39 results about "Circular DNA" patented technology

The application discloses an ecDNA recognition method based on scATAC-seq data. The ecDNA recognition method provided by the application can accurately detect copy number amplification, breakpoint quantity and ecDNA feature quantity on the whole cell level. The method provided by the application can recognize specific circular oncogenes carried in tumors, analyze the distribution characteristics of the circular oncogenes in the genome, and reveal the overall composition of all circular DNAs in a single cell and the specific composition of a single circular DNA. In addition, the method can also verify the existence of the circular DNA through a staining experiment, so that the reliability of the detection result is ensured.

This invention relates to a method for preparing a functional DNA gel and its applications. Addressing the problems of existing methods for immobilizing functional nucleic acids requiring DNA modification, which increases immobilization costs, and the reduction of functionality due to chemical fixation, this invention mixes functional circular DNA with a gel monomer solution, and adds an initiator and a coagulant to initiate a cross-linking polymerization reaction. During the reaction, the gel monomers pass through the functional circular DNA, polymerizing into long chains and then cross-linking into a network structure. This allows the functional circular DNA to be enriched on the network structure in a suspended, fixed form, resulting in a functional DNA gel. The functional circular DNA is circular DNA containing functional nucleic acid sequences. This invention immobilizes functional circular DNA on the gel network structure without chemical modification, and the preparation method is simple. It can be used for the efficient and specific removal of pollutants such as heavy metal ions, toxins, veterinary drugs, and pesticides from solutions, as well as the enrichment of small molecule nucleic acids in solutions.

This invention discloses a method and kit for detecting RNA using a three-stage amplification melting curve based on isothermal self-circulation. The method includes designing a first primer, a second primer, and a circular amplification probe targeting the target nucleic acid sequence. The first primer consists of an RNA polymerase promoter sequence and a target nucleic acid-specific binding sequence, sequentially from the 5' end to the 3' end. The circular amplification probe is a circular DNA segment whose sequence consists of three parts: the first part completely contains a continuous sequence composed of the tag sequence and the nicking enzyme-specific recognition sequence from the second primer; the second part is a self-cleaving DNase sequence. In the technical solution used in this invention, a cascaded nucleic acid amplification system is constructed, achieving exponential signal amplification of the target nucleic acid through the synergistic effect of rolling circle amplification and RNA polymerase-mediated transcriptional amplification.

Disclosed herein is a method of purifying circular DNA from a sample by exposure of the sample to a nanoclay that preferentially adsorbs multiple contaminants, including non-circular DNA, RNA, proteins, and endotoxins, while leaving circular DNA substantially unbound in solution. The elution-free method also includes a step of separating nanoclay-contaminant complexes from the enriched circular DNA fraction. Also disclosed herein are compositions and systems operable for purifying circular DNA from a sample. Also disclosed herein are methods of refining, formulating or modifying a nanoclay for use in purifying circular DNA from a sample.

This invention discloses a bisphosphonate-nucleic acid nanoparticle, its preparation method, and its applications. The preparation method involves adding divalent metal ions to a rolling circle amplification (RoBA) reaction solution, followed by the addition of bisphosphonate, to perform a RoBA reaction to obtain the bisphosphonate-nucleic acid nanoparticles. The RoBA reaction solution contains a circular DNA template. The preparation method of this invention has the advantages of high speed, precise synthesis, and simplicity, realizing multiple reactions—RoBA, ion coordination, crystallization, and co-assembly of DNA and bisphosphonate—in the same reaction system, greatly simplifying the reaction process and improving reaction efficiency. The obtained bisphosphonate-nucleic acid nanoparticles exhibit dual-pathway therapeutic effects and excellent ribozyme stability, showing promising application prospects.

The application discloses a kind of for sweat glucose concentration monitoring method, it is related to sweat monitoring field, comprising the following steps: step 1, enzyme-fixed chain conjugate synthesis: glucose dehydrogenase is reacted with bilirubin oxidase respectively with bifunctional crosslinking agent and is purified, obtain enzyme-crosslinking agent intermediate;It is clicked with azide modified DNA fixed chain chemistry reaction again, and after purification, obtain GDH-fixed chain and BOx-fixed chain conjugate;Step 2, preparation of circular DNA template: after annealing hybridization of phosphorylated template chain and primer chain, it is connected into ring by T4 DNA ligase;Step 3, rolling circle amplification self-assembly package enzyme DNA hydrogel;The present application uses super-flexible DNA hydrogel as flexible electrode material, realizes the efficient fixation and continuous stable catalysis of GDH and BOx, and constructs flexible sweat-based EBFC self-powered sensor as flexible bioelectrode, to realize continuous non-invasive blood glucose monitoring.

The present invention is in the field of biomedicine and drug delivery as well as pest and vector control. The present invention relates to a novel family of silicon-introduced ionizable cationic lipids belonging to the trademark LipexSill second generation lipids wherein the tail is linked to the head group via a biodegradable silicon-based acetal linker. Lipids containing a silicon-based acetal linker are the most advanced, and are effective as ionizable cationic lipids in formulating empty or loaded lipid nanoparticles (LNPs). The novel linker according to the present invention is [WO2022129966] designed using a proprietary borane catalyst. The present invention describes the synthesis of lipids of formula (I), the formation and characterization of nanoparticles, and biological experiments that prove that lipid nanoparticles prepared with these novel lipids can effectively deliver their cargo receptors (cargo) (e.g., RNA, DNA, mRNA, siRNA, dsRNA, pDNA, microRNA, circular DNA, small bioactive molecules) into cells.

Provided herein are improved DNA vectors (e.g., circular DNA vectors and / or non-viral DNA vectors) that can provide features including enhanced expression, persistence, safety, and manufacturability. Also provided herein are methods of producing such DNA vectors, host cells (e.g., engineered bacterial cells useful for producing such DNA vectors) containing such DNA vectors, methods of using such DNA vectors (e.g., engineered bacterial cells useful for producing such DNA vectors), and methods of using such DNA vectors. Methods of expressing therapeutic sequences in target cells by administering such DNA vectors and methods of treating diseases or conditions by administering such DNA vectors), as well as pharmaceutical compositions containing such DNA vectors.

A method for producing deoxyribonucleic acid (DNA) includes providing a circular DNA template. The method also includes conducting in vitro an amplification reaction on the circular DNA template to generate a DNA product that is rolling circle amplified, wherein a reaction fold-amplification is minimized to enhance protein expression from the DNA product, and wherein a rolling circle amplification reaction is conducted with at least one of (i) the rolling circle amplification reaction being conducted at 15 degrees Celsius to less than 25 degrees Celsius to minimize the reaction fold-amplification, (ii) the rolling circle amplification reaction being conducted with an input concentration of the circular DNA template being greater than 1 nanogram per milliliter to minimize the reaction-fold amplification, or (iii) the rolling circle amplification reaction being conducted with input concentrations of deoxyribonucleoside triphosphates being titrated to 1.2 millimolar or less per deoxynucleotide to minimize the reaction-fold amplification.

The invention relates to a method and a kit for generating nucleic acid for target capture. The method involves binding of a circ-olio sequence to at least one target oligonucleotide to generate a single stranded circular DNA. The single stranded circular DNA is bidirectionally amplified with the sense and antisense oligonucleotides, followed by biotinylation to obtain for a double-stranded concatemer of linear sequence. The double-stranded concatemer of linear sequence with the restriction endonucleases produces a target capture nucleic acid. The method offers a scalable targeted capture of pr eselected genomic regions, and is cost effective. The method of generating nucleic acid for target capture has application in the field of oncology hematology, inherited malignancies, germline disorders, microbiology and prenatal diagnosis. The invention generates baits over high GC-rich regions of the genome.

