34 results about "RNase H" patented technology

The invention provides a macro virus group sequencing library construction method for removing host ribosome RNA through an enzyme method and a kit, and belongs to the technical field of biology. The method comprises the following steps: hybridizing total RNA (Ribonucleic Acid) of a sample with a DNA (Deoxyribose Nucleic Acid) probe aiming at host ribosome RNA to form a DNA / RNA hybrid chain; rNase H is used for digesting RNA in the hybrid chain, and DNase I is used for digesting the remaining DNA probe in the system; purifying to obtain virus RNA (Ribonucleic Acid); carrying out fragmentation treatment on the virus RNA; carrying out reverse transcription on the fragmented RNA to synthesize a first chain cDNA, then synthesizing a second chain cDNA, and carrying out terminal repair and dA tail addition to obtain double-chain cDNA; connecting the linker with the dT tail with the double-chain cDNA; and carrying out PCR amplification and purification on the connection product to obtain the macro virus group sequencing library. According to the construction method and the kit, the problem that the full-length sequence of the virus cannot be obtained from a low-load sample can be solved.

The invention provides a method for eliminating ribosomal RNA in a mouse total RNA sample, which comprises the following steps: carrying out reverse transcription on the total RNA in the mouse sample into cDNA, and amplifying the cDNA by adopting a primer sequence group to obtain a DNA fragment covering all sequences of the mouse ribosomal RNA; carrying out amplification by taking the DNA fragment as a template and adopting a single primer in the primer sequence group to obtain a single-stranded DNA probe; hybridizing the total RNA of the mouse sample by adopting a single-stranded DNA probe, and performing RNase H digestion to obtain a digestion product; sequentially carrying out DNase I digestion and RNA purification on the digestion product to obtain a target product; wherein the primer sequence group comprises upstream primers from the first to the nineteenth and downstream primers from the first to the nineteenth, and the primer sequence group has the characteristics of low cost, stable performance and high reduction efficiency.

The invention discloses antisense oligonucleotides for reducing XDH gene expression in a targeted manner and application of the antisense oligonucleotides, and belongs to the technical field of biological medicines. The antisense oligonucleotide mediates the degradation of the target mRNA through an RNase H (RNase H) dependent cleavage mechanism, and the mRNA expression of the XDH gene is horizontally knocked down after transcription; the antisense oligonucleotide molecule has a specific nucleic acid sequence, is combined with chemical modification design, can specifically bind XDH mRNA molecules in a targeting manner, promote XDH mRNA degradation and inhibit XDH protein synthesis, so that the expression level of XDH in cells is reduced, uric acid synthesis is reduced, the purpose of reducing uric acid is finally achieved, and cell experiments and animal model tests show that the antisense oligonucleotide molecule has a good application prospect. The antisense oligonucleotide molecule disclosed by the invention has a good uric acid reducing effect, and is long in drug effect duration and high in drug safety.

The present invention provides oligonucleotides referred to as circular prodrug nucleic acid (“CPN”) as described herein, compositions comprising same, and methods of using same. This design of circular prodrug nucleic acids maintains a circular form until the circularizing domain is cleaved in situ by RNase H or Dicer or other intracellular factors.

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes that comprise a ribonucleotide position that is susceptible to endoribonuclease (e.g., RNase H) cleavage in the presence of target nucleic acid molecules. Probes of the embodiments may also comprise non-natural nucleotide linked to a reporter and / or quenching moiety.

The purpose of the present invention is to provide an immunogenic composition containing a new HBV polypeptide or polynucleotides encoding the polypeptide. The immunogenic composition contains at least one polypeptide selected from among polypeptides a) through c) or polynucleotides encoding the polypeptide, and can be used for treating or preventing HBV infection. a) an HBV surface protein polypeptide including a specific amino acid substitution; b) an HBV polymerase polypeptide including an inactivated reverse transcriptase domain and an inactivated ribonuclease H domain, and including a specific amino acid substitution; and c) an HBV core protein polypeptide including a specific amino acid sequence.

