46 results about "Argonaute" patented technology

The present disclosure relates in some aspects to methods for analyzing target nucleic acids and their spatial locations in a biological sample. In some aspects, the presence / absence, amount, and / or identity of 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 situ in the sample. Also provided are compositions and kits for use in accordance with the methods.

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.

The invention discloses an application of a gene GmAGO5 of a soybean ARGONAUTE family. The gene GmAGO5 of the soybean ARGONAUTE family is shown The nucleotide sequence of the soybean GmAGO5 protein coding gene GmAGO5 is as shown in SEQ ID NO. 1. The invention also discloses a preparation method of the soybean GmAGO5 protein coding gene The constructed plant gene editing knockout vector pSCM-GmAGO5 is transferred into an acceptor material Tianlong No.1 by using a cotyledonary node transformation method. The GmAGO5 gene is knocked out, so that the plant height of the gene edited soybean is reduced, the branch included angle is reduced, and the protein content of the soybean seeds is remarkably increased. Therefore, the soybean GmAGO5 protein coding gene GmAGO5 disclosed by the invention can be applied to reducing soybean plant height, reducing branch included angle and improving the protein content of soybean seeds through a genetic engineering means.

The invention provides characterization and application of novel programmable endonuclease Argonaute (Ago), and particularly provides a detection system for directly detecting target nucleic acid molecules, the system comprises: (a) guide DNA (gDNA) and / or guide RNA (gRNA); (b) a programmable endonuclease Argonaute (Ago), wherein the programmable endonuclease Argonaute (Ago) is a programmable endonuclease Argonaute (Ago) which is used for guiding the shearing of the target DNA and / or the target RNA by taking the DNA and / or the RNA as a guide; (c) a reporter nucleic acid; wherein the target nucleic acid molecule is a target DNA and / or a target RNA, the invention also accidentally finds that the nuclease MaAgo provided by the invention can be used for independently detecting the target DNA or the target RNA and also can be used for simultaneously detecting the target DNA and the target RNA, and has a shearing characteristic different from that of the shearing DNA when the RNA is sheared, so that a new enzyme resource is expected to be provided for nucleic acid detection and in-vivo editing.

The present disclosure relates to methods of detecting and / or quantifying one or more target nucleic acid molecules in a sample, comprising: a) contacting the sample with a reaction mix comprising: i) reagents for amplifying the target nucleic acid molecules; ii) an Argonaute (Ago) enzyme, one or more guide single stranded uncleic acid molecules, and one or more reporter nucleic acid molecules; and iii) a pyrophosphatase (PPase) for regenerating Mg2+ in the reaction mix; b) partitioning the mixture of the sample and the reaction mix into a plurality of compartments; c) amplifying the one or more target uncleic acid molecules in each compartment to obtain amplified target nucleic acid molecules; and d) detecting a signal in each compartment based on cleavage of the reporter nucleic acid molecules by the Ago enzyme in the presence of the amplified target nucleic acid molecules and guide single stranded nucleic acid molecules.

Nucleotide sequences of interest, which may be as yet unknown sequences, comprised in a biological sample may often be present in diminishingly small amounts, meaning there are difficulties in detecting, sequencing and identifying these sequences. A method of enrichment of the sequences of interest prior to sequencing of a sample overcomes the problem. In such a method a library of nucleic acid guides is generated from a portion of the sample itself, and the guides are used with a guide-dependent endonuclease such as an Argonaute, usually a prokaryotic Argonaute (pAgo) in a reaction which cleaves the nucleic acids recognised by the guides in another portion of the same sample, but which spares the low abundance sequences for which no guides have been generated. In this way a sample enriched for rarer or low abundance sequences is provided and used in subsequent steps of detection of sequences present, including sequencing of the enriched sample. The method has wide range of application in scientific research of all kinds and forensics where is it necessary to detect the presence of nucleotide sequences and where needed to allow for the detection and / or sequencing of these.

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.

