamous cell carcinoma (cSCC) in a subject having a tumor by intratumoral administration of chemically-modified double stranded nucleic acid molecules directed against a gene encoding PD- 1.

A nanostructure comprising: a) a base plate sheet comprising a single-duplex layer comprised of DNA origami configured in a planar arrangement, b) a pore configured within the center of the base plate sheet, and c) a multi-duplex layer-thick wall comprising a single-duplex layer comprised of DNA origami that extends downward from the base plate sheet on the periphery of the pore, wherein the base plate sheet comprises a single-stranded DNA region on the bottom surface for lipid membrane anchor modification.

The invention belongs to the technical field of microorganisms, and relates to a riemerella anatipestifer phage and application thereof. The riemerella anatipestifer bacteriophage provided by the invention is RA-XHX1, is preserved in the China Center for Type Culture Collection (CCTCC), has the preservation number of CCTCC NO: M 20241268, and is classified and named as the riemerella anatipestifer bacteriophage. The bacteriophage is double-stranded DNA (Deoxyribose Nucleic Acid) and myuroviridae, can form transparent plaques on a solid culture medium, has no halo ring around and is clear and regular in edge; the bacteriophage has a relatively good killing effect on riemerella anatipestifer in a space environment, particularly high-pathogenicity riemerella anatipestifer, and can realize a broad-spectrum antibacterial effect when being applied to preparation of medicines for preventing and treating riemerella anatipestifer infection.

A double-stranded oligonucleotide and a use thereof in inhibiting the expression of angiotensinogen (AGT). The double-stranded oligonucleotide comprises a sense strand and an antisense strand; the antisense strand contains a nucleotide sequence comprising at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides of any sequence in SEQ ID NO: 225-SEQ ID NO: 448, or contains a nucleotide sequence which differs from the contiguous nucleotides by 1, 2, or 3 nucleotide(s).