45 results about "Double strand dna" patented technology

A nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a nucleotide sequence comprising a cardiovascular disease associated gene to be silenced and wherein said cardiovascular gene is proprotein convertase subtilisin kexin 9 (PCSK9) there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ end of said sense strand; and ii) a second nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a different cardiovascular disease associated gene to be silenced wherein said cardiovascular gene is lipoprotein (a) (Lp(a)) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ end of said sense strand wherein the single stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single stranded deoxyribonucleic acid (DNA) molecule set forth in i) above and anneals by complementary base pairing to form a double stranded DNA linker that links the first and second double stranded inhibitory ribonucleic acid (RNA) molecules.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The disclosure provides, for example, a double stranded DNA (dsDNA) molecule comprising chemically modified nucleotides. In some embodiments, the dsDNA molecule comprises an upstream DNA end form which is a closed end, a downstream DNA end form which is a closed end, and a double stranded region comprising a sense strand and an antisense strand, in which the sense strand comprises one or more chemically modified nucleobases, and the antisense strand is free of chemically modified nucleobases.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.

The present disclosure provides compositions and kits for the tagmentation of double stranded DNA. In some embodiments, the compositions and kits for the tagmentation of double stranded DNA include one or more histone-like proteins and / or one or more transposition systems. The present disclosure also provides methods for the tagmentation of double stranded DNA in the presence of one or more histone-like proteins.

The disclosure provides, for example, a double stranded DNA (dsDNA) molecule comprising chemically modified nucleotides. In some embodiments, the dsDNA molecule comprises an upstream DNA end form which is a closed end, a downstream DNA end form which is a closed end, and a double stranded region comprising a sense strand and an antisense strand, in which the sense strand comprises one or more chemically modified nucleobases, and the antisense strand is free of chemically modified nucleobases.

The present invention relates to enzymes having DNA polymerase and 3′-5′ exonuclease activities. In particular, the present invention relates to a heat labile enzyme possessing a DNA polymerase II activity and a 3′-5′ exonuclease activity of marine origin. Furthermore, the present invention relates to a DNA polymerase primarily exerting a 3′-5′ activity, i.e. where the polymerase activity is absent. The present invention furthermore relates to the use of the exonuclease activity to degrade the 3′-5′ strand of double stranded DNA to perform single stranded overhang, e.g. in recombinant cloning processes, or in processes for removal of contaminating nucleic acid molecules.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

Described herein are, among other things, in vitro methods of identifying double stranded DNA sequences that are cleaved by a nuclease. For example, by providing a library of defined linear dsDNA oligonucleotides of known sequences, incubating the library in the presence of a nuclease, ligating DNA adapters, amplifying cleaved oligonucleotides, and determining the sequence(s) of the amplified fragments.

The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.

Mismatch anchoring compounds (MACs) are disclosed that recognize and bind to specific base-pair mismatches (mmBP) present within single stranded or double stranded DNA, RNA, and oligonucleotide sequences, and enable the ex vivo identification of DNA / RNA point mutations (DNAPM / RNAPM), including disease-associated, rare, and low abundance DNAPM / RNAPM, and the isolation and elimination of mmBP-positive cells. The use of MACs in vitro and in vivo (a) blocks mmBP-positive DNA replication and gene transcription / expression, (b) inhibits mmBP-positive cell proliferation, (c) reverses, prevents, and / or treats mmBP-rnediated disease, aging, and age-related disorders, (d) blocks mmBP-positive RNA and / or RNA-DNA duplex translation and inhibits the production of abnormal disease-causing proteins, (e) silence mmBP-positive genes, and (f) blocks intracellular pathogen replication. MAC-conjugates and their uses are disclosed, including MACs radiolabeled with SPECT / PET / particle-emitting isotopes for radioimaging and / or radiotherapy of mmBP-positive diseases.

Described is a linear double stranded DNA product comprising a first adaptor molecule ligated to a first end and a second adapter ligated to a second end. The first adaptor is a linear nucleic acid co

The present invention is concerned with linear double stranded DNA, which is coupled to a single support or a tag at the 3′ end of its non-coding strand and methods for producing said linear double stranded DNA. The present invention further relates to the use of said linear double stranded DNA in an RNA in vitro transcription reaction and also to a method for producing RNA in vitro. The present invention also relates to a bioreactor for RNA in vitro transcription.

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.

