28 results about "Argonaute" patented technology

The invention discloses an application of a soybean ARGONAUTE family gene GmAGO5 in soybean resistance to soybean mosaic virus diseases. The invention also discloses application of the soybean ARGONAUTE family gene GmAGO5 as shown in SEQ ID NO.1 and SEQ ID NO.2 in genetic engineering modification of soybean mosaic virus resistance. The soybean ARGONAUTE family gene GmAGO5 can negatively regulate and control the resistance of soybeans to diseased soybean mosaic virus diseases, and the antiviral response of the soybeans can be activated due to function deficiency of the soybean ARGONAUTE family gene GmAGO5. The gene is knocked out through a CRISPR / Cas9 gene editing technology, so that the resistance of soybeans to the soybean mosaic virus disease can be remarkably improved.

The present disclosure relates in some aspects to methods for analyzing target nucleic acids and their spatial locations in a biological sample using Argonaute proteins. In some aspects, a barcode probe library comprising a plurality of probes each comprising a plurality of barcode subunits that identifies a target analyte is detected in situ in the sample. Also provided are compositions and kits for use in accordance with the methods.

Disclosed herein are methods and compositions for normalizing polynucleotide concentration. Normalizing may be accomplished using polynucleotide binding proteins (e.g., catalytically inactive CRISPR protein or catalytically inactive Argonaute proteins). The polynucleotide binding proteins may bind to the adapter sequences of a target polynucleotide. Thus, adding the same amount of a polynucleotide binding proteins to different samples and then extracting the polynucleotide protein is shown herein to extract similar amounts of target polynucleotides from the different samples.

The application discloses a circRNA detection method based on Argonaute protein and rolling circle amplification. The method comprises the following steps: mixing a sample containing target circRNA to be detected with a specific reverse transcription primer and reverse transcriptase to obtain long-chain cDNA; mixing the obtained long-chain cDNA with pfAgo, gDNA and a DNA nucleic acid probe to carry out a reaction, and calculating the content of the target circRNA according to the fluorescence signal of the reaction mixture; the nucleotide sequence of the gDNA is specifically complementary to the cDNA sequence obtained by reverse transcription of the circRNA; the sequence of the DNA nucleic acid probe is specifically complementary to the sequence of the cDNA after one cutting of the cDNA by the pfAgo; and the DNA nucleic acid probe is a single-stranded DNA probe with a fluorescent label. The application can realize high-sensitivity, high-specificity and multiple target simultaneous analysis and detection of potential diagnostic biomarkers of liver cancer such as hsa_circ_0001445 and hsa_circ_0001141.

The application discloses an application of a crenarchaeota-derived Argonaute protein or a coding gene thereof in RNA interference. The Argonaute protein is named as HrAgo1, and the amino acid sequence is shown as SEQ ID No. 1. The application finds a new crenarchaeota-derived Argonaute protein, which is named as HrAgo1. Different from known prokaryote Argonaute proteins, the HrAgo1 has two characteristics. First, the HrAgo1 can use short-chain RNA as a guide chain to cut mRNA paired with the sequence. Therefore, a gene interference method based on the HrAgo1 is obviously different from other applications based on prokaryote Argonaute proteins. Second, the HrAgo1 can exert the function at 9-71.1 DEG C, and can be applied to cells growing at different temperatures.

Guide RNA can be engineered and used with an Argonaute (AGO) molecule. An AGO molecule can interact within a target nucleic acid, and this interaction can be used in many biotherapeutic and diagnostic methods. For example, gene expression of a target nucleic acid can be regulated using an AGO molecule.

A method for enriching or amplifying a target nucleic acid including providing a system having a guide nucleic acid, and a Cas or Argonaute protein or a variant thereof. The guide nucleic acid contains a target-specific nucleotide region substantially complementary to a region of the target nucleic acid, and contacting the target nucleic acid with the system to form a complex.

The application discloses application of a soybean ARGONAUTE family gene GmAGO5. A soybean GmAGO5 protein coding gene GmAGO5 has a nucleotide sequence as shown in SEQ ID NO. 1. A plant gene editing knock-out vector pSCM-GmAGO5 is constructed and transformed into a receptor material Tianlong No. 1 by using a cotyledon node transformation method. By knocking out the GmAGO5 gene, the gene edited soybean plant height is reduced, the branch angle is reduced, and the soybean seed protein content is significantly improved. It can be seen that the soybean GmAGO5 protein coding gene GmAGO5 can be applied to reducing the soybean plant height, reducing the branch angle and improving the soybean seed protein content by means of genetic engineering.

