33 results about "Repair enzymes" patented technology

A process for assembling a series of DNA fragments generated by PCR into an ordered circular arrangement for replication and genetic work in cells. The PCR fragments are made with a modified nucleotide in the primers that can be removed with a DNA excision repair enzyme to generate a 3′ overhang. The 3′ overhangs are designed to allow directional annealing and thus sequential PCR fragments can be assembled by annealing the overhangs and subsequent ligation. Sequential addition of PCR fragments is facilitated by growing the chain on a solid support, and the assembled chain can be removed with a site specific recombinase if the first and last primers contain the recombinase site. The circularized assembled fragment can be directly used for cell transformation if the appropriate sequences are included, such as an origin of replication and a selectable marker.

The present invention pertains generally to the field of therapeutic compounds. More specifically the present invention pertains to 5-[[4-[[morpholin-2-yl]methylamino]-5-(trifluoromethyl)-2-pyridyl]amino]pyrazine-2-carbonitrile compounds (referred to herein as “TFM compounds”) which, inter alia, inhibit Checkpoint Kinase 1 (CHK1) kinase function. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to inhibit CHK1 kinase function, and in the treatment of diseases and conditions that are mediated by CHK1, that are ameliorated by the inhibition of CHK1 kinase function, etc., including proliferative conditions such as cancer, etc., optionally in combination with another agent, for example, (a) a DNA topoisomerase I or II inhibitor; (b) a DNA damaging agent; (c) an antimetabolite or a thymidylate synthase (TS) inhibitor; (d) a microtubule targeted agent; (e) ionizing radiation; (f) an inhibitor of a mitosis regulator or a mitotic checkpoint regulator; (g) an inhibitor of a DNA damage signal transducer; or (h) an inhibitor of a DNA damage repair enzyme.

The present invention provides methods relating to chemotherapeutic treatment of a cell proliferative disorder. In particular, a method is provided for predicting the clinical response to certain types of chemotherapeutic agents. Alkylating agents, used for the treatment of certain types of tumors including tumors of the nervous system and lymph system, are efficacious agents when the damage they do to tumor cell DNA is not repaired by cellular DNA repair mechanisms. The present invention provides a method for determining the activity of a gene encoding a DNA repair enzyme, thus providing a prediction of the clinical response to alkylating agents.

Disclosed are a phthalic hydrazide (phthalazine ketone) compound, and a pharmaceutical composition comprising the same. As a DNA repair enzyme poly (ADP-ribozyme) polymerase inhibitor, the compound and the pharmaceutical composition can effectively treat diseases involving PARP enzymatic activity, including cancer, neural degenerative diseases, inflammation and the like.

Disclosed are a phthalic hydrazide (phthalazine ketone) compound, and a pharmaceutical composition comprising the same. As a DNA repair enzyme poly (ADP-ribozyme) polymerase inhibitor, the compound and the pharmaceutical composition can effectively treat diseases involving PARP enzymatic activity, including cancer, neural degenerative diseases, inflammation and the like.

The following invention concerns a method for investigating cytosine methylation by means of DNA repair enzymes. Here, the DNA is first converted so that unmethylated cytosines are converted to uracil, while 5-methylcytosine remains unchanged. Then the DNA is hybridized to oligonucleotides, whereby hybrids will be formed with or without erroneous base pairings, in each case depending on the methylation status of the DNA. Following this, the erroneously paired hybrids will be cleaved by repair enzymes. Then the methylation status of the DNA can be determined in different ways. The method according to the invention is particularly suitable for the diagnosis and prognosis of cancer disorders and other diseases associated with a change of the methylation status as well as for predicting undesired drug effects.

