209 results about "Ribozyme" patented technology

According to the invention, a structural domain reaction substrate sequence consisting of Exon1 (E1), P1 and Exon2 (E2) sequences of ribozyme is mutated, and E1, P1 and E2 sequences are mutated under the condition of maintaining the structural stability, so that the Azoarcus group I intron ribozyme still has enzyme activity and can maintain the capability of forming circular RNA (Ribose Nucleic Acid). The invention discloses a flexible vector construction method for preparing circular RNA (Ribonucleic Acid) without limitation of a substrate sequence, which comprises the following steps: determining a target to-be-cyclized site sequence NNUNNNN, and segmenting the target to-be-cyclized site sequence NNUNNNN into E1: NNU and E2: NNNN; with E1 and E2 sequences as references, designing IGS sequences to respectively form complementary pairing with E1 and E2; and 5'and 3 'homologous arms, IRES, CDS and other elements are respectively added. The method provided by the invention can be used for preparing the circular RNA for any target sequence, has no residual sequence, and has relatively high cyclization efficiency. The FlexCirc cyclization system designed on the basis of Azoarcus group I intron ribozyme can form the circular RNA, the cyclization substrate sequence has the characteristic of flexible design, and the cyclization efficiency can realize a relatively high cyclization proportion.

The invention discloses a deoxyribozyme probe for identifying carbapenem-resistant klebsiella pneumoniae and application of the deoxyribozyme probe, and belongs to the technical field of deoxyribozyme probes. The method comprises the following steps: firstly, mixing a phosphorylated library chain, a substrate chain and a connecting chain, then, carrying out covalent linkage to construct a DNA library, firstly, carrying out reverse screening, separating and purifying a DNA band of an uncut substrate RNA site, constructing a chain-shaped DNA library by using the obtained DNA band, carrying out forward screening, separating and purifying the DNA band of the cut substrate RNA site, carrying out PCR (Polymerase Chain Reaction) amplification, and recovering a positive-sense chain, so as to obtain the DNA library. The deoxyribozyme probe is obtained by performing repeated screening and enriching, and the probe can recognize carbapenem-resistant klebsiella pneumoniae with high specificity and has the potential of exerting advantages in the aspect of biosensing application.

The invention discloses vibrio parahaemolyticus deoxyribozyme screening and label-free biosensor development. The vibrio parahaemolyticus deoxyribozyme screening and label-free biosensor development comprises the following steps: (1) obtaining a vibrio parahaemolyticus deoxyribozyme sequence; (2) optimizing reaction conditions of the vibrio parahaemolyticus label-free biosensor; and (3) detecting the vibrio parahaemolyticus. The detection principle of the sensor is as follows: when vibrio parahaemolyticus exists, deoxyribozyme is triggered to be self-cleaved, a generated cleaved short chain can be used as a primer for starting rolling circle amplification (RCA), a large number of long-chain DNA products with repeated series G-quadruplex sequences are generated by amplifying a designed RCA annular template, G-quadruplex and ThT can form a G-quadruplex / ThT compound, and the long-chain DNA products can be used for detecting the vibrio parahaemolyticus. The fluorescence is generated under the excitation wavelength of 439 nm, and the higher the concentration of the vibrio parahaemolyticus is, the higher the fluorescence intensity of the system is, so that the simple, convenient, rapid, low-cost and high-sensitivity detection of the vibrio parahaemolyticus is realized.

The present invention relates to a MeCP2 gene-specific trans-splicing ribozyme, and a use thereof. The trans-splicing ribozyme according to the present invention replaces the RNA of a mutant MeCP2 gene, which causes Rett syndrome, with a wild type MeCP2 RNA, so as to reduce the expression of the mutant MeCP2 protein and increase the expression of the wild type MeCP2 protein, and enables regulated expression so that wild type MeCP2 expression can be prevented from exceeding physiological levels, and thus can be effectively used for preventing or treating Rett syndrome.

