24 results about "Substrate recognition" patented technology

The application discloses a covalent polypeptide inhibitor targeting protein kinase A, a conjugate, a kit, a pharmaceutical composition and application. The general formula of the covalent polypeptide inhibitor is Z1-Seq-Z2; wherein Seq is an amino acid sequence; the C-terminal of Seq comprises a pseudo-substrate recognition motif recognized by protein kinase A, a non-natural amino acid with a covalent reaction group, and can be covalently crosslinked with a Cys200 residue on a catalytic subunit of protein kinase A; Z1 is selected from hydrogen, an acyl group, a fluorescent group, biotin, an isotopic tag or a biological ortho-reaction group; and Z2 is selected from a hydroxyl group, an amino group, a cell-penetrating peptide or a stabilization modification group. The covalent polypeptide inhibitor can strongly and irreversibly inhibit PKA activity, and can be used as an active probe to specifically label active PKA in a complex biological sample, and is used for biological research and drug development.

The invention discloses an in-vitro ubiquitination screening system of E3 ubiquitin ligase Smurf1, belongs to the technical field of molecular biology and protein modification research, and provides an efficient and standardized Smurf1 in-vitro ubiquitination screening system construction method which can be used for screening E3 ubiquitin ligase Smurf1 through multi-step accurate condition control and system verification. The specificity and the repeatability of the ubiquitination reaction are ensured, and a new technical platform is provided for researching a substrate recognition mechanism and function regulation and control of Smurf1. The method can be used for high-throughput screening of Smurf1 ubiquitinated substrate proteins, and is used for mechanism research and novel drug research and development.

The application relates to the field of medicinal chemistry and provides a use of a diphenylacetonitrile compound and a protein-targeting hydrolytic chimeric compound. The application finds that a diphenylacetonitrile compound shown in formula (I) directly interacts with FEM1B, thereby serving as an inhibitor of a human E3 ubiquitin ligase substrate recognition receptor FEM1B. The application also provides a compound shown in formula (II), and the compound shown in formula (II) is a protein-targeting hydrolytic chimeric (PROTAC) drug, wherein Q2 is a target protein ligand molecule, Q1 is a FEM1B inhibitor, Q2 is combined with a target protein, thereby inducing E3 ligase FEM1B combined with Q1 to approach the target protein, leading to ubiquitination and degradation of the target protein. Experimental results show that the PROTAC drug shown in formula (II) provided by the application can specifically degrade a target protein in cells. Formula (I); formula (II).

The invention belongs to the technical field of organic synthetic chemistry, and discloses a method for efficiently synthesizing methyl 2-chloro-3-oxovalerate. According to the method, high-selectivity chlorination is performed on a beta-site C-H bond of methyl 3-oxovalerate under a mild condition by taking sodium chloride as a chlorine source and adopting a dual-catalysis system consisting of engineered halogenase and a visible light-sensitive catalyst under visible light irradiation. The engineered halogenase is a HalB triple mutant, and the affinity and regioselectivity of a substrate are remarkably improved; the photosensitive catalyst is a ruthenium bipyridine complex and synergistically drives generation of chlorine free radicals. According to the invention, a brand new biological-photochemical synergistic catalysis platform is constructed by organically combining the substrate recognition capability of the engineered halogenase and an electron transfer mechanism driven by visible light catalysis, so that the contradiction among selectivity, mildness and atomic efficiency of a traditional chlorination method is solved; and the method can be expanded to precise synthesis of other beta-functionalized ketone ester compounds.

The invention relates to a deoxyribozyme binding arm, a kit, a biosensor and a nucleic acid detection method. The deoxyribozyme binding arm is of an annular structure and comprises a catalytic core A sequence of deoxyribozyme, a monomer amplicon recognition region, a sequence of a fragment containing a detection probe and a deoxyribozyme substrate recognition region; wherein the catalytic core part sequence of the deoxyribozyme comprises a part of sequence in any one of 8-17 type deoxyribozyme, E6 type deoxyribozyme or 10-23 type deoxyribozyme. The invention provides a deoxyribozyme binding arm and a multifunctional DNA nano-machine obtained by combining the deoxyribozyme binding arms, which integrate cutting enhanced rolling circle amplification, self-cutting DNAzyme and rolling circle amplification technologies, construct a self-cascade amplification nano-machine, and can perform molecular signal amplification on the existence of a formulated sequence. The system exhibits an increase in cascade reaction efficiency due to the minimization of the number of independent molecular diffusion / collision events.

The invention relates to the cross technical field of artificial intelligence and bioinformatics, in particular to a multi-modal enzyme kinetic parameter prediction method of an adaptive protein language model. Aiming at the problems that an existing processing method is lack of enzymatic reaction dynamic mechanism modeling and insufficient in three-dimensional structure utilization, the invention provides the following technical scheme: step 1, acquiring and preprocessing multi-modal data; 2, protein language model and molecular fingerprint feature extraction; 3, carrying out substrate recognition feature fusion based on cross attention; step 4, extracting conformation adaptive features based on the hybrid expert network; 5, correcting enzyme-substrate distribution alignment characteristics; step 6, kinetic parameter regression prediction; step 7, constructing a multi-objective loss function; and step 8, model training and parameter optimization. Through an enzyme reaction bridging adapter and an enzyme-substrate distribution alignment technology, high-precision prediction of enzyme kinetic parameters is realized, and cross-data-set accuracy and robustness are remarkably improved.

The invention relates to the field of medicinal chemistry, and provides application of a diphenylacetonitrile compound and a protein targeted hydrolysis chimera compound. The research finds that the diphenylacetonitrile compound shown in the formula (I) directly interacts with FEM1B, so that the diphenylacetonitrile compound can be used as an inhibitor of a human E3 ubiquitin ligase substrate recognition receptor FEM1B. The invention further provides a compound shown in the formula (II), the compound shown in the formula (II) is a protein targeted hydrolysis chimera (PROTAC) medicine, Q2 is a target protein ligand molecule, Q1 is an FEM1B inhibitor, Q2 is combined with target protein, and then E3 ligase FEM1B combined with Q1 is induced to approach the target protein, so that ubiquitination and degradation of the E3 ligase FEM1B are caused. Experimental results show that the PROTAC medicine as shown in the formula (II) provided by the invention can specifically degrade target protein in cells. # imgabs0 # formula (I); # imgabs 1 # formula (II).

The invention provides a nucleic acid in-situ dual-channel fluorescence detection probe group and system based on cleavage type DNAzyme and application of the nucleic acid in-situ dual-channel fluorescence detection probe group and system. The probe group comprises at least two probes, each probe comprises a recognition activation region and a signal generation region, the recognition activation region sequentially comprises a target nucleic acid recognition arm, a catalytic core sequence and a substrate recognition arm, and each probe can be paired with at least another probe; a substrate recognition arm and a catalytic core sequence on the paired probe are two cleavage type DNAzyme subunits formed by DNAzyme, and the DNAzyme with catalytic activity is formed by self-assembly in the presence of target nucleic acid; the signal generation region comprises at least one substrate of DNAzyme, two ends of the substrate are respectively modified with a fluorophore and a quenching group, the substrate is complementary with a substrate recognition arm, and the fluorophore modified on the substrate of each probe is different. According to the invention, in-situ real-time dynamic imaging, high-specificity and high-sensitivity detection and targeted regulation of target nucleic acid are realized.

DCAF15 is a substrate recognition (adaptor) protein of E3 ubiquitin ligase. Disclosed herein are compounds that recruit ubiquitin ligase CRL4DCAF15, to a target RNA recognition motif (RRM), causing its degradation. Also disclosed herein are compositions and methods of use in treating associated disorders and diseases.

