58 results about "RNase H" patented technology

The present disclosure relates in some aspects to methods, systems, and kits for analyzing a biological sample comprising generating a rolling circle amplification product (RCP) using a target ribonucleic acid (RNA) as a primer. In some aspects, RNase H and a nucleic acid oligonucleotide are used to generate a free 3′ end of the target RNA to prime RCA.

The invention belongs to the technical field of molecular biology, and particularly discloses heat-resistant MMLV reverse transcriptase and application thereof. According to the heat-resistant MMLV reverse transcriptase provided by the invention, T246K mutation is introduced into a DNA polymerase active structural domain of a wild type MMLV reverse transcriptase, so that the heat stability and reverse transcription activity of the enzyme are remarkably improved. E286R, E302K and / or L435R mutations are further introduced into a thumb domain and a connecting structural domain, and the heat resistance of the enzyme can be improved by cooperating with T246K mutations. Meanwhile, D524G, E562Q and / or D583N mutations are introduced into an RNase H active structural domain, so that the RNase H activity of the enzyme can be reduced or removed. Compared with a commercial enzyme, the heat-resistant MMLV reverse transcriptase has the optimal reaction temperature higher than 50 DEG C, is suitable for high-temperature reverse transcription reaction, and has wide application prospects and higher economic value in the fields of molecular diagnosis and medical examination.

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.

A method of reducing the level of a transcription product in a cell comprising contacting with the cell a composition comprising a double-stranded nucleic acid complex comprising a first nucleic acid strand annealed to a second nucleic acid strand, wherein: (i) the first nucleic acid strand hybridizes to the transcription product and comprises (a) a region consisting of at least 4 consecutive nucleotides that are recognized by RNase H when the strand is hybridized to the transcription product, (b) one or more nucleotide analogs located on 5′ terminal side of the region, (c) one or more nucleotide analogs located on 3′ terminal side of the region and (d) a total number of nucleotides and nucleotide analogs ranging from 8 to 35 nucleotides and (ii) the second nucleic acid strand comprises (a) nucleotides and optionally nucleotide analogs and (b) at least 4 consecutive RNA nucleotides.

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.

The invention provides application and a method for repairing growth defects of neuronal dendrites by using ribonuclease RNase H. In particular, ribonuclease RNase H can be used as an R-loop degradation agent for treating and / or preventing neuron dendritic growth defects and related diseases thereof. A Rett syndrome cell model is utilized, and it is found that growth of dendrites can be restored by expressing ribonuclease RNase H of mammals. The method disclosed by the invention provides a very significant means for treating and preventing the Rett syndrome and other neuronal dendritic growth defect related diseases.

The invention relates to a method for carrying out capping rate analysis on mRNA (messenger ribonucleic acid) by using an unmarked RNase H probe and LC-MS (liquid chromatography-mass spectrometry), and belongs to the technical field of biology. A DNA-RNA probe is designed according to a to-be-detected RNA sequence, RNA molecules are cut by utilizing the specificity of RNase H, short segments are separated by virtue of specific adsorption of a silica gel column, and the molecular weight and relative proportion of a cut RNA product are detected by utilizing an LC-MS method, so that the capping efficiency is determined, quantitative detection of the RNA capping efficiency is realized, radioactive contamination can be avoided, and the detection accuracy is high. The operation is simple and the cost is low.

Provided are the use of a ribonuclease RNase H in repairing growth defects of neuron dendrites, and a method therefor. Specifically, the ribonuclease RNase H can be used as an R-loop degradation agent for treating and / or preventing growth defects of neuron dendrites and related diseases thereof. By means of utilizing a Rett syndrome cell model, it is found that expressing the mammalian ribonuclease RNase H can restore the growth of dendrites, thus providing means of great significance for treatment and prevention of the Rett syndrome and other diseases related to growth defects of neuron dendrites.

The invention relates to the technical field of biological medicine, in particular to a molecular marker of alveolar rhabdomyosarcoma and application of the molecular marker, the molecular marker is a circPAX3R-loop site, and a DNA-RNA hybridization region of the molecular marker is located on chr2: 2230 94913-2230 95853 of a human genome. According to the application disclosed by the invention, a large number of R-loop structures exist in an alveolar rhabdomyosarcoma cell through DRIP-seq, a plurality of circRNAs exist in a cell nucleus of the alveolar rhabdomyosarcoma through circRNA sequencing, the existence of circPAX3R-loop is proved through qPCR after a DRIP product is subjected to RNase R and RNase H treatment, and the result shows that circPAX3R-loop exists in the cell nucleus of the alveolar rhabdomyosarcoma in the cell nucleus of the alveolar rhabdomyosarcoma in the cell nucleus of the alveolar rhabdomyosarcoma. The important clinical application value is realized on developing a reagent or a kit for diagnosing the alveolar rhabdomyosarcoma, a medicine for preventing and / or treating the alveolar rhabdomyosarcoma or a gene treatment strategy aiming at the target spot.