The invention provides a preparation method of dendritic mesoporous silicon bionic nanoparticles loaded with RNA (Ribonucleic Acid) molecules, which comprises the following steps: preparing dendritic mesoporous silicon nanoparticles DMSNs by adopting a double-phase layering method, dissolving a surfactant CTAC and a catalyst TEA in water to serve as a water phase solution, dissolving ethyl silicate TEOS in cyclohexane to serve as an oil phase, slowly dropwise adding the oil phase into the water phase, and reacting for 2-4 hours to obtain the dendritic mesoporous silicon bionic nanoparticles loaded with the RNA molecules. The DMSNs nanoparticles are obtained; amino groups are introduced to the surfaces of DMSNs through gamma-aminopropyltriethoxysilane APTES so as to enhance electrostatic attraction to nucleic acid, and after a surfactant template is removed, the DMSNs are mixed with RNA molecules in ribozyme-free water; meanwhile, a cytoplasmic membrane is harvested by using a hypotonic method, and the dendritic mesoporous silicon bionic nanoparticles loaded with the RNA molecules are prepared through ultrasonic treatment and polycarbonate film extrusion. The method is simple and convenient, and has a wide application prospect in RNA delivery.

The invention belongs to the field of biological materials, and particularly relates to a multifunctional DNA nanometer material, a preparation method and application of the multifunctional DNA nanometer material in treatment of traumatic brain injury secondary encephaledema. The multifunctional DNA nano material comprises a plurality of deoxyribozyme (DNAzyme) targeting C1q mRNA (messenger Ribonucleic Acid) and a repeating unit which is integrally connected with G-quadruplex / Hemin in series, wherein the G-quadruplex / Hemin is labeled by streptavidin SA and biotin and is used for scavenging free radicals. The DNAzyme for silencing the C1q gene of the mouse is designed on the basis of a bioinformatics method, and the C1q gene overexpressed in an inflammation model can be knocked down to an approximate normal level. The RCA technology mediated self-assembled nano material SA-B / H has excellent enzyme mimetic activity, can effectively remove free radicals such as H2O2 and O2 <.->, and has the potential of in-vivo oxidation resistance. In-vitro experiments prove that SA-B / H can effectively relieve high-level oxidative stress of cells caused by H2O2 stimulation.

The invention discloses a universal method for preparing circular RNA (Ribonucleic Acid) and application of the circular RNA. Specifically, an optimized ribozyme recognition site is determined based on a specific local secondary structure, and a ribozyme sequence is designed correspondingly, so that the circular RNA precursor molecule is constructed, and the invention provides a method for preparing the circular RNA based on the precursor molecule and application of the circular RNA prepared by the method. The cyclic RNA is high in cyclization efficiency, any sequence except the target cyclic RNA sequence is not introduced into the cyclization product, the cyclization efficiency is not influenced by different sequences of the cyclized RNA, and the method is a universal method for preparing the cyclic RNA. The circular RNA prepared by the method can be used in various biological medicine fields such as diagnosis, prevention and treatment of human or animal diseases.

The present disclosure provides compositions, systems, and methods for promoting trans-splicing. In some examples, provided herein is a composition comprising or encoding a trans-splicing nucleic acid molecule comprising a 3' domain disclosed herein. In some embodiments, the 3' domain comprises one or more of the following domains: a nuclear retention domain, a stabilization domain, and a cleavage domain. In some embodiments, the 3' domain comprises a sequence or structure that functions as any one, any two, or all three of a nuclear retention domain, a stabilization domain, and a cleavage domain. In some embodiments, the 3' domain comprises: (i) a sequence or structure derived or isolated from a ribozyme (e.g., Twister or Twister Sister), and (ii) optionally a structured sequence or domain (e.g., a G-quadruplex, a pseudoknot, and / or a triplex), in the 3' to 5' direction.

The invention relates to compositions and methods for RNA editing. In particular, ribozymes can be utilized to enhance RNA trans-splicing.

This invention relates to methods for producing a circular gene of interest. A nucleic acid molecule is provided that comprises, in the 5' to 3' direction, a first bridge sequence, and a gene of interest. The first bridge sequence comprises a sequence corresponding to a 3' portion of a ribozyme. Also provided is a modified ribozyme comprising a truncated ribozyme that does not comprise a 3' portion of a corresponding wild-type ribozyme. The nucleic acid molecule and the modified ribozyme are combined under conditions suitable for circularization to occur. Modified ribozymes, nucleic acid molecules and kits are also provided.

The invention discloses an efficient gene editing system for streptomyces as well as a construction method and application of the efficient gene editing system. The system comprises a TnpB nuclease which can be expressed in streptomyces and a guide RNA, wherein the guide RNA comprises an RNA skeleton, a gene targeting section and a gene sequence of hepatitis delta virus (HDV) ribozyme. Wherein the gene targeting segment is located at the 3'end of the RNA skeleton and is a nucleic acid fragment with the length of 12-40bp after a TAM sequence (5 'TTGAT) on a target gene; the hepatitis D virus (HDV) ribozyme is used for stabilizing the RNA skeleton-gene targeting section structure. According to the streptomyces mini-gene editing tool provided by the invention, in the presence of a homologous repair template, the gene editing efficiency can reach 70-100% in streptomyces, and the gene editing efficiency under the action of the guide RNA after rational design can reach 100%.