The invention discloses a glyphosate fluorescence detection method based on RNase H cycle amplified magnetic bead-aptamer-Cas9 / deoxyribozyme, belongs to the technical field of environmental monitoring and biological detection, and can realize high-sensitivity and rapid quantitative detection of glyphosate residues in soil, water bodies and agricultural products. According to the method, the glyphosate is combined with an aptamer to release complementary DNA (cDNA) combined with the aptamer, then signal cascade amplification is realized through RNase H-mediated cDNA cyclic utilization, the anti-interference advantage brought by magnetic bead separation is combined, and the known characteristics of Cas9 endonuclease and DNAzyme in the aspects of nucleic acid recognition and cutting are used as signal amplification and transduction means, so that the detection sensitivity of the glyphosate is improved, and the detection sensitivity of the glyphosate is improved. Therefore, trace detection of glyphosate is realized. According to the invention, sensitive detection of other targets can be realized by replacing nucleic acid aptamers capable of specifically recognizing other targets and correspondingly adjusting the sequence of cDNA to maintain base pairing.

The invention provides sets of RNA depletion probes, short DNA oligos that hybridize along the length of a target RNA and mediate digestion of the target RNA by RNase H to remove super-abundant RNA molecules from a sample. Depletion probes according to the invention are designed foremost based on biochemistry and the biophysical properties of the probes so that all of the depletion probes of a set exhibit substantially uniform, consistent behavior in binding to a target RNA in a sample. Probes are principally designed to specific performance targets and biophysical properties, yielding probe sets with irregular, even apparently random, spacing along a target RNA molecule.

The present invention describes that a pre-mRNA molecule, which temporarily exists before polyadenylation-site induced cleavage occurs, can be targeted by RNase H recruiting antisense oligonucleotides downstream of a transcripts 3' terminus to reduce expression levels of target genes from which the RNA polymerization originates.

The present invention relates to the technical field of biomedicine, and provides an antisense oligonucleotide targeting IL-17A mRNA, a use thereof and a pharmaceutical composition comprising same. The nucleotide sequence of the antisense oligonucleotide targeting IL-17A mRNA provided in the present invention is as shown in SEQ ID NO. 1, 3, 5-18, 20-23, 26-28 or 31. The antisense oligonucleotide can specifically bind to IL-17A mRNA, and upon binding, recruits RNase H, thereby achieving the degradation of IL-17A mRNA, and preventing the synthesis of IL-17A protein; the antisense oligonucleotide can be used in the preparation of drugs for treating IL-17A-related diseases.

Methods as well as related compositions and kits for recombinase-mediated nucleic acid amplification, such as recombinase-polymerase amplification (RPA), of a nucleic acid template using at least one blocked primer that contains a 5′ domain, at least one nucleotide that is cleavable by an RNase H enzyme, a 3′ domain, wherein the primer is not extendable by a polymerase, and wherein the 3′ domain has a length of 7-100 nucleotides, for example 10-30 nucleotides. These methods and the use of a blocked primer reduce or eliminate non-specific amplification products, such as primer dimers, which are generated in RPA reactions.

Provided are systems / kits and methods for the preparation of cDNA from a sample of total RNA, in which excess RNA species such as rRNA and tRNA are depleted from the cDNA products via prevention of reverse transcription through competitive binding of DNA oligonucleotide probes that prevent binding and / or extension of randomer reverse primers. This process allows downstream high-throughput sequencing on a library prepared from the cDNA to efficiently profile RNA expression without significant loss of accuracy due to excess RNA species occupying too many reads. Compared to other approaches for excess RNA depletion, such as those based on RNAse H systems, the systems and methods provided herein neither require additional enzymes beyond reverse transcriptase, nor extra purification before reverse transcription.

The invention discloses an SERS (Surface Enhanced Raman Scattering) sensor for detecting ribonuclease H activity. The SERS sensor comprises a gold electrode, a single-chain RNA (Ribonucleic Acid), an initiation probe tDNA (Deoxyribose Nucleic Acid), a hairpin chain H1, a capture probe S1, Exo III and an SERS tag, the SERS tag is prepared by assembling a signal probe S2 and a signal molecule 4-MBA on the surface of a gold nano cube together. According to the SERS sensor, a target object RNase H is converted into a strong Raman signal through the assistance of Exo III and the good SERS performance of a gold nano cube; s0 with an N3 functional group and a signal probe S2 with a DBCO functional group on the SERS tag can be subjected to a click chemical reaction, and the SERS tag is assembled on the surface of a gold electrode, so that an SERS signal is detected on the surface of a substrate. The SERS sensor can efficiently and selectively detect the activity of RNase H, and the detection limit is 3.89 * 10 <-4 > U / mL (S / N = 3) and is lower than the reported value of most literatures.