The present invention discloses a DEAR nucleic acid manipulation system based on RNA ribozyme and its application. The present invention provides a DEAR nucleic acid manipulation system, wherein the DEAR nucleic acid manipulation system comprises an RNA molecule of a C-type second-class intron derived from bacteria, the RNA molecule comprises a substrate recognition region that hybridizes with a target sequence in a target nucleic acid, and the C-type second-class intron is a C-type second-class intron in which there is no open reading frame encoding an intron-encoded protein in the IV domain. The RNA ribozyme-based DEAR nucleic acid manipulation system provided by the present invention avoids the problems existing in the CRISPR-Cas system, such as the large protein molecule affecting the transfection efficiency, and the potential immunogenicity brought about by the Cas protein. The RNA ribozyme-based DEAR nucleic acid manipulation system provided by the present invention can realize the cutting of DNA and RNA.

The invention discloses application of an FBXO45 inhibitor in preparation of a medicine for treating psoriasis, and belongs to the technical field of biological medicine. According to the application disclosed by the invention, the specific high expression of the FBXO45 in skin lesion tissues of psoriasis patients and imiquimod-induced mouse model skin lesion is found for the first time, and the phenotypes (such as epidermal thickening, inflammatory factor expression and immune cell infiltration) of the psoriasis can be obviously relieved by specifically knocking out the FBXO45 from the skin, so that the FBXO45 is proved to be a potential therapeutic target of the psoriasis. Through virtual screening and molecular docking based on an FBXO45 protein structure, it is found that CD437 can be specifically combined with FBXO45, and the substrate recognition function of the FBXO45 is inhibited. In-vitro experiments show that the CD437 can inhibit the expression level of FBXO45 protein in HaCaT cells, reduce the expression of inflammatory factors such as TNF-alpha and the like, and induce cell apoptosis; in an imiquimod-induced mouse model, the skin lesion symptom (such as reduction of PASI score and reduction of epidermal thickness) is remarkably relieved through local external application of CD437. Therefore, the invention provides a novel FBXO45-targeted small molecule treatment strategy for the psoriasis.

The present invention relates to a DICER cleavage site motif that is a sequence determinant of dsRNA processing by DICER. Using the DICER cleavage site motif according to the present invention can strongly promote the processing of dsRNA by DICER, and thereby promote RNA interference. In addition, the DICER cleavage site motif according to the present invention is an integrated and conserved determinant of substrate recognition by DICER, and can be applied to any technique capable of generating siRNA through DICER processing. Thus, the present invention can greatly contribute to future studies using DICER processing, for example, studies on the biological or therapeutic use of small RNAs.

The invention discloses a molecular cage-based co-assembly catalyst driven by cation-pi action and containing a bionic pocket as well as a preparation method and application of the molecular cage-based co-assembly catalyst, and relates to the technical field of supramolecular self-assembly. The catalyst is a cage-based co-assembly formed by co-assembly of a molecular cage and positive ions under the surface-surface three-site positive ion-pi effect, wherein the molecular cage and the positive ions are subjected to surface-surface three-site positive ion-pi effect; by designing a co-assembly with a bionic pocket, accurate regulation and control of a reaction path in CO2RR are realized. The cage can form a self-adaptive size cavity similar to an induced fitting mechanism in protein-substrate recognition by combining the expansion and contraction behaviors of the large-size cage with the adaptability of cation-pi action, the assembly is allowed to stabilize various photocatalytic intermediates, and reaction paths are selectively guided to respectively generate CH4 and CO.