The invention provides a pathogen viable bacteria detection kit based on endogenous protein activated deoxyribozyme. The kit comprises a deoxyribozyme recognition element and a signal transduction element. The deoxyribozyme recognition element is composed of a substrate chain and an enzyme chain, and the signal transduction element is composed of two DNA reporter probes (Sub-F and Sub-Q), wherein the tail ends of the two DNA reporter probes are respectively marked with a fluorescence staining group and a fluorescence quenching group. The deoxyribozyme recognition element can recognize specific endogenous protein of target pathogenic viable bacteria and activate an enzyme chain to cut a substrate chain to generate two independent substrate chain fragments. And then a signal transduction element is introduced to realize specific output of a fluorescence signal. The kit provided by the invention can realize detection of living salmonella by targeting endogenous protein, can sensitively detect 190 CFU / mL of living salmonella with abundance as low as 0.1%, and has a good application prospect in the directions of food safety detection and clinical examination.

The invention relates to a multiple miRNA (micro Ribonucleic Acid) detection method based on deoxyribozyme or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / The detection method provided by the invention comprises the following steps: designing a probe 1 and a probe 2 matched with different targets according to the different targets; when a target exists, the probe 1 and the probe 2 are connected to miRNA through T4DNA liguase, then DNA polymerase is introduced for amplification of a DNA chain, the released miRNA is further hybridized with a new probe, the next round of reaction is activated, and signals are circularly amplified. The S1 duplex body generated after amplification can be subjected to multiple characterization through two design modes: the first mode is based on deoxyribozyme detection; and the second mode is to carry out detection based on CRISPR / Cas12a. The method provided by the invention can realize detection of multiple tumor miRNAs, is short in detection time, and provides an important theoretical basis for treatment of tumor diseases.

The invention relates to compositions and methods for RNA editing. In particular, ribozymes can be utilized to enhance RNA trans-splicing.

The present disclosure provides compositions and methods for inserting heterologous payload sequences into a target-site in a host cell genome. The compositions and methods use non-LTR retrotransposon reverse transcriptase proteins that bind template RNAs comprising a payload sequence that encodes a protein or regulatory RNA. The template RNA can comprise modified uridines that are not cleavable by a ribozyme. The incorporation of modified uridines increases the efficiency of integration and expression of the payload sequence and decreases cellular toxicity.

The invention discloses a small molecule compound for regulating and controlling the catalytic activity of tetrahymena ribozyme and a screening method and application thereof, based on FRET (Fluorescence Resonance Energy Transfer) high-throughput entity screening, virtual screening, a biological membrane interference technology, a freezing electron microscope structure analysis technology and other technologies are combined to form a set of complete and efficient RNA (Ribonucleic Acid) targeted small molecule compound extraction method; the tetrahymena ribozyme is specifically subjected to small molecule compound screening; aiming at research and regulation of tetrahymena ribozyme; meanwhile, the invention aims to develop a high-throughput and systematic small-molecule compound screening method which is used for rapidly and efficiently screening potential small-molecule inhibitors so as to accelerate the discovery and research and development process of new drugs.

The invention discloses allosteric DNA ribozyme and application thereof. The nucleotide sequence of the allosteric DNA ribozyme is selected from SEQ ID NO.01 to SEQ ID NO.10. The invention further discloses a preparation method of the allosteric DNA ribozyme The allosteric DNA ribozyme provided by the invention adopts a DNA structure, and compared with RNA ribozyme, the allosteric DNA ribozyme has higher chemical stability, is not easily degraded by nuclease, can complete theophylline detection within 60 minutes, does not need complex pretreatment or expensive instruments, is simple and convenient to operate, is suitable for bedside detection and household monitoring scenes, and greatly improves the detection efficiency.

The present invention relates to compositions and methods for activating expression from the paternally-inherited allele of UBE3A in human Angelman's Syndrome neurons using viral vector delivery of short hairpin RNAs, ribozymes, and / or microRNAs.