The present application relates to the field of genetic engineering. Specifically, the present application relates to a PERV reverse transcriptase-based prime editing system and a method for gene editing by the system. More specifically, the present application provides: 1. A prime editing system using PERV-NC fusion RNase H structure deletion and carrying multiple mutation sites of PERV-RT; 2. A pegRNA fused with a truncated Csy4 (tCsy4-pegRNA); 3. One or more small molecule compounds for treating cells and combinations thereof; to improve the efficiency of prime editing in mammals.

An antisense oligonucleotide against IFN-γ mRNA, a use thereof and a pharmaceutical composition thereof, relating to the technical field of biomedicine. The nucleotide sequence of the antisense oligonucleotide against IFN-γ mRNA is as shown in SEQ ID Nos: 1-12, 15, 17, 19, 24 or 25. The antisense oligonucleotide can specifically bind to IFN-γ mRNA and recruit RNase H upon binding, thereby achieving degradation of IFN-γ mRNA and preventing synthesis of IFN-γ protein, and the antisense oligonucleotide can be used for preparing medicaments for treating IFN-γ related diseases.

The invention provides a method for eliminating ribosomal RNA in a bovine total RNA sample, which comprises the following steps: reversely transcribing the total RNA in the bovine sample into cDNA, and amplifying the cDNA by using a primer sequence group to obtain a DNA fragment covering all sequences of the bovine ribosomal RNA; carrying out amplification by taking the DNA fragment as a template and adopting a single primer in the primer sequence group to obtain a single-stranded DNA probe; hybridizing the total RNA of the cattle sample by adopting a single-stranded DNA probe, and performing RNase H digestion to obtain a digestion product; sequentially carrying out DNase I digestion and RNA purification on the digestion product to obtain a target product; wherein the primer sequence group comprises first to seventeenth upstream primers and first to seventeenth downstream primers, and the primer sequence group has the characteristics of low cost, stable performance and high reduction efficiency.

The application belongs to the technical field of biological detection, and relates to a miRNAs quantitative detection kit based on probe connection and a detection method. The detection kit comprises a first probe, a second probe, a primer F, a primer R, a reaction enzyme and magnetic beads. The reaction enzyme comprises a Splint-R ligase and an RNase H enzyme. The miRNA to be detected is miRNA159, miRNA122 or miRNA24. According to the target to be detected, the application designs a responding probe sequence, and has the advantages of fast detection speed, short measurement time, high connection efficiency, high sensitivity and strong specificity.

The invention discloses an anti-degradation antisense nucleic acid based on a D-type DNA structure and a preparation method and application thereof, and belongs to the technical field of genetic engineering. Compared with the traditional antisense nucleic acid, the anti-degradation antisense nucleic acid (D-ASO) based on the D-type DNA structure has the advantages that the design of coupling a single-chain string and a double-chain bow is adopted, the nuclease degradation resistance is remarkably improved, and the half-life period in serum is prolonged to 24 hours (only 2 hours in the traditional ASO). The target mRNA triggers the replacement of a protection chain (P) of D-ASO to form a DNA-RNA hybrid which is degraded by RNase H, and then the P chain is recombined to realize cyclic utilization (more than or equal to 5 times). According to the innovative structure, the binding rate of non-specific RNA is reduced by 90% or above. The invention solves the problems of stability and off-target toxicity of ASO while maintaining efficient gene silencing, and is suitable for treatment of tumors and genetic diseases.

The invention provides a method for eliminating ribosomal RNA in a human total RNA sample, which comprises the following steps: carrying out reverse transcription on the total RNA in a human blood sample into cDNA, and amplifying the cDNA by adopting a primer sequence group to obtain a DNA fragment covering all sequences of the human ribosomal RNA; carrying out amplification by taking the DNA fragment as a template and adopting a single primer in the primer sequence group to obtain a single-stranded DNA probe; hybridizing the total RNA of the human blood sample by adopting a single-stranded DNA probe, and performing RNase H digestion to obtain a digestion product; sequentially carrying out DNase I digestion and RNA purification on the digestion product to obtain a target product; wherein the primer sequence group comprises upstream primers from the first to the nineteenth and downstream primers from the first to the nineteenth, and the primer sequence group has the characteristics of low cost, stable performance and high reduction efficiency.

The invention provides a method for eliminating ribosome RNA (Ribosome Ribonucleic Acid) in a pig total RNA (Ribosome Ribonucleic Acid) sample, which comprises the following steps: reversely transcribing the total RNA in the pig sample into cDNA (Complementary Deoxyribonucleic Acid), and amplifying the cDNA by adopting a primer sequence group to obtain a DNA fragment covering all sequences of the pig ribosome RNA; carrying out amplification by taking the DNA fragment as a template and adopting a single primer in the primer sequence group to obtain a single-stranded DNA probe; hybridizing the total RNA of the pig sample by adopting a single-stranded DNA probe, and performing RNase H digestion to obtain a digestion product; sequentially carrying out DNase I digestion and RNA purification on the digestion product to obtain a target product; wherein the primer sequence group comprises first to fifteenth upstream primers and first to fifteenth downstream primers, and the primer sequence group has the characteristics of low cost, stable performance and high reduction efficiency.

To provide designed oligonucleotides, compositions and methods thereof.SOLUTION: In one embodiment, provided oligonucleotide compositions improve single stranded RNA interference and / or RNaseH-mediated knockdown. In particular, it encompasses the recognition that structural elements of an oligonucleotide can significantly affect the properties and activities (e.g., RNA interference (RNAi) activity, stability, delivery, etc.) of the oligonucleotide, such as base sequence, chemical modifications (e.g., sugar modifications, base modifications, and / or internucleotide linkage modifications) or patterns thereof, conjugation with additional chemical moieties, and / or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotide linkages)) and / or patterns thereof. The oligonucleotide compositions can also be used, for example, to effect RNA interference and / or RNaseH-mediated knockdown.SELECTED DRAWING: Figure 1A

The present invention relates to a bioswitchable, embedded miRNA-loaded tetrahedral framework nucleic acid, and a preparation method therefor and the use thereof. In the present invention, miRNA is embedded into a tetrahedral framework nucleic acid (tFNA), and a ribonucleotide is extended at the end of each miRNA strand as a biological switch. Such enzyme-responsive biological switches can be triggered by intracellular RNase H to realize the disassembly of a three-dimensional structure. Moreover, the miRNA comprises a toehold-mediated strand displacement domain, which facilitates the binding of miRNA to an intracellular target, thereby achieving specific cell recognition. The miRNA delivery platform provided by the present invention can effectively treat tumors such as uveal melanoma, and diseases such as acute liver injuries and acute kidney injuries.

The application discloses a preparation method of an mRNA molecule containing a threose nucleic acid (TNA) modified poly(A) tail, and belongs to the field of mRNA modification, and comprises the following steps: preparing mRNA by using a T7 RNA polymerase in vitro transcription; using TNA polymerase 10-92 to enzymatically synthesize RNA-TNA chimeric single strands by taking RNA with a monophosphate group at the 5' end as a primer; using cell lysate to test the anti-degradation of the chimeric single strands to prove the superior biological stability of the chimeric single strands; using T4 RNA ligase 1 to connect the chimeric single strands to the 3' end of the mRNA; and verifying the connection efficiency by RNase H enzyme cutting analysis. The application provides a new strategy for optimizing the molecular design of mRNA drugs by improving the stability of mRNA molecules, and helps to expand the application prospect of TNA in the field of nucleic acid drugs.