The invention discloses a molecular docking-based glycosyl transferase substrate catalytic specificity multi-dimensional evaluation method, system, equipment and medium, and belongs to the technical field of protein structure simulation evaluation, and the method comprises the following steps: determining glycosyl transferase, and predicting a corresponding protein three-dimensional structure; performing molecular docking simulation based on the predicted protein three-dimensional structure; the catalytic specificity of different substrates is evaluated according to the difference between receptor substrate channels and ligands; and displaying a docking result through visual software. According to the method, a set of complete standardized operation program is formed, a clear methodology guide is provided for similar research, on the basis of traditional docking scoring, three-dimensional space size measurement analysis of the acceptor and the ligand is creatively used as a key screening index, multi-dimensional evaluation from the bonding strength to the space suitability is achieved, and the evaluation accuracy is improved. A new visual angle is provided for understanding an enzyme substrate recognition mechanism.

The application provides a preparation method of an engineered DEAR nucleic acid manipulation system, and the preparation method comprises at least one selected from the following: lengthening a substrate recognition region in a RNA molecule of an original DEAR nucleic acid manipulation system to have a length of 7-14 nucleotides; adding a recruitment sequence to a 3' end of a RNA molecule of the original DEAR nucleic acid manipulation system; respectively arranging a first dimerization motif and a second dimerization motif in a III domain in a first RNA molecule and a second RNA molecule from the original DEAR nucleic acid manipulation system, so that the first RNA molecule and the second RNA molecule form a heterodimer, and the like. The preparation method of the engineered DEAR nucleic acid manipulation system provided by the application further improves the specificity and cleavage activity of the DEAR nucleic acid manipulation system based on an RNA ribozyme.

The invention relates to the technical field of biology, in particular to an LbCas12a variant and application thereof in nucleic acid detection. According to the invention, a series of LbCas12a mutants with differentiated enzyme digestion activities are obtained by modifying key amino acid sites related to substrate recognition and conformation activation in LbCas12a protein. Compared with an existing method, a one-tube nucleic acid detection system based on the mutant has better system stability, sensitivity, specificity and universality, and high-sensitivity one-tube nucleic acid detection can be achieved under the condition that exogenous addition is not needed, and special consumables or complex equipment are not needed. The mutant set provides a more advantageous tool for the on-site application of a CRISPR (clustered regularly interspaced short palindromic repeats) detection system.

The invention discloses a hydroxylase involved in the biosynthesis of benzylisoquinoline alkaloids and its application, and relates to the field of medicinal plant genetic engineering, wherein the amino acid sequence is SEQ ID NO.1, or a protein having hydroxylase function obtained by substitution and / or deletion and / or addition of one or more amino acid residues thereof; or a protein having 90% or more identity therewith and having the function of hydroxylating benzylisoquinoline alkaloids; a recombinant expression vector, a recombinant microorganism; a yeast system composed of the enzyme and a reductase StCPR, and the application of the hydroxylase and its recombinant expression vector in the in vitro synthesis of heterologously expressed StCYP80B protein, and the in vivo synthesis of benzylisoquinoline compounds by recombinant microorganisms and the yeast system. The hydroxylase StCYP80B provided by the invention expands the substrate recognition range and is an effective enzymatic tool for hydroxylating monobenzylisoquinoline alkaloids of different structures.

The invention relates to the technical field of medicines, in particular to application of Cullin4A and DCAF7 in preparation of a product for mediating HDAC7 ubiquitination degradation. It is found that knock-down of the Cullin4A gene can obviously up-regulate the HDAC7 content and promote proliferation and transfer of NSCLC cells, and over-expression of the Cullin4A can obviously increase the ubiquitination level of HDAC7 molecules. Therefore, the Cullin4A can mediate the ubiquitination degradation of the HDAC7 and inhibit the malignant development of the NSCLC. It is also found that a substrate recognition molecule DCAF7 of the Cullin4A interacts with HDAC7, after exogenous overexpression of DCAF7, the protein ubiquitination level of HDAC7 is increased, the protein content is reduced, and up-regulation of the DCAF7 gene significantly inhibits proliferation and metastasis of NSCLC cells. The result shows that the DCAF7 is used as a substrate recognition molecule to mediate the ubiquitination degradation of the CRL4A on the HDAC7.

This invention discloses an RNA ribozyme-based DEAR nucleic acid manipulation system and its applications. The invention provides a DEAR nucleic acid manipulation system comprising a bacterial-derived C-type group II intron RNA molecule, the RNA molecule comprising a substrate recognition region that hybridizes with a target sequence in a target nucleic acid. The RNA ribozyme-based DEAR nucleic acid manipulation system provided in this invention avoids problems in the CRISPR-Cas system, such as reduced transfection efficiency due to protein molecule oversize and potential immunogenicity caused by the Cas protein. The RNA ribozyme-based DEAR nucleic acid manipulation system provided in this invention is capable of cleaving DNA and RNA.

The application discloses a preparation method of an engineered DEAR nucleic acid manipulation system. The application provides a preparation method of an engineered DEAR nucleic acid manipulation system, and the preparation method comprises at least one of the following: lengthening a substrate recognition region in a RNA molecule of an original DEAR nucleic acid manipulation system to have a length of 7-14 nucleotides; adding a recruitment sequence to a 3' end of a RNA molecule of the original DEAR nucleic acid manipulation system; setting a first dimerization motif and a second dimerization motif in III domains in a first RNA molecule and a second RNA molecule from the original DEAR nucleic acid manipulation system respectively, so that the first RNA molecule and the second RNA molecule form a heterodimer. The preparation method of the engineered DEAR nucleic acid manipulation system provided by the application further improves the specificity and cleavage activity of the DEAR nucleic acid manipulation system based on an RNA ribozyme.

The present application relates to the field of tumor immunotherapy, discloses a kind of ribozyme for targeted inhibition of PD-L1 gene expression and its preparation method and application, specifically relates to a kind of engineered Pistol ribozyme and its use in targeted inhibition of PD-L1 (CD274) gene expression.The ribozyme is RNA molecule connected by ribonucleotide monomer, contains the substrate recognition region of the conserved structural framework maintaining catalytic activity and PD-L1 transcript complement;Conserved skeleton covers P1, P2, P3 region and false node area, and contains catalytic related conserved base site, substrate recognition region can be designed as required, can recognize and cut the targeted site containing GU dinucleotide in PD-L1 transcript, reduce the PD-L1 expression of tumor cell in post-transcriptional level.The PD-L1 transcript includes mature mRNA, precursor mRNA, intron retention transcript and variable splicing transcript.Different from existing immune checkpoint blockade therapy, the present application can sustainably inhibit endogenous PD-L1 in tumor cell, relieve immune suppression, enhance anti-tumor immune response, and also provide the preparation method of the ribozyme and its pharmaceutical composition, and application prospect is good.

Provided are a mutated V-type CRISPR enzyme and use thereof. By means of the protein mutation rational mutagenesis technology, the restriction on the recognition and cleavage of DNA substrates by RuvC in Cas12a or Cas12b is removed, so that RNA substrates can be efficiently recognized and cleaved, and the problem of insufficient RNA substrate recognition efficiency in practical applications is solved. The reaction efficiency of Cas12 with the RNA substrates after the activation of trans-cleavage activity is improved, and the reporting efficiency of Cas12 using an RNA probe is improved.

The present invention relates to the modification of effector proteins (e.g., E3 ligase) to alter their activity (e.g., induced functional obtainment). The present invention relates to methods of modifying effector proteins by inserting peptide motifs or libraries of peptide motifs into effector proteins, libraries and methods of screening with modified effector proteins to identify new protein functions, i.e., new protein-protein interactions. For example, the present invention relates to a method of identifying a modified effector protein (e.g., an E3 ligase) having an induced change in substrate recognition activity, identified by measuring a change in substrate binding activity and / or degradation of a target protein. For example, the present invention enables the identification of new E3 ligases: Protein Of Interest (POI) interfaces, which can be used in drug discovery and development, such as monovalent degrading agent drugs such as molecular glue.