The present invention relates to a method for producing a circular gene of interest. A nucleic acid molecule comprising in the 5' to 3' direction a first bridging sequence and a gene of interest is provided. The first bridging sequence comprises a sequence corresponding to the 3' part of a ribozyme. A modified ribozyme comprising a truncated ribozyme which does not comprise the 3' part of the corresponding wild type ribozyme is also provided. The nucleic acid molecule and the modified ribozyme are combined under conditions suitable for circularization to occur. The invention also provides a modified ribozyme, a nucleic acid molecule and a kit.

The application provides a plasmid containing modified satellite RNA and a construction method and application, and belongs to the field of plant virology and molecular biology. Each copy number of the modified satellite RNA is composed of a modified satRNA TA-Tb fragment and an HDV ribozyme fragment, and the copy number of the modified satellite RNA in the plasmid is 10. The nucleotide sequence of the modified satellite RNA is shown in SEQ No. 3. After the plasmid of the application is inoculated by agrobacterium injection, the symptoms caused by cucumber mosaic virus on different strains and the same strain (CMV Fny ) on different hosts can be significantly reduced. An effective control measure for field control of cucumber mosaic virus is provided.

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly and multiply detecting breast cancer related protein markers based on cas12a and application of the method. HER2, CEA, CA15-3 and CD63 are taken as protein markers, are fixed on magnetic beads through biotin and streptavidin and are used for recognizing and capturing proteins, meanwhile, protein detection is converted into deoxyribozyme recognition of a cyclic sequence, and signal amplification is performed through cas12a; the method provided by the invention can be used for detecting four breast cancer protein markers in a system in a short time, is simple to operate, does not need to heat up, has high accuracy and specificity, accelerates the progress of breast cancer diagnosis and postoperative and prognostic diagnosis, and provides reference for subsequent treatment of clinicians.

The invention provides an RNA (Automatous Circulating RNA) capable of being self-cyclized in a cell and a precursor RNA (pre-RNA) thereof, and the precursor RNA (pre-RNA) sequentially comprises a ribozyme splicing element designed on the basis of human tRNA at the 5'end, a ribozyme splicing element designed on the basis of human tRNA at the 3 'end, a ribozyme splicing element designed on the basis of human tRNA at the 5' end, and a ribozyme splicing element designed on the basis of human tRNA at the 3 'end from the 5' direction to the 3 'direction. An RNA sequence having biological activity; the self-cyclized RNA (acRNA) comprises a ribozyme splicing element designed on the basis of human tRNA at the 5'end and a ribozyme splicing element designed on the basis of human tRNA at the 3 'end in sequence from the 5'to the 3' direction: a human tRNA sequence with hydroxyl formed at the 5 'end; an RNA sequence having biological activity; a human tRNA sequence of 2 ', 3'-cyclic phosphate is formed at the 3'end; the invention also provides a nucleic acid vector for preparing the precursor RNA and a method for generating the circular RNA.

The present disclosure is directed to a split ribozyme biosensor system. Additionally, a genetically modified plant, plant tissue, or plant cell comprising the split ribozyme biosensor system is described. Methods for examining in vivo RNA expression in plants, plant cells or plant tissues are disclosed. Lastly, disclosed herein is a kit comprising the split ribozyme biosensor system.

The invention relates to a nucleic acid molecule complex for pseudouridylation of a target RNA in a mammalian cell comprising a guide RNA (gRNA) that at one end is flanked by a first twister ribozyme and at its other end by a second twister ribozyme to generate a circular gRNA, wherein the nucleic acid molecule complex has the capability to generate circular gRNA which is characterized by a secondary structure comprising two pseudouridylation pockets that harbor antisense guide sequences. More specifically, the first twister ribozyme is a P3 twister ribozyme and the second twister ribozyme is a P1 twister ribozyme, wherein each twister ribozyme is separated from the gRNA by a linker. In a further aspect, the invention relates to a method for generating circular gRNA. The invention furthermore relates to a nucleic acid molecule complex as defined herein for use in the treatment of a genetic disease caused by a premature stop codon. It furthermore relates to a pharmaceutical composition, and a delivery vector to express the construct in mammalian cells to generate circular gRNA.

The invention relates to ribozyme for labeling biotin on target RNA (Ribonucleic Acid) as well as a screening method and application thereof. The RNA labeling technology is one of core tools for modern molecular biology research and clinical diagnosis. A specific marker is covalently linked to a target RNA molecule, so that accurate tracking, efficient detection and functional analysis of RNA can be realized. A biotin-streptavidin system becomes one of the technical routes which are most widely applied due to extremely high affinity and signal amplification capability of the biotin-streptavidin system. At present, common biotin-labeled RNA technical methods mainly comprise a chