31 results about "Methyl cytosine" patented technology

The invention belongs to the technical field of biological detection, and particularly relates to a bisulfite-free detection kit for specific site 5-methylcytosine and a detection method thereof. According to the method, a TAPbeta treatment method is adopted, 5hmC is sealed through beta-GT, 5mC oxidation is carried out through TET, and 5mC is reduced into dihydrouracil (DHU) through pyridine borane, so that the apparent difference between 5mC and C and 5hmC is converted into single nucleotide polymorphism difference. In addition, a flow microsphere technology based on PNA-assisted click chemical connection starting is established to detect DNA methylation specific sites. A PNA clip is innovatively introduced into click chemical connection, and non-specific connection is inhibited. Target sequence enrichment, signal amplification and signal acquisition are carried out through surface-functionalized magnetic nanoparticles, and the detection sensitivity is improved to fM. The invention provides a new tool for high-specificity and high-sensitivity epigenetic marker detection.

The invention provides a method for the analysis of a biological sample to determine multiple types of information therefrom in a streamlined, combined workflow, where all information is obtained in a sequencing-based analysis. The information includes the presence and concentration of specific plasma proteins in a blood sample: the number, location, and types of histone modifications associated with cell-free DNA obtained from the same sample: the sequence of cfRNA and cfDNA in the cell-free DNA sample; and epigenetic information pertaining to the cell-free DNA, such as hydroxy methylation and methylation profiles, i.e., the distribution of 5-hydroxymethylcytosine (5hmC) and 5-methylcy tosine (5mC) residues, respectively. The invention additionally pertains to a classical sequencing-based method for analyzing a biological sample to determine one or more non-classical sequence features of the sample. Compositions, kits, and related methods are also provided, including an embodiment in which truncated sequencing adapters are used in conjunction with barcoded PCR primers.

The invention relates to the technical field of biology, in particular to 5-hydroxymethylcytosine and application of a prognosis model of 5-hydroxymethylcytosine in nasopharynx cancer prognosis evaluation. According to the method, the prognosis scoring model capable of layering the outcome of the patient is constructed, and the model shows strong distinguishing performance in a training set and a verification set. When it is integrated into a prognostic model together with tumor staging and EBV status, the 5hmC score results in higher calibration accuracy and net clinical benefit in decision curve analysis. According to the findings, a 5hmC prognosis model is determined as a noninvasive marker rich in biological information, can be used for prognosis risk layering of NPC patients, and has potential significance for individualized management and improvement of outcome prediction.

Methods, compositions, and systems are provided for detecting 5-methylcytosine modifications in nucleic acids, particularly DNA samples. One or more templates are sequenced using single molecule real-time sequencing. A feature vector is generated using the particular feature. In some aspects, feature vectors with reduced feature sets are provided, and these feature vectors are input into a deep learning model.

A method of determining percentages of twelve blood cell subtypes in a blood specimen, includes steps of: (a) obtaining genomic DNA from the blood specimen of a human subject, (b) observing unmethylated CG loci, in the genomic DNA of the human subject, wherein said observing includes performing a bisulfate conversion process on the genomic DNA of the human subject so that cytosine residues in the genomic DNA of the human subject are transformed to uracil, while 5-methylcytosine residues in the genomic DNA of the human subject are not transformed to uracil, (c) comparing the unmethylated CG loci observed in (b) to unmethylated CG loci observed in genomic DNA collected from a reference group of human individuals and (d) correlating the unmethylated CG loci observed in (b) with the unmethylated CG loci observed in the reference group of human individuals to determine the percentage of the twelve blood cell subtypes in the blood specimen.

Protein complexes including a cytidine deaminase and a helicase. In some embodiments, the cytidine deaminase is an altered cytidine deaminase. In some embodiments, the protein complex converts 5 methylcytosine to thymine. Kits, compositions, and methods of use for the protein complexes including a cytidine deaminase and a helicase are also described.

The present disclosure is concerned with modified proteins, methods, compositions, and kits for mapping of methylation status of nucleic acids, including 5-methylcytosine. In some embodiments, the proteins are altered cytidine deaminases, such as altered APOBEC3A proteins. In some embodiments, the proteins selectively act on cytosines and have significantly reduced activity on modified cytosines, such as 5-methylcytosine.