The invention provides an application and a method for repairing neuronal dendritic growth defects by using ribonuclease RNase H. In particular, ribonuclease RNase H can be used as an R-loop degradation agent for treating and / or preventing neuron dendritic growth defects and related diseases thereof. By using a Rett syndrome cell model, it is found that the growth of dendrites can be restored by expressing ribonuclease RNase H of mammals. And a very significant means is provided for the treatment and prevention of Rett syndrome and other neuronal dendritic growth defect related diseases.

The present disclosure relates in some aspects to methods, systems, and kits for analyzing a biological sample comprising generating a rolling circle amplification product (RCP) using a target ribonucleic acid (RNA) as a primer. In some aspects, RNase H and a nucleic acid oligonucleotide are used to generate a free 3′ end of the target RNA to prime RCA.

The invention belongs to the technical field of biological medicines, and particularly relates to embedded miRNA-carried tetrahedral framework nucleic acid as well as a preparation method and application thereof. The miRNA is embedded into a tetrahedral framework nucleic acid (tFNA), ribonucleotide extends from the tail end of each miRNA chain to serve as a biological switch, the enzyme-responsive biological switch can be triggered by RNase H in a cell to realize opening of a three-dimensional structure, meanwhile, a foothold-mediated strand displacement reaction domain is arranged on the miRNA, combination of the miRNA and a target in the cell is facilitated, and the targeting effect of the miRNA is improved. The specific recognition on the cells is realized. Therefore, the invention provides a miRNA delivery platform with high carrying efficiency, high stability and good biological compatibility, and the miRNA delivery platform can effectively treat acute liver injury and acute kidney injury and has a good application prospect.

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 invention relates to a protein for guiding editing and application thereof, the protein is formed by fusion of a first protein and a second protein, the amino acid sequence of the first protein is one of SEQ ID No. 6, SEQ ID No. 28, SEQ ID No. 30 and SEQ ID No. 32, and the amino acid sequence of the second protein is one of SEQ ID No. 6, SEQ ID No. 28, SEQ ID No. 30 and SEQ ID No. 32. And the amino acid sequence of the second protein is as shown in SEQ ID No. 26. After the fragment with reverse transcriptase activity and the fragment with RNase H are fused, the editing efficiency of guiding editing of the fragment with the length larger than or equal to 40 bp is obviously improved. Furthermore, site-directed mutagenesis is carried out on the reverse transcriptase active fragment, so that the editing efficiency of guiding editing of long fragments greater than or equal to 40bp is obviously improved again.

The invention discloses an oligonucleotide pool for specifically digesting a virus transcript in pitaya and application of the oligonucleotide pool. Belongs to the technical field of biology. At present, a technology capable of specifically eliminating RNA of a specific species in mixed RNA of multiple species does not exist. According to the invention, transcripts of virus types in pitaya are assembled and analyzed, and a set of independent oligonucleotide pool with universality in pitaya is designed. By applying the oligonucleotide pool, RNase H and DNase I to pitaya mixed RNA, high-purity pitaya RNA can be obtained for next RNA sequencing, and the invention of the technology has important significance on molecular biology research of pitaya.

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.

Provided herein are methods and compositions for a simplified and cost effective method for removing unwanted nucleic acids from a sample using RNase H.

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a (CRISPR / CRISPR / Cas12a)-based ultra-sensitive detection method for miRNA (micro Ribonucleic Acid) single nucleotide polymorphism. A fluorescence sensor comprises an A chain, a B chain, SplintR ligase, RNase H, Cas12a protease / crRNA and FQ. According to the sensor and the sensing method thereof disclosed by the invention, accurate quantification of miRNA with mutated single base can be realized, the distinguishing rate of low-abundance mutation reaches 0.005%, and the sensor and the sensing method are a simple, rapid and ultra-sensitive miRNA-SNPs detection technology.

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 invention discloses a fluorescent cross-linked RNase H mutant conjugate, a preparation method and use thereof, a kit containing the same, and a method for RNA detection using the RNase H mutant conjugate. The RNase H mutant conjugate, such as RNase Hv-(L x ‑I‑F) n RNase Hv is an RNase H mutant that can bind to RNA or RNA-DNA hybrids but cannot cleave RNA; see the detailed definition herein. The fluorescent cross-linked RNase H mutant conjugate of the present invention can directly recognize DNA / RNA hybrids and convert them into a detectable signal without the need for PCR amplification, making it suitable for sensitive and rapid RNA detection.

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.