The invention discloses an ultramicro nucleic acid sample library building method applied to an NGS platform. The ultramicro nucleic acid sample library building method comprises the following steps: adding nucleic acid repair enzyme into an ultramicro nucleic acid sample for nucleic acid repair before DNA extraction of the ultramicro nucleic acid sample; then carrying out DNA extraction on the ultramicro nucleic acid sample containing the nucleic acid repair enzyme; and carrying out library construction on the extracted DNA to obtain a sequencing library of the ultramicro nucleic acid sample. According to the ultramicro nucleic acid sample library building method, nuclease repair is carried out on sample DNA before DNA extraction; in addition, nucleic acid repair enzyme is added in the DNA extraction process; DNA damage can be effectively repaired, reaction false positive is reduced, DNA extraction quality is improved, and the constructed library can be better suitable for NGS sequencing. According to the library building method, the problems that the DNA extracted from the FFPE sample is poor in quality and low in total quantity, and the library cannot be effectively built are solved.

The invention relates to a contaminated site remediation method which is mainly used for remedying a contaminated site mainly containing halohydrocarbon pollutants, in particular to remediation adopting the reductive dechlorination enzyme. According to the specific efficiency of the reductive dechlorination enzyme on the halohydrocarbon pollutants, one method for improving the activity of the reductive dechlorination enzyme is proposed, so that the halohydrocarbon DNAPL (dense non-aqueous phase liquid) pollutants in the deep position of the contaminated site are rapidly and thoroughly removed. The method for remedying the halohydrocarbon contaminated site through the reductive dechlorination enzyme comprises the step that biological enzyme is injected to the underground part of the halohydrocarbon contaminated site and transferred to a pollution source area for pollutant degradation, and is characterized in that the biological enzyme is the reductive dechlorination enzyme, and the reductive dechlorination enzyme is prepared to form a liquid enzyme preparation containing the reductive dechlorination enzyme, diethyl aminoethyl synthetic plasma and polymeric alcohol. The activity of the enzyme in the reductive dechlorination enzyme preparation prepared with the method can keep longer than one week, and the practicability of the preparation for site remediation is guaranteed.

The invention discloses Aspergillus niger 6-4 photolyase with site-specific metagenesis and a construction method thereof. The Aspergillus niger 6-4 photolyase with site-specific metagenesis has a nucleotide sequence shown as SEQ ID NO: 1. A 6-4 photolyase gene sequence acquired from existing WT Aspergillus is subjected to artificial codon optimization; a metagenesis primer is designed to acquirea metagenetic gene fragment; the gene fragment is linked with pET22b plasmid to construct a recombinant expression plasmid; the recombinant expression plasmid is converted to Escherichia coli competent cells to construct a genetically engineered bacterium for photolyase expression, which is well expressed under 20 DEG C and 1 mM IPTG. Compared with WT photolyase, the mutant enzyme S60N expressed herein has higher activity which is stably maintained longer, can better restore DNA damage caused by ultraviolet, and can be more easily used as cosmetics, and pharmaceutical additives in people's daily lives.

The invention provides a biosensor for detecting repair glycosylase and a detection method and application of the biosensor. The biosensor comprises a stem ring DNA template and linear primer probes;the linear primer probes are LAMP primer probes, at least four primer probes are arranged, and comprises two front positive outer and inner primers, namely FIP and FOP, and two rear positive outer andinner primers BIP and BOP. A target base to be detected to repair glycosylase is designed in a stem region of the stem ring DNA template; and the stem ring DNA template is further provided with a region complementary to or identical to a primer probe sequence. The biosensor prepared by the invention actually constructs a self-directed replication system, and can be used for zero background, highsensitivity and high specificity detection of repair glycosylase activity. A simple, robust and universal platform is provided for repairing enzyme related biomedical research and early clinical diagnosis.

Noncovalent small-molecule inhibitors of the enzyme OGG1, methods of their manufacture, and applications for their administration are provided. Small molecule inhibitors were shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and it displayed no toxicity in two human cell lines. The inhibitors provide a tool for the study of the role of OGG1 in multiple disease-related pathways, and a therapeutic target for the treatment thereof.