The invention provides a targeted ALKBH2 small-molecule inhibitor and application thereof, and belongs to the field of medicinal chemistry. The structure of the small-molecule inhibitor is shown as a formula I. The compound provided by the invention has good affinity activity on ALKBH2, can competitively inhibit recognition of the ALKBH2 on a substrate in a targeted manner, and specifically targets ALKBH2 instead of other AlkB family proteins. The compound provided by the invention can be used for effectively targeting ALKBH2 in cells and increasing the modification abundance of N3-methylcytosine (m3C) in the cells. The compound provided by the invention not only can effectively inhibit proliferation of brain glioma cells, but also can effectively inhibit migration of the brain glioma cells. The compound provided by the invention can be used for preparing ALKBH2 inhibitors and medicines for preventing or treating ALKBH2 expression abnormity related diseases (such as brain tumor, non-small cell lung cancer, rectal cancer, bladder cancer and gastric cancer), and has a wide application prospect.

The present invention provides oligonucleotides useful for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy, compositions containing oligonucleotides, and methods for prevention, treatment, and / or delay. [Solution] An oligonucleotide is provided that comprises a 2'-O-methylRNA monomer and a phosphorothioate skeleton, and also comprises 5-methyluracil and / or 5-methylcytosine and / or a 2,6-diaminopurine base.

The application discloses a data analysis method in the field of epigenetics: a quantitative analysis method of RNA 5-methylcytosine modification on splicing isomers. The method can solve the problem of how to quantitatively analyze RNA 5-methylcytosine modification on the level of gene splicing isomers, and the technical points include: converting the data set generated by the RNA-BSseq sequencing technology into a virtual RNA-Seq data set and marking the data in which RNA 5-methylcytosine modification occurs; estimating the expression amount of the splicing isomer in the virtual data set, and calculating the mean and variance thereof by using the bootstrap technology; estimating the RNA 5-methylcytosine modification level of the splicing isomer, and calculating the mean and variance of the RNA 5-methylcytosine modification level of the splicing isomer by using the Delta technology; and based on the normal distribution, testing whether there is a significant difference in the estimated RNA 5-methylcytosine modification level of the splicing isomer under different conditions. The application helps to reveal the regulation mechanism of RNA 5-methylcytosine modification in physiological processes and pathological changes.

The present disclosure relates generally to the enzymatic conversion of methylated nucleic acids in order to differentiate methylated cytosine from unmethylated cytosine in DNA, and more particularly to improved methods and compositions for enzymatic methylation sequencing. In one aspect, various compositions and methods for improving the recovery of methylation signals are provided. The methods include one or more of a nick repair step, methylation signal restoration, use of a modified methylcytosine nucleic acid adapter, and use of a helicase, an ssDNA binding protein, an engineered DNA ligase, or a combination thereof.

The present disclosure is concerned with modified proteins, methods, compositions, and kits for mapping of methylation status of nucleic acids, including 5-methylcytosine and 5-hydroxymethyl cytosine (5hmC). The proteins selectively act on certain modified cytosines of target nucleic acids and include one or more substitution mutations that enhance the selectivity of the proteins for certain modified cytosines, enhance the stability of the proteins, or enhance both selectivity and stability. Also provided are compositions and kits that include one or more of the proteins and methods for using one or more of the proteins.

The invention relates to the technical field of biochemical detection, in particular to a sample pretreatment method, a method for detecting the ratio of 5-methylcytosine to cytosine and a kit. The sample pretreatment method comprises the following steps: providing a sample containing substances to be detected, wherein the substances to be detected comprise 5-methylcytosine and cytosine; the method comprises the following steps: mixing magnetic beads containing sulfonic acid groups with a sample, carrying out adsorption treatment, and then carrying out leaching treatment by using an alcoholic solution to obtain a to-be-detected substance-magnetic bead compound; eluting the to-be-detected substance-magnetic bead compound by using the eluent to obtain a to-be-detected sample; the eluent comprises at least one of a formic acid-alcohol solution and an ammonia water-alcohol solution. The sample pretreatment method is simple in process and low in cost, the to-be-detected sample containing the 5-methylcytosine and the cytosine can be efficiently obtained, the ratio of the 5-methylcytosine to the cytosine can be accurately quantified in a high-sensitivity mode on the basis of the electrospray high-efficiency ion mobility spectrometry technology subsequently, and the method has good application prospects.