The invention relates to the technical field of molecular biology, in particular to Argonaute protein derived from normal-temperature prokaryotes and gene editing application of the Argonaute protein. The amino acid sequence of the Argonaute protein is as shown in SEQ ID No.1, or the Argonaute protein has more than 90%, more than 95%, more than 98%, preferably more than 99% of identity with the sequence as shown in SEQ ID No.1, has the same function and comes from normal-temperature prokaryotes. The prokaryote Ago protein and PLD nuclease AgaP coupled with the prokaryote Ago protein are used for endowing a host with a virus defense function for the first time, and a strain with antiviral capacity can be constructed; furthermore, on the basis of the function of the Ago protein, the NpAgo can be used for remarkably improving the gene editing efficiency in escherichia coli, and the practical application of bacterial gene editing is effectively improved.

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.

The present invention discloses a mesophilic endonuclease mutant protein, its preparation method, and application. Through extensive and in-depth research and extensive screening, the present invention unexpectedly obtained, for the first time, a mutant protein with significantly improved KmAgo enzyme activity. The mutant protein is based on the mesophilic nuclease Argonaute (KmAgo, wild-type sequence shown in SEQ ID NO. 1) from Kurthia massiliensis, or its active fragments, variants, and derivatives. Compared with the wild-type, the mutant protein of the present invention has significantly improved enzymatic activity by up to 7 times; it also maintains excellent thermal stability; and has potential applications in nucleic acid detection technology and the development of gene editing tools.

An efficient synthesis and assembly method for the large fragment DNA based on the programmable nuclease Argonaute, specifically includes: constructing and treating antibiotic resistance gene reconstructed vectors with linearization, dividing a target DNA into multiple small DNA fragments and then synthesizing the small DNA fragments, followed by loading the synthesized small DNA fragments to the antibiotic resistance gene reconstructed vectors; and the SLIC and resistance gene reconstruction are used to achieve assembly of the target DNA. The method combines the SLIC with a resistance gene reconstruction strategy, allowing for the assembly of 5-6 small fragments in a single resistance gene reconstruction, which is more efficient and time-saving. Moreover, the number of the resistance gene reconstructions can be flexibly chosen according to the length of the DNA fragments. Mutations are not introduced caused by PCR, and the reconstructed large fragment do not need a second sequencing, saving time and costs.

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.

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.

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 discloses application of Argonaute protein derived from archaea or a coding gene of the Argonaute protein in RNA (Ribonucleic Acid) interference. The Argonaute protein is named as HrAgo1, and the amino acid sequence of the Argonaute protein is as shown in SEQ ID No. 1. The invention discloses a novel Argonaute protein derived from archaea, the novel Argonaute protein is named as HrAgo1, the novel Argonaute protein is different from a known prokaryotic organism Argonaute protein, and the HrAgo1 has two characteristics. Firstly, HrAgo1 can use short-chain RNA as a guide chain to shear mRNA paired with the sequence of HrAgo1. Therefore, the gene interference method based on the HrAgol is obviously different from other applications based on the prokaryote Argonaute protein. And secondly, the HrAgo1 can play the function at 9-71.1 DEG C, and can be suitable for cells growing at different temperatures.

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.

An efficient large DNA fragment synthesis and assembly method based on a novel programmable nuclease Argonaute. The method specifically comprises: constructing a resistance gene reconstruction vector and using Argonaute to perform linearization processing on the resistance gene reconstruction vector, fragmenting a target DNA into a plurality of small DNA fragments and synthesizing the small DNA fragments, loading the small DNA fragments into the resistance gene reconstruction vector, and using SLIC cloning and resistance gene reconstruction to implement assembly of the target DNA. SLIC cloning and a resistance gene reconstruction strategy are integrated, and 5-6 small fragments can be assembled by reconstructing a resistance gene once, thereby achieving higher efficiency and shorter cycle; and the frequency of reconstructing the resistance gene can be flexibly selected on the basis of the length of fragments of a DNA, mutation caused by PCR is not introduced, and the reconstructed large fragment does not need to undergo secondary sequencing, thereby saving time and reducing costs, and providing new ideas for the synthesis and assembly technology of large DNA fragments.