The present disclosure relates to methods for analyzing target nucleic acids in a biological sample using Argonaute proteins and single cell RNA-templated ligation probe chemistry. Variant sequences (e.g., single nucleotide variations such as SNPs or point mutations) in a plurality of target nucleic acids in a cell or tissue sample are analyzed in the sample. The application is drawn to assay methods, and further drawn to compositions and kits for use in accordance with the methods. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.

The invention provides a method for constructing a multi-copy eukaryotic expression vector based on a programmable enzyme Argonaute and application of the multi-copy eukaryotic expression vector based on the programmable enzyme Argonaute, and belongs to the technical field of biological medicines.The method comprises the following operation steps that DNA assembly is conducted on recombinant plasmids In-pc-CDs based on Argonaute and resistance gene reconstruction, and expression units in the plasmids obtained through assembly are transferred to plasmids Lamp; in R-Arm, the multi-copy expression vector is obtained. A set of in-vitro seamless DNA assembly technology which is low in mutation rate, convenient to operate, easy to screen, short in assembly period, wide in base sequence universality and low in both economic cost and time cost is developed by starting from a novel programmable enzyme Argonaute and combining a resistance reconstruction strategy, and the in-vitro seamless DNA assembly technology is used as an eukaryotic expression platform DNA assembly tool to realize the in-vitro seamless DNA assembly of the eukaryotic expression platform. The invention provides a method for rapidly constructing a multi-copy expression vector and a signal peptide expression plasmid library, so that the construction of the CHO stable transfection cell strain is realized.

Disclosed herein are methods and compositions for normalizing polynucleotide concentration. Normalizing may be accomplished using polynucleotide binding proteins (e.g., catalytically inactive CRISPR protein or catalytically inactive Argonaute proteins). The polynucleotide binding proteins may bind to the adapter sequences of a target polynucleotide. Thus, adding the same amount of a polynucleotide binding proteins to different samples and then extracting the polynucleotide protein is shown herein to extract similar amounts of target polynucleotides from the different samples.

The invention discloses a novel nucleic acid detection method based on Argonaute protein and application of the novel nucleic acid detection method, and a reaction system comprises the Argonaute protein; a nucleic acid sample to be detected; a single-stranded DNA signal molecule; the nucleic acid sequence of the single-stranded DNA signal molecule is complementary to the sequence of the nucleic acid sample to be detected. The novel method provided by the invention has the advantages of simple system, convenience in construction and low cost, has superiority compared with the existing Argonaute protein-based nucleic acid detection method depending on the guide nucleic acid, and is expected to be widely applied.

The invention relates to ApsA and ApsB proteins, including ApsA-like and ApsB-like proteins, and to their involvement in the mobilisation of the carbapenemase blaOXA-48 gene from the pOXA-48 plasmid into the chromosome. The ApsA and ApsB proteins combine a nuclease / helicase protein and a novel type of Argonaute-like protein to cleave DNA. The invention encompasses compositions comprising ApsA and ApsB proteins, including ApsA-like and ApsB- like proteins, antibodies against these proteins, nucleic acids encoding these proteins, vectors expressing these proteins and cells and kits comprising these nucleic acids, vectors and / or proteins. The invention further encompasses methods for making and using these compositions for diagnostic assays and for genetic engineering.

The present invention relates to the technical field of biochemical analysis. Disclosed are a guide DNA for enhancing the cleavage activity of an Argonaute protein and a use thereof in target detection. In the present invention, a metal-dependent DNAzyme fragment is combined with gDNA, to construct gDNAzyme which can significantly improve the enzymatic cleavage efficiency of CbAgo, show an obvious synergistic effect and maintain the targeting specificity of CbAgo. An aptamer sensor medicated by CbAgo further activated by gDNAzyme achieves DNA-extraction-and-amplification-free, highly sensitive and simultaneous detection of three pathogenic bacteria, and has sensitivity and a detection limit that are significantly better than those of other methods. In addition, detection results of the sensor for clinical samples are consistent with those of a gold-standard microbial culture method, indicating that the gDNAzyme designed in the present invention has broad-spectrum applicability and practicability.