The present invention belongs to the field of biomedicine and drug delivery as well as pest and vector controls. The invention relates to a novel ionizable cationic lipid family incorporating silicon, which belongs to the trademark LipexSil® second generation lipids, wherein the tail is connected to the headgroup with biodegradable silyl acetal linker. Lipids containing silyl acetal linker(s) are state-of-the-art and are effective as ionizable cationic lipids in the formulation of empty or loaded lipid nanoparticles (LNPs). The novel linkers according to the invention are designed by means of proprietary borane catalysts [WO2022129966]. The invention describes the synthesis of the lipids of formula (I), formation and characterization of nanoparticles and biological experiments demonstrating that the lipid nanoparticles prepared with these novel lipids can efficiently deliver their cargo (e.g. RNA, DNA, mRNA, siRNA, dsRNA, pDNA, micro RNA, circular DNA, small biologically active molecules) into the cells.

The present invention relates to methods of generating one or more transgenic cells using circular single stranded DNA (CiSSD) as a donor template, as well as target genome modification. The methods include transferring one or more DNA polynucleotides into a cell for site-specific nuclease-mediated DNA repair, and selecting one or more cells into which the transferred DNA is introduced in the genome of the cell.

The present invention relates to a method for producing a desired linear combined DNA molecule, linear or circular DNA plasmid using at least one genetically altered E. coli strain, wherein linear DNA fragments are converted into a linear combined DNA molecule or circular DNA plasmid. The method can be performed in vivo or in vitro, in particular as a single step method. The invention further relates to a method for cloning a nucleic acid fragment of interest, comprising the method according to the present invention, specific genetically altered E. coli strains, a kit for performing the method according to the present invention, and the use of the genetically altered E. coli strains, or the kit for producing a linear combined DNA molecule or circular DNA plasmid or cloning a nucleic acid fragment of interest.

The invention discloses a single-stranded circular DNA Marker as well as a preparation method and application thereof, and relates to the technical field of biology. The DNA Marker prepared by the invention has the characteristics of uniform and sharp bands and obvious brightness among the bands during use, can be widely applied to molecular weight indication in a CssDNA electrophoresis process, can be stored at room temperature for a long time, and is convenient to use. The preparation method of the CssDNA Marker is high in stability, repeated production between batches can be guaranteed, and therefore large-scale production is achieved.

The application discloses a bovine viral diarrhea virus chimeric virus expressing porcine circovirus type 3 capsid protein and a preparation method thereof. In the experiment, a gene sequence for encoding a porcine circovirus type 3 capsid protein and a His tag fused with the C terminal of the protein is inserted into different sites of a bovine viral diarrhea virus genome through a circular polymerase extension reaction, and the chimeric virus is prepared through virus rescue of the obtained circular DNA. The His tag sequence in the gene sequence and the bovine viral diarrhea virus genome are connected through a 2A peptide sequence, so as to realize the target of effectively releasing the antigen through the co-expression of the heterologous protein. The chimeric virus prepared by the application retains the infectivity and replication capacity of the parent virus, can stably replicate in cells and efficiently express the target antigen, and provides an efficient and flexible virus carrier platform for the research and development of a porcine circovirus type 3 vaccine.

Current minicircle production methods are slow, expensive, and difficult to perform under GMP conditions because, in most cases, the product is derived from bacteria. In contrast, HTLA-and CHTLA-based synthetic circular supercoiled DNA production can be done completely in a test tube, using chemically or enzymatically synthesized oligonucleotides, long single stranded DNA, and / or double stranded DNA.

The invention relates to the technical field of phytopathogen molecule detection, and discloses a rolling circle replication-based high-sensitivity fusarium pseudograminearum detection method which comprises the following steps: extracting a to-be-detected genome of a sample; preparing closed-loop DNA (deoxyribonucleic acid); carrying out mixed reaction on the closed-loop DNA and exonuclease to obtain a purified circular DNA template; mixing the purified annular template, a rolling circle amplification primer, Phi29 DNA polymerase, dNTPs and a buffer solution, and carrying out rolling circle replication to obtain super-long single-stranded DNA; and mixing the rolling circle replication product, Cas12b protein, sgRNA, an ssDNA fluorescence report probe and a buffer solution, and carrying out trans-cleavage to release a fluorescence signal. According to the invention, the necessary in-vitro transcription step in the traditional isothermal amplification-CRISPR coupling technology is eliminated, the detection time is shortened, and meanwhile, the reagent cost and the operation error risk are reduced.

The application provides a biotin-labeled exogenous circular DNA and a construction method and application in protein interaction thereof, and belongs to the technical field of gene interaction. The application provides a construction method of the biotin-labeled exogenous circular DNA, wherein a target sequence is amplified by using a biotin-labeled primer to obtain linear DNA containing a preset restriction enzyme cutting site; the obtained linear DNA is subjected to single enzyme cutting treatment by using a corresponding restriction endonuclease; and the DNA after enzyme cutting is subjected to a self-ligation reaction to generate closed circular DNA. The preparation process of the biotin-labeled circular DNA is stable and controllable, and has high repeatability; the biotin labeling of the circular DNA can be combined with a streptavidin system to realize high-affinity and specific enrichment of a DNA-protein complex; and the circular DNA can be stably delivered in cells and maintain the structural integrity of the circular DNA, thereby providing an effective means for truly reflecting the interaction between the circular DNA and the protein in a physiological environment.

The invention discloses a chromosome outer circular DNA sequence identification method based on deep learning, and relates to the technical field of genome biological information, and the method comprises the following steps: obtaining a known chromosome outer circular DNA sequence as a positive sample; randomly selecting a genome segment as a negative sample, wherein the overlapping degree of the genome segment and any known chromosome outer circular DNA region is lower than 20% from the reference genome according to an equal length distribution and chromosome distribution principle; cleaning and standardizing the positive and negative samples, and dividing the positive and negative samples into a training set, a verification set and a test set; converting the positive and negative sample sequences into feature representation which can be processed by a neural network through a multi-modal coding strategy, and training a one-dimensional convolutional neural network architecture by using a training set; when the number of the training samples is insufficient, introducing a deep convolutional generative adversarial network to perform data enhancement; and processing a DNA sequence to be detected through a multi-modal coding strategy, inputting the processed DNA sequence to the trained model, outputting a probability score of existence of the circular DNA outside the chromosome, and completing classification judgment according to a preset threshold value.

The invention discloses an immunodetection probe, a kit and a detection method based on proximity ligation and nucleic acid amplification in the technical field of biological detection. The immunodetection probe comprises an antibody modified probe A, an antibody modified probe B and a splint probe, the antibody modification probe A comprises a first antibody and a first DNA chain, the antibody modification probe B comprises a second antibody and a second DNA chain, and the splint probe sequentially comprises a front end chain, a primer complementary chain and a rear end chain from the 5'end to the 3 'end; the first antibody and the second antibody can be specifically combined with different epitopes of the same target antigen to form a sandwich compound. By designing a double-hairpin closed probe with a synergistic effect, non-specific hybridization and connection are greatly inhibited from a molecular stress level by utilizing self-closing of a hairpin structure of the probe in an uncombined state and a specific open-loop mechanism under adjacent triggering, so that a background signal is remarkably reduced; the near-quantitative and high-efficiency formation of the circular DNA template is realized, and the uniformity and stability of signal generation are guaranteed.

The invention discloses double-stranded circular DNA and application thereof in biological prevention and control of cotton bollworm, and relates to the technical field of genetic engineering and agricultural biological prevention and control, the two circular DNA (recombinant plasmids: pBE-cDNA4-lhAChE and pBE-cDNA4-dsAChE) stably generate double-stranded RNA and long hairpin RNA in an insect body and activate RNA interference (RNAi) effect by targeting a key gene of the cotton bollworm, so that the target gene is silenced; and the compound can also have synergistic interaction with viruses (HaNPV), so that the growth and development of cotton bollworms are inhibited. The invention has the advantages of high stability, strong delivery efficiency, environmental friendliness and the like, and is suitable for biological control of cotton bollworm. The technology can reduce the use of chemical pesticides and promote the sustainable development of agriculture.