A slow release compound platinum drug is a slow release injection which is composed of slow release microspheres and a dissolvant. The slow release microspheres comprise anticancer active components and a slow release adjuvant, and the dissolvant is a common dissolvant or a special dissolvant containing a suspending agent. The anticancer active components are platinum drugs such as ormaplatin, hetaplatin, lobaplatin, nedaplatin or oxaliplatin, and the like, and / or platinum drug synergists selected from a phosphoinositide-3-kinase inhibitor, pyrimidine analogue and / or a DNA repair enzyme inhibitor; the slow release adjuvant is selected from the copolymer of polylactic acid, EVAc, polifeprosan, sebacic acid copolymer, and the like; the viscosity of the suspending agent is 100cp-3,000cp (at the temperature of 20-30 DEG C), and the suspending agent is selected from sodium carboxymethyl cellulose, and the like. The slow release microspheres can be also made into a slow release implant. By injecting the slow release injection or placing in tumors or around the tumors, the general toxic reaction of the drug can be reduced, the local drug concentration of the tumors can be selectively improved, and the curative effects of non-operative therapies such as radiotherapy, chemotherapy, and the like, can be improved.

Three recombinant E. coli strains produce the enzymes CPD-photolyase, 6,4-bifunctional photolyase and 6,4-photolyase, from bacterial Antarctic isolates of the genus Hymenobacter the first one and Sphingomonas the others. It is also disclosed a process of production and purification of the recombinant enzymes with high performance, high degree of purity and high catalytic repair activity, having applications in, but it is not limited to, cosmetics and pharmaceutical industry. A fast, cheap and qualitative method is provided for the determination of the CPD photolyase activity.

The invention discloses a fusion protein and a design method thereof. The design method comprises the following steps: fusing a recombinant helper protein with the DNA binding function and a protein with the DNA cleavage function. When the fusion protein with the DNA cleavage function performs cleavage, the recombinant helper protein provides a DNA recombinant repair template near a cleavage site,and recruits recombinant repair enzyme, so that the recombinant repair efficiency is greatly improved. The fusion protein has the advantage that the recombination repair efficiency is improved.

The following invention concerns a method for investigating cytosine methylation by means of DNA repair enzymes. Here, the DNA is first converted so that unmethylated cytosines are converted to uracil, while 5-methylcytosine remains unchanged. Then the DNA is hybridized to oligonucleotides, whereby hybrids will be formed with or without erroneous base pairings, in each case depending on the methylation status of the DNA. Following this, the erroneously paired hybrids will be cleaved by repair enzymes. Then the methylation status of the DNA can be determined in different ways. The method according to the invention is particularly suitable for the diagnosis and prognosis of cancer disorders and other diseases associated with a change of the methylation status as well as for predicting undesired drug effects.

Noncovalent small-molecule inhibitors of the enzyme OGG1, methods of their manufacture, and applications for their administration are provided. Small molecule inhibitors were shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and it displayed no toxicity in two human cell lines. The inhibitors provide a tool for the study of the role of OGG1 in multiple disease-related pathways, and a therapeutic target for the treatment thereof. Various embodiments include substituted 1,2,3,4-tetrahydroquinolines, such as compounds of the of the formula ABCDE:

The invention discloses a preparation method of cyclobutane pyrimidine dimmer photolyase liposome, which comprises the following steps: an expression of cyclobutane pyrimidine dimmer photolyase, a purification of cyclobutane pyrimidine dimmer photolyase, and a preparation of cyclobutane pyrimidine dimmer photolyase liposome; the method employs a Q column for separating the cyclobutane pyrimidine dimmer photolyase, and the medium price is low, moreover, the effects for separating and removing glutathione-S-transferase are better, and the method is suitable for industrialization production of cyclobutane pyrimidine dimmer photolyase. The cyclobutane pyrimidine dimmer photolyase is prepared into a liposome according to the invention, thereby the cyclobutane pyrimidine dimmer photolyase can penetrate the barrier of cell membrane or skin and enter into cells, thereby substantially improving restoration effect of DNA damage.