The application provides an Argonaute-mediated chain displacement exponential amplification method and application. First, TtAgo enzyme is specifically recognized and cut under the guidance of gDNA, the cutting site is located in the complementary fragment formed by gDNA and a target gene or a DNA fragment, a pair of amplification primers are extended to form a double strand under the action of a polymerase, the double strand is continuously recognized and cut by the protein due to the existence of the cutting site, and the single strand replaced can be used as a substrate of cycle two; the sequence replaced in the extension process of cycle one can be combined with the amplification primer in a complementary manner and is extended under the action of the polymerase, the product extended also has a cutting site, and a single strand formed under the action of TtAgo enzyme can continue to be used as a substrate of cycle two to participate in the cycle. The application realizes efficient amplification, significantly shortens the time required for the amplification reaction, and gradually detects in the amplification process, thereby realizing significant distinction of single-base differences on DNA.

Disclosed herein is a method and composition for normalizing polynucleotide concentration.Normalization can be achieved by using polynucleotide binding protein (for example, catalytically inactive CRISPR protein or catalytically inactive Argonaute protein).Polynucleotide binding protein can bind to the adapter sequence of target polynucleotide.Therefore, it is shown herein that by adding the same amount of polynucleotide binding protein to different samples, and then extracting polynucleotide protein, the same amount of target polynucleotide can be extracted from different samples.

Constructs comprising Argonautes and neighboring genes are disclosed for use in gene editing. Disclosed are also compositions and methods utilizing these Argonautes and neighboring genes. Also disclosed are the methods of making and using the Argonautes and neighboring genes in treating various diseases, conditions, and cancer.

This invention discloses guide DNA that enhances the cleavage activity of Argonaute protein and its application in target detection, belonging to the field of biochemical analysis technology. This invention constructs gDNA by combining a metal-dependent DNA zyme fragment with gDNA. zyme It can significantly improve the cleavage efficiency of CbAgo, while exhibiting a significant synergistic effect and maintaining the targeting specificity of CbAgo. Further utilization of gDNA... zyme The activated CbAgo-mediated aptamer sensor enables highly sensitive simultaneous detection of three pathogenic bacteria without DNA extraction and amplification. Its sensitivity and detection limit are significantly superior to other methods. Furthermore, the sensor's detection results for clinical samples are consistent with the gold standard microbial culture method, demonstrating the effectiveness of the gDNA design of this invention. zyme It has broad applicability and practicality.

The invention relates to the technical field of molecular biology and biological detection, in particular to a concentration dependent type dual-mode nucleic acid cleavage system and a concentration dependent type dual-mode nucleic acid cleavage method for mesophilic Clostridium perfringens Argonaute protein. The system is based on the cleavage behavior difference of CpAgo protein under different target single-stranded DNA (ssDNA) concentrations. When the concentration of the target ssDNA is micromole or above, the CpAgo shows non-specific ssDNA cleavage activity independent of a guide chain, and has wide compatibility to the length, sequence and various chemical modifications of a substrate; when the concentration of the target ssDNA is nanomole or below, the cleavage behavior of the target ssDNA is converted into a specific mode which strictly depends on a complementary guide chain. The dual-mode mechanism of the CpAgo and the efficient non-specific cutting capacity of the CpAgo at normal temperature are verified through electrophoresis, fluorescence kinetic analysis and comparison experiments with other prokaryotic Ago proteins. The system can be used for rapidly degrading exogenous nucleic acid or realizing high-specificity nucleic acid detection, and has the advantages of single-enzyme dual-mode, concentration gating, strong substrate compatibility and the like.

Methods, kits and compositions, in some embodiments, may include a thermostable DNA guided Argonaute protein for example TtAgo, a thermostable single-stranded DNA binding protein (SSB) for example, extreme thermostable single-stranded DNA binding protein (ET SSB), and, optionally, a strand-displacing polymerase. A SSB may allow (a) Argonaute / guide DNA complexes to substantially enhance cleavage efficiency of single- and double-stranded DNA substrates; (b) the use of longer guide DNAs (e.g., guide DNAs that are at least 24 nucleotides in length) and / or (c) increases in the sequence specificity of Argonaute-mediated binding and cleavage reactions.

Disclosed is a room-temperature Argonaute protein-based multiplex nucleic acid amplification-free digital encoding-decoding detection method. The present invention pertains to the field of nucleic acid molecule detection. The method comprises extracting DNA from a target analyte to be tested, accurately cleaving a target by means of dual-guide DNA mediated by a room-temperature Argonaute enzyme, performing rapid site amplification on the surface of magnetic nanoparticles on the basis of a tyramide system, encoding the magnetic nanoparticles with a biotin-modified fluorophore, and applying an artificial intelligence-based digital readout algorithm.