The application belongs to the technical field of biological medicine, and particularly relates to a cordycepin nano delivery body and a preparation method and application thereof. The method utilizes rolling circle amplification technology, and connects the cordycepin on the circular DNA through base complementary pairing to obtain the cordycepin nano delivery body. The prepared cordycepin nano carrier is a nanoparticle, and the particle size is 100-120 nm. The application utilizes the characteristics that the cordycepin can form unconventional base complementary pairing with DNA, and first and successfully adopts the biological macromolecule DNA as the cordycepin carrying carrier to obtain the cordycepin nano delivery body with high efficient carrying, controllable slow release and high biological safety, and the cordycepin nano delivery body has good targeting property to melanoma. The cordycepin nano delivery body has mild and safe preparation condition, and the preparation process is simple and easy to operate, and is beneficial to the industrialized production of the nano carrier and the wide clinical application.

The invention discloses a method for enzymatic cyclization of threose nucleic acid. The method comprises the following operation steps: connecting 5 '-terminal monophosphate of DNA with 2'-terminal hydroxyl of TNA in an intramolecular manner by using CircLigase to complete cyclization of a single chain; the generation of a circular DNA-TNA product is confirmed by exonuclease in combination with mass spectrometry; 5 '-terminal monophosphate and 2'-terminal hydroxyl sequences with 16 different base combinations are prepared, the universality of cyclized connection of TNA is evaluated, and the accurate requirements of the sequences for connection site bases are defined; the cyclization efficiency of the CircLigase on a TNA single chain containing a single DNA nucleotide fragment and a DNA oligonucleotide fragment is tested; the cyclic TNA containing single DNA nucleotide can effectively resist degradation of S1 endonuclease and maintain the cyclic structure of the S1 endonuclease. According to the invention, not only is the universality of the method to different sequences tested, but also the influence of endonuclease on the stability of the cyclized TNA is explored, so that the types of tool enzymes for operating the TNA are enriched, and the optimization of the molecular performance of the functional TNA and the expansion of the application field of the TNA are facilitated.

The present invention belongs to the field of biomedicine and drug delivery. The invention relates to LipexSil® lipid-containing nanoparticle ("LNP") compositions, that permit preferential targeting of spleen versus liver, where preferential targeting is defined as a luminescence intensity ratio of spleen and liver greater than 9 after administration of a luminescent cargo, when the different weights of spleen and liver are taken into account. In the LNP composition, a silicon-containing ionizable lipid is combined with a certain structural lipid, a helper lipid, a shield lipid and a fifth lipid component. The invention describes the production and characterization of the lipid nanoparticles and in vivo experiments demonstrating that the corresponding formulations with LipexSil® lipids as described in WO2024 / 023174 are superior to the current approach, delivering their cargo (e.g. RNA, DNA, mRNA, microRNA, siRNA, ceDNA, pDNA, circular DNA, small biologically active molecules) preferentially to the spleen.

This invention relates to novel ionizable amine lipids with one or more sulfur atoms introduced into their tails. These lipids can be used in combination with other components to form lipid nanoparticles with oligonucleotides. The invention describes the synthesis, nanoparticle formation, and characterization of lipids of formula (I), as well as biological experiments demonstrating that lipid nanoparticles prepared from these novel lipids can efficiently deliver their carriers (e.g., RNA, DNA, mRNA, siRNA, miRNA, pDNA, circular DNA, dsRNA, small bioactive molecules) into cells.

The application discloses a method for continuous editing of multi-site chromosomes in eukaryotic cells. The application provides a method for integrating the genome of eukaryotic cells, comprising: using a knock-in site-specific recombination site (such as noxM) in the genome of eukaryotic cells, realizing integration of a large fragment circular DNA molecule at a specific site of the genome under the action of a knock-in site-specific recombinase (such as Nigri), and simultaneously using an adjacent knock-out site-specific recombination site (such as loxP or a mutant thereof), rearranging the related sites under the action of a knock-out site-specific recombinase (such as Cre), so as to realize continuity of integration. The novel editing platform of high-efficiency site-specific recombination provided by the application can realize directional, continuous and multi-site integration of a large fragment DNA in eukaryotic cells, and the application has important application prospects in the field of biological technologies including plant breeding, animal cell line development, fungus engineering and the like.

The present invention addresses the problem of providing a novel method for removing an unnecessary functional sequence contained in unnecessary linear covalently-closed DNA that is generated as a by-product during the production of target linear covalently-closed DNA.　Provided is a double-stranded circular DNA vector which includes a pair of protelomerase recognition sequences, wherein a first region that constitutes one of two regions constituting the double-stranded circular DNA vector between the pair of protelomerase recognition sequences includes two or more endonuclease recognition sequences and a functional sequence disposed between the two or more endonuclease recognition sequences, and a second region that constitutes the other of the two regions includes a target nucleic acid sequence and does not include the endonuclease recognition sequence.

The invention discloses a nucleic acid composition, application thereof and a Parkinson's disease detection kit. The nucleic acid composition comprises: a single-stranded circular nucleic acid comprising a DNA sequence as shown in SEQ ID No.1; and a single-stranded oligonucleotide capable of complementarily hybridizing to the single-stranded cyclic nucleic acid and capable of specifically binding to a monomer or oligomer of alpha-synuclein. The nucleic acid composition can be applied to nanopore detection of the exclusion volume of the alpha-synuclein oligomer. The alpha S oligomers in different aggregation states in a sample can be specifically captured through a designed annular DNA frame structure, and due to the fact that unique via hole signals with sub-peaks are generated through capturing of the annular structure of the frame, quantitative measurement of the volume of the alpha S oligomers can be achieved through statistical analysis of the sub-peak signals. The invention can provide a new thought and strategy for early diagnosis of Parkinson's disease.

This invention discloses a PSA detection method based on a black phosphorus / bismuth vanadate composite material, comprising: S1, coating a microplate with a PSA monoclonal antibody, blocking it, adding the PSA sample to be tested and incubating it, then incubating it with a PSA polyclonal antibody, a thiolized oligonucleotide sequence 1, and a gold nanoparticle conjugate to form a sandwich immune complex; S2, adding a circular DNA template, DNA ligase, and other reagents, and incubating to complete RCA amplification; S3, adding a thiolized oligonucleotide sequence 2 and a conjugate with alkaline phosphatase, and adding a substrate for hydrolysis to generate ascorbic acid; S4, using a biosensor to perform photoelectrochemical testing under simulated sunlight and voltage, recording the oxidation current signal to quantify the PSA concentration. The PSA detection method disclosed in this invention, using a biosensor modified with this composite material and employing a signal amplification strategy, achieves highly sensitive and stable detection of PSA, which has significant clinical implications for the early diagnosis of prostate cancer.

The invention provides an amplification library building method for high-throughput sequencing verification of synthetic genes. The amplification library building method comprises the following steps: obtaining a joint connection product of which two ends are connected with double-chain joints after a to-be-detected synthetic gene sequence, and carrying out first PCR (Polymerase Chain Reaction) amplification by adopting a first gene specific primer by taking the joint connection product as a template to obtain a first amplification product; taking the first amplification product as a template, and carrying out second PCR amplification by adopting a second gene specific primer to obtain a second amplification product; performing thermal denaturation single-chain separation and single-chain cyclization on the second amplification product to obtain single-chain circular DNA; the preparation method comprises the following steps: carrying out DNB preparation on single-stranded circular DNA to obtain DNB nanospheres; and sequencing the DNB nanospheres to obtain synthetic gene sequence information. The method verifies that the synthesized gene is high in flux, and the flux requirement of large-scale gene synthesis can be met.