A primer for detection of a mutation in a microsatellite contained in a target sequence of a double stranded DNA molecule, wherein the mutation is a frameshift of the microsatellite as compared with the corresponding wild type microsatellite sequence. The primer comprises a region of at least 10 nucleotides that is complementary to the target sequence of the antisense or the sense strand of the DNA molecule containing the microsatellite having a frameshift mutation, except that the primer includes between one and four, or one and three, nucleotides which are mismatched to the target sequence containing the mutation in the microsatellite and which are also mismatched to a corresponding sequence containing the wild type microsatellite.

Described herein are various methods of removing uracils due to the deamination of unmethylated cytosines in an assay using engineered cytosine deaminases to deaminate methylated cytosines. The Chemoenzymatic Uracil Replacement of Nucleobases (ChURN) method includes providing a sample comprising single stranded DNA library fragments in which a cytosine deaminase has deaminated methylated cytosines; contacting the sample with an uracil DNA glycosylase (UDG) to deglycosylate uracil residues to form abasic sites having a hemiacetal formation within the single stranded DNA library fragments; contacting the sample with a reactive cytosine nucleobase analog to install a cytosine at abasic sites thru a noncanonical linkage; and subjecting the sample to polymerase chain reaction (PCR) amplification resulting in double stranded DNA corrected library fragments. The Uracil Enzymatic Removal and Substitution at Errors (U-ERASE) method includes providing a sample comprising single stranded DNA library fragments in which a cytosine deaminase has deaminated methylated cytosines, synthesizing double stranded DNA library fragments in which the second strand is tagged to facilitate its downstream degradation, treating the double stranded DNA library fragments with an uracil DNA glycosylase and an endonuclease resulting in the removal of uracil bases and single nucleotide gaps at those sites, and repairing the single nucleotide gaps through treatment with a polymerase, dCTP, and a ligase, resulting in replacement of false positive uracil bases with a mismatched base, such as cytosine. Subsequently, the second strand is selectively degraded, allowing for selective amplification of the original DNA strand via PCR.

The methods, compositions, and kits of the present disclosure provide a new method for whole genome, i.e., unbiased DNA analysis, that can be performed on limited amounts of DNA. It can be used to analyze DNA to determine its modification status. Aspects of the present disclosure relate to methods for amplifying a bisulfite treated deoxyribonucleic acid (DNA) molecule, the method comprising: (a) ligating an adaptor to the DNA molecule, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite protected cytosines; (b) treating the ligated DNA molecule with bisulfite; (c) hybridizing the bisulfite treated DNA molecule to a primer; (d) extending the hybridized primer to produce double stranded DNA; and (e) in vitro transcribing the double stranded DNA to produce RNA.

The present invention provides engineered wide-range nuclease enzymes derived from I-CreI having substitutions at specific positions that increase the activity of the nuclease on recognition sequences comprising certain central sequences. The invention also provides methods of cleaving double stranded DNA using such engineered wide range nuclease enzymes. The invention further provides methods for improving the activity of engineered broad-range nuclease enzymes against recognition sequences comprising certain central sequences.

The disclosed invention relates to a linear double stranded DNA product with enhanced exonuclease resistance due to the presence of protected nucleotides, preferably phosphorothioated nucleotides. The

The present invention provides a method for producing double stranded DNA (dsDNA) molecules. In particular, the invention provides a method that utilises a plurality of single stranded DNA (ssDNA) molecules to form a dsDNA complex in which the ssDNA molecules are ligated to produce the dsDNA molecule.

The invention relates to an in vitro method for modifying double stranded DNA (dsDNA) in a eukaryotic cell. The method comprises: a) introducing a base editor system into the cell, said system comprising i) a base editor, comprising an RNA guided DNA nickase linked to a single-stranded DNA nucleobase modifying enzyme, or a nucleic acid encoding said base editor, and ii) at least two guide RNA (sgRNA) molecules, comprising a first sgRNA that hybridizes to a first target sequence in the dsDNA, and a second sgRNA that hybridizes to a second target sequence in an opposing strand of the dsDNA, b) generating at least two single-strand breaks (SSBs), in opposing strands, of the dsDNA, wherein a first base editor complex, comprising the first sgRNA, introduces a nick cleavage of one strand of the dsDNA in the first target sequence, and a second base editor complex, comprising the second sgRNA, introduces a nick cleavage of the opposite strand of the dsDNA in the second target sequence, thereby producing a 5' or 3' overhang, c) inserting an exogenous DNA repair template sequence in proximity to the two single strand breaks (SSBs). The invention further relates to a genetically modified eukaryotic cell obtained by the method according to the present invention. The invention also relates to a kit for use in the in vitro method.