The invention provides an exponential rolling circle amplification method based on Argonaute protein. The exponential rolling circle amplification method comprises the following steps: providing a 5'end phosphorylated single-chain gDNA; the method comprises the following steps: carrying out hybridization annealing on target RNA (Ribonucleic Acid) and a Padlock probe to form an RNA-Padlock compound; under the action of ligase, cyclizing the RNA-Padlock compound to form a circular DNA (Deoxyribose Nucleic Acid) template; the gDNA and TtAgo are subjected to pre-incubation, and a TtAgo / gDNA compound is formed; the method comprises the following steps: mixing a circular DNA template, a TtAgo / gDNA compound, DNA polymerase, dNTPs and a fluorescent dye in a same reaction system, and carrying out an exponential rolling circle amplification reaction. According to the exponential rolling circle amplification method based on the Argonaute protein, accurate shearing can be achieved at any preset position of an RCA product, a specific enzyme cutting site does not need to be introduced, system construction is greatly simplified, and exponential amplification is achieved.

The invention discloses a method for detecting food-borne pathogenic bacteria based on Argonaute protein in combination with loop-mediated isothermal amplification and electrochemical biosensing, and belongs to the technical field of microbiological detection and biosensing. The integrated detection method based on Argonaute protein, loop-mediated isothermal amplification and electrochemical biosensing, which is high in sensitivity, strong in specificity, simple and convenient to operate and suitable for on-site rapid detection, is provided aiming at the problems that an existing food-borne pathogenic bacterium detection method is insufficient in sensitivity, depends on complex instruments, is difficult to apply on site and the like. Target nucleic acid is subjected to exponential amplification through LAMP (loop-mediated isothermal amplification), then Argonaute-mediated two-stage specific recognition and signal transduction are performed, and finally reading is performed through a high-sensitivity electrochemical platform, so that the detection limit of salmonella typhimurium as high as 1 CFU / mL is realized, and the detection range can cover 100-108 CFU / mL.

Argonaute proteins from prokaryotes (pAgos) and applications thereof are provided, the pAgo is from a mesophilic prokaryote of Kurthia massiliensis and named as KmAgo, the pAgos and applications thereof are capable of site-specific modification of intracellular and extracellular genetic material, and thus can be effectively applied to many fields of biotechnology, such as nucleic acid detection, gene editing and gene modification, and provide a new tool for gene editing, modification and molecular detection of the pAgo polypeptides.

The present invention relates to a method for generating ssDNA guides from dsDNA using an Argonaute protein. Said guides may be useful in applications, like selectively reducing repetitive sequences in sequencing libraries using Argonaute proteins. The invention further relates to a method for cleaving a nucleic acid molecule, a method for reducing the number of nucleic acid molecules comprising a target sequence and a method for selectively enriching and / or sequencing nucleic acid molecules in a sample, like a DNA sequencing library.

The invention belongs to the technical field of microbiological detection, and discloses an Argonaute-based biosensor for helicobacter pylori detection, a method and application, the biosensor comprises a TtAgo-LAMP one-pot reaction system, and every 25 [mu] L of the reaction system comprises TtAgo, guide DNAmix, Buffer I, dNTPs mix, a 10 * primer mix-vacA, BSA with a mass concentration of 2%, Bst DNA polymerase, a helicobacter pylori genome to be detected, a fluorescent probe report and ddH2O. The invention designs a biosensor which is based on an Argonaute system and is combined with an LAMP (Loop-Mediated Isothermal Amplification) assisted one-pot method detection sensing platform, so that signal transduction and amplification are realized. The method is used for detecting effective targets in helicobacter pylori and is used for rapidly and sensitively detecting and applying the helicobacter pylori.

The present invention belongs to the field of synthetic biology and biotechnology. Disclosed are a high-temperature prokaryotic Argonaute nuclease having both DNA and RNA cleavage activity and the use thereof. The thermophilic-bacterium-derived Argonaute nuclease is specifically A1) or A2) below: A1) a protein having an amino acid sequence as shown in SEQ ID NO: 1; and A2) a fusion protein obtained by means of linking a tag to the N-terminus or / and C-terminus of A1). The enzyme can mediate the cleavage of a single-stranded nucleic acid target by using both 5'-phosphorylated gDNA and 5'-hydroxylated gDNA; is resistant to high temperatures of 50-85°C, and has high specificity; and is conducive to advancing the development of tools for DNA and RNA manipulation in vivo and in vitro.