This invention discloses a method and kit for isothermal cascade amplification melting curve analysis to detect RNA. The method includes designing a first primer, a second primer, a circular amplification probe, and a detection probe based on the sequence of the circular amplification probe. Isothermal amplification is performed in an amplification system containing the first primer, the second primer, the circular amplification probe, the detection probe, the sample, a nicking enzyme, RNA-dependent DNA polymerase, ribonuclease H, RNA polymerase, and strand displacement polymerase. This invention ingeniously combines an isothermal detection system, RNA polymerase-mediated transcription amplification technology, and a melting curve analysis system, avoiding the heating delay caused by the large heat capacity of the metal module in traditional thermal cyclers, thus significantly reducing the required time and enabling rapid detection.

Among other things, the present disclosure provides designed APOC3 oligonucleotides, compositions, and methods thereof. In some embodiments, provided oligonucleotide compositions provide improved single-stranded RNA interference and / or RNase H-mediated knockdown. Among other things, the present disclosure encompasses the recognition that structural elements of oligonucleotides, such as base sequence, chemical modifications (e.g., modifications of sugar, base, and / or internucleotidic linkages) or patterns thereof, conjugation with additional chemical moieties, and / or stereochemistry [e.g., stereochemistry of backbone chiral centers (chiral internucleotidic linkages)], and / or patterns thereof, can have significant impact on oligonucleotide properties and activities, e.g., RNA interference (RNAi) activity, stability, delivery, etc. In some embodiments, the present disclosure provides methods for treatment of diseases using provided oligonucleotide compositions, for example, in RNA interference and / or RNase H-mediated knockdown.

The present invention provides oligonucleotides referred to as cyclic prodrug nucleic acids ("CPNs") as described herein, compositions comprising the same, and methods of using the same. This design of cyclic prodrug nucleic acids maintains its cyclic form until the cyclic domain is cleaved in situ by RNase H or Dicer or other intracellular factors.

The application provides a preparation method and application of an anti-L1CAM antibody drug conjugate, the anti-L1CAM antibody drug conjugate comprising an anti-L1CAM single-chain antibody, a DNA / RNA hybrid chain molecule and a cytotoxin DM1; the anti-L1CAM single-chain antibody is covalently connected to one end of the DNA / RNA hybrid chain molecule; the cytotoxin DM1 is covalently connected to the other end of the DNA / RNA hybrid chain molecule; and the DNA / RNA hybrid chain molecule comprises an RNase H sensitive segment. The technical scheme of the application specifically couples the anti-L1CAM single-chain antibody and the DM1 through the DNA / RNA hybrid chain, so that the obtained antibody drug conjugate integrates targeting, intelligent release and high-efficiency killing, has the effect of inhibiting retinoblastoma proliferation, and can effectively inhibit retinoblastoma proliferation and metastasis.

The application discloses an oligonucleotide pool for specifically digesting viral transcripts in pitaya and application thereof, and belongs to the technical field of biotechnology.At present, there is no technology capable of specifically eliminating RNA of a specific species in mixed RNA of multiple species.The application assembles and analyzes viral transcripts in pitaya, and designs an independent and universal oligonucleotide pool in pitaya.By applying the oligonucleotide pool, RNase H and DNase I to mixed RNA in pitaya, high-purity pitaya RNA can be obtained for next-step RNA sequencing, and the application of the technology is of great significance to molecular biology research of pitaya.

The present disclosure provides, inter alia, designed oligonucleotides, compositions, and methods thereof. In some embodiments, the provided oligonucleotide compositions provide improved single stranded RNA interference and / or RNase H mediated knock-down. The present disclosure encompasses, among other things, the recognition of structural elements of oligonucleotides, such as base sequences, chemical modifications (e.g., modifications of sugars, bases and / or inter-nucleotide linkages), or patterns thereof, conjugation to additional chemical moieties and / or stereochemistry [e.g., stereochemistry of the backbone chiral center (chiral inter-nucleotide linkages)], and / or patterns thereof, the oligonucleotide has significant influence on properties and activity of the oligonucleotide, such as RNA interference (RNAi) activity, stability, delivery and the like. In some embodiments, the disclosure provides methods of treating disease using the provided oligonucleotide compositions (e.g., in RNA interference and / or RNase H mediated knock-down).

Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes that comprise a ribonucleotide position that is susceptible to endoribonuclease (e.g., RNase H) cleavage in the presence of target nucleic acid molecules. Probes of the embodiments may also comprise non-natural nucleotide linked to a reporter and / or quenching moiety.