The application provides a nucleic acid aptamer-drug conjugate, a preparation method thereof and application thereof in preparing an anti-tumor drug, and belongs to the technical field of biological medicines. The nucleic acid aptamer-drug conjugate comprises a nucleotide sequence, an HG9 nucleic acid aptamer as shown in SEQ ID NO:1 and an extended chain at the end of the HG9 nucleic acid aptamer; the extended chain is loaded with 5-hydroxymethylcytosine and / or 5-formylcytosine. The application further provides a preparation method of the nucleic acid aptamer-drug conjugate. By means of PCR amplification technology, an epigenetic modification nucleoside with cytotoxicity is introduced into a nucleic acid aptamer with specific targeting capability, the nucleic acid aptamer-drug conjugate constructed has the binding capability to target cells, and can release the modified nucleoside in the cells, cause DNA damage and significantly inhibit the activity of the target cells. The method is simple in operation and high in efficiency, and the obtained conjugate has potential value in drug development.

Disclosed herein are compositions for preparing a target nucleic acid molecule comprising one or more 5-formylcytosine bases or adducts of 5-formylcytosine. Also disclosed herein are methods of efficiently synthesizing a nucleic acid molecule comprising one or more 5-formylcytosine bases from a target nucleic acid molecule comprising one or more 5-hydroxymethylcytosine bases. The disclosure also provides methods of detecting epigenetic modifications in a target nucleic acid molecule, such as those characterized by the methylation of cytosine at position 5 (e.g., 5-methylcytosine, 5-hydroxymethylcytosine).

The invention provides a nucleic acid aptamer-drug conjugate as well as a preparation method and application thereof in preparation of anti-tumor drugs, and belongs to the technical field of biological medicines. The nucleic acid aptamer-drug conjugate comprises an HG9 nucleic acid aptamer with a nucleotide sequence as shown in SEQ ID NO: 1 and an extension chain at the end part of the HG9 nucleic acid aptamer; 5-hydroxymethylcytosine and / or 5-formyl cytosine are / is loaded in the extension chain. The invention further provides a preparation method of the nucleic acid aptamer-drug conjugate, apparent modified nucleoside with cytotoxicity is introduced into the nucleic acid aptamer with specific targeting capacity through a PCR amplification technology, and the constructed nucleic acid aptamer-drug conjugate has the binding capacity to target cells, can release the modified nucleoside in the cells, and can be used for preparing the nucleic acid aptamer-drug conjugate. DNA damage is triggered; and the activity of target cells is remarkably inhibited. The method is simple and convenient to operate and high in efficiency, and the obtained conjugate has potential drug development value.

Methods for inhibiting respiratory viruses (i.e., viruses associated with respiratory conditions, e.g., influenza A, influenza B, RSV, and the like) are provided. Aspects of the methods include contacting a sample comprising viral RNA (vRNA) having a target motif with an effective amount of an agent that specifically binds to the target motif to inhibit the respiratory virus. Methods for treating or preventing respiratory virus infection in a subject are also provided. Pharmaceutical compounds and compositions comprising an oligonucleotide sequence complementary to a target region of the vRNA used in the methods are also provided. The oligonucleotides provided may include a 5-methylcytosine.

The present invention provides methylcytosine selective deaminases, compositions, kits, and methods of using the thereof, including sequencing methods involving: (a) contacting a DNA substrate comprising cytosine (C) and at least one methylcytosine nucleobase selected from 5-methylcytosine (5mC) and 5-hydroxymethylcytosine, or comprising C and both 5mC and 5hmC, with a methylcytosine selective deaminase to produce a deamination product, wherein the methylcytosine selective deaminase (i) is capable of deaminating 5mC to thymidine (T) and / or deaminating 5hmC to hydroxymethyluridine (hmU), and (ii) preferentially deaminating at least one methylcytosine nucleobase relative to cytosine (C); and (b) sequencing the deamination product, or amplifying the deamination product to produce an amplification product, and sequencing the amplification product to produce a sequence read in each case, wherein the position of C and the position of at least one methylcytosine nucleobase in the DNA substrate are determined based on the sequence read.

The invention relates to the technical field of biology, in particular to a tissue sample space DNA hydroxymethylation sequencing library construction and sequencing method, a kit and application. The library construction method comprises the following steps: breaking DNA in a fixed tissue slice in situ, preparing a double-stranded DNA fragment, and adding a linker on the DNA fragment in situ; carrying out in-situ bar code coding on the DNA fragments, so that pixel points are formed in the region where the DNA fragments containing the same bar code in the tissue slice are located; separating a DNA (Deoxyribonucleic Acid) fragment; filling in the deleted nucleotide residues between the linker and the bar code of the separated DNA fragment; and converting cytosine bases in the DNA fragments into other base types by using an enzymatic reaction system. According to the construction and sequencing method of the sequencing library, high-precision detection of the 5-methylcytosine basic group and the 5-hydroxymethylcytosine basic group in the genome is realized on the premise of retaining the tissue structure and spatial information.

The present disclosure relates to modified proteins, methods, compositions and kits for mapping the methylation status of nucleic acids comprising 5-methylcytosine and 5-hydroxymethylcytosine. In some embodiments, the modified proteins have been altered to increase protein stability. In some embodiments, a protein selectively acts on certain modified cytosines of a target nucleic acid and includes one or more substitution mutations that enhance the protein's selectivity for certain modified cytosines, optionally enhance the stability of the protein, or optionally enhance both selectivity and stability. Also provided are compositions and kits comprising one or more of the proteins, as well as methods of using one or more of the proteins.

This disclosure provides compositions for use in the preparation of target nucleic acid molecules, comprising one or more 5-formylcytosine bases or adducts of 5-formylcytosine. Also disclosed herein is a method for efficiently synthesizing nucleic acid molecules comprising one or more 5-formylcytosine bases from target nucleic acid molecules comprising one or more 5-hydroxymethylcytosine bases. This disclosure also provides a method for detecting epigenetic modifications in target nucleic acid molecules, such as epigenetic modifications characterized by methylation of cytosine at position 5 (e.g., 5-methylcytosine; 5-hydroxymethylcytosine).

Provided herein are methods and systems related to obtaining and processing sequence reads. The methods may comprise obtaining cell-free DNA sequences from a subject and obtaining sequencing reads from the cell-free DNA sequences that identify cell-free DNA sequences, 5-methylcytosines, and 5-hydroxymethylcytosines. The methods may involve applying a trained classifier to the sequencing reads.

The present disclosure is concerned with modified proteins, methods, compositions, and kits for mapping of methylation status of nucleic acids, including 5-methylcytosine and 5-hydroxymethyl cytosine. In some embodiments, the modified proteins have been altered to increase protein stability. In some embodiment, the proteins selectively act on certain modified cytosines of target nucleic acids and include one or more substitution mutations that enhance the selectivity of the proteins for certain modified cytosines, optionally enhance the stability of the proteins, or optionally enhance both selectivity and stability. Also provided are compositions and kits that include one or more of the proteins and methods for using one or more of the proteins.

Provided herein are methods of processing DNA of a sample. The methods can comprise steps of (a) partitioning the DNA into a plurality of subsamples by contacting the DNA with an agent that recognizes methyl cytosine in the DNA, (b) contacting the DNA of the sample or a subsample thereof with a methyltransferase in the presence of an azide donor or an amine donor, thereby labeling unmethylated CpGs in the DNA with azide or an amine and providing azide-labeled DNA or amine-labeled DNA, respectively, and (c) tagging the azide-labeled DNA or the amine-labeled DNA, and separating the tagged, azide-labeled DNA from DNA of the second subsample that is not azide-labeled or separating the tagged, amine-labeled DNA from DNA of the second subsample that is not amine-labeled. In some embodiments of the disclosed methods, these steps are performed in a different order.

A polynucleotide comprising an indexing barcode incorporated at a first end of the polynucleotide only and a label bound site-specifically to a nucleotide. Preferably the nucleotide is between 100 – 500 nucleotides and the label is not fluorescent The label comprises a tag from a cofactor analogue, a high affinity binding protein or antibody or a DNA binding protein that has been crosslinked to a nucleotide residue Also disclosed is an amplified sequencing library comprising a first indexing barcode, a first subset of polynucleotides comprising a site-specifically bound label and a second subset of unlabelled polynucleotides. Further disclosed is a method of preparing a polynucleotide sample for sequencing, where the polynucleotide is suitable for determining the status of nucleotide residues and the presence of a genetic mutation The label is a methyl-CpG binding domain protein, a high affinity antibody specific for, 5-methylcytosine, 5-hydroxymethy[cytosine, N4-methylcytosine or N6-methylcytosine. Further disclosed is an in vitro method of diagnosing a disease in a subject, based on the profile obtained by the method and a kit for determining the status of nucleotide residues and the presence of a genetic mutation in a polynucleotide sample.

The present invention provides antisense oligonucleotides (ASO) for inhibiting ACSL4 gene expression. The oligonucleotide sequences are represented by SEQ ID NO.3 and SEQ ID NO.7. The antisense oligonucleotide sequences are fully phosphorothioated, and the ribose of the initial and termina five bases at each end is modified with a 2′-O-Methoxyethyl group. Additionally, all cytosines are modified with methylcytosine. Cell experiments and animal model studies conducted in this invention have demonstrated that the ACSLA ASO can effectively alleviate the ferroptosis damage inflicted on neural cells during the process of stroke. Moreover, modulation of ACSLA expression is beneficial for a range of medical disorders including acute ischemic strokes, cancer-related diseases, metabolic disorders, neurodegenerative diseases and ferroptosis related diseases, thus presenting a bright future for clinical utilization.

Provided herein are methods and systems related to obtaining and processing sequence reads. The methods may comprise obtaining cell-free DNA sequences from a subject and obtaining sequencing reads from the cell-free DNA sequences that identify cell-free DNA sequences, 5-methylcytosines, and 5-hydroxymethylcytosines. The methods may involve applying a trained classifier to the sequencing reads.

The invention relates to the technical field of biological detection, in particular to a kit for detecting methylation of colorectal cancer related genes and application of the kit. According to the kit provided by the invention, by utilizing the characteristic that methylation sensitive restriction enzyme (MSRE) can specifically recognize and cut unmethylated DNA sequences but cannot cut methylated (such as 5 '-methylcytosine, 5mC) sites, a sample to be detected is subjected to enzyme digestion reaction in combination with a specific primer and a probe designed for a target gene, so that the methylated DNA sequences can be specifically recognized and cut, and the methylated (such as 5'-methylcytosine, 5mC) sites can be detected. The Ct difference value (namely delta Ct) between the target gene and the reference gene is calculated through qPCR reaction, so that the methylation occurrence state of the sample to be detected can be determined.

The invention discloses a method for DNA methylation detection by using a Cy7 labeled nucleic acid probe, which comprises one of the following two detection paths: path I: hybridization and hydrolysis mediated real-time fluorescence quantitative detection, comprising the following steps of: bisulfite treatment: treating extracted target DNA with bisulfite to convert unmethylated cytosine into uracil, and detecting the methylation of the target DNA; the methylated cytosine is kept unchanged; the method has the beneficial effects that background fluorescence interference is reduced by near-infrared optical characteristics of Cy7, PCR amplification or antibody signal enrichment is matched, the detection sensitivity can reach the almole level, and methylated DNA with extremely low concentration can be detected; a probe sequence is designed aiming at a methylation target sequence, and an antibody specifically recognizes 5-methylcytosine, so that methylated DNA and unmethylated DNA are effectively distinguished, and non-specific signal interference is avoided; the path I is suitable for high-throughput clinical sample quantitative detection, and multiple detection can be realized; the second path is suitable for in-situ imaging of cells or tissues and supports in-vivo deep tissue observation.