Mismatch anchoring compounds (MACs) are disclosed that recognize and bind to specific base-pair mismatches (mmBP) present within single stranded or double stranded DNA, RNA, and oligonucleotide sequences, and enable the ex vivo identification of DNA / RNA point mutations (DNAPM / RNAPM), including disease-associated, rare, and low abundance DNAPM / RNAPM, and the isolation and elimination of mmBP-positive cells. The use of MACs in vitro and in vivo (a) blocks mmBP-positive DNA replication and gene transcription / expression, (b) inhibits mmBP-positive cell proliferation, (c) reverses, prevents, and / or treats mmBP-rnediated disease, aging, and age-related disorders, (d) blocks mmBP-positive RNA and / or RNA-DNA duplex translation and inhibits the production of abnormal disease-causing proteins, (e) silence mmBP-positive genes, and (f) blocks intracellular pathogen replication. MAC-conjugates and their uses are disclosed, including MACs radiolabeled with SPECT / PET / particle-emitting isotopes for radioimaging and / or radiotherapy of mmBP-positive diseases.

Described is a linear double stranded DNA product comprising a first adaptor molecule ligated to a first end and a second adapter ligated to a second end. The double stranded DNA molecule is closed at

The disclosed invention relates to a linear double stranded DNA product with enhanced exonuclease resistance due to the presence of protected nucleotides, preferably phosphorothioated nucleotides. The

The present disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising a chemically modified uridine nucleotide. In some embodiments, the dsDNA molecules comprise a therapeutic load sequence. In some embodiments, the dsDNA molecules are resistant to endonuclease digestion and / or to immunosensor recognition, and support the expression of a therapeutic load encoded in the dsDNA molecules. The present disclosure also provides, for example, pharmaceutical compositions comprising dsDNA molecules comprising a chemically modified uridine nucleotide.

An effective gene therapy technique that can transfect a gene to a cell in an organ or a tissue in a subject in a safe, effective, and durable manner is disclosed herein. The methods disclosed herein can induce expression of a nucleic acid payload in target cell(s) of a mammalian subject by administering to the subject a linear double stranded DNA (dsDNA) construct covalently closed at each of its ends by DNA loops, the linear dsDNA construct comprising the nucleic acid payload; administering to the subject a plurality of microbubbles; and administering to the subject, in proximity to the target cell(s) an effective amount of an ultrasound energy, thereby inducing the formation of pores the target cell(s) by disrupting the plurality of microbubbles to result in introduction of the dsDNA into the target cell(s) and expression of the nucleic acid payload in the target cell(s).

It has been established that one or more large double stranded DNA fragments (each 2,000 to 40,000 base pairs in size) can be captured and isolated from genomic DNA fragments using sequence specific PNA hybridization probes. Compositions and methods for enrichment of a multiplicity of long DNA sequences selected from the genome of any eukaryote are provided. Capture is performed using multiple PNA molecules with gamma-modified chiral backbones, comprising a mixture of neutral and positive chemical groups. Two or more PNA probes with covalently bound haptens, preferably biotin, target each DNA domain of interest for capture, isolation, and subsequent sequencing analysis of the multiplicity of enriched targets, including DNA methylation sequencing. The methods include enhancement of probe-DNA binding specificity through single strand binding proteins (SSB).

The present invention is concerned with linear double stranded DNA, which is coupled to a single support or a tag at the 3′ end of its non-coding strand and methods for producing said linear double stranded DNA. The present invention further relates to the use of said linear double stranded DNA in an RNA in vitro transcription reaction and also to a method for producing RNA in vitro. The present invention also relates to a bioreactor for RNA in vitro transcription.

The present disclosure provides a method for performing a template conversion reaction on a nucleic acid sample comprising at least one double stranded DNA and at least one RNA. The method comprises subjecting the nucleic acid sample to a first template conversion reaction in the absence of at least one dNTP selected from the group consisting of dATP, dCTP, dGTP and dTTP, thereby forming a first nucleic acid product comprising the double-stranded DNA having at least one extended 3'end complementary to a first template conversion oligonucleotide. The method further includes performing a second template conversion reaction on the nucleic acid sample to form a second nucleic acid product comprising a first primer extension product complementary to at least a portion of the RNA, the first primer extension product having an extended 3'end complementary to the second template conversion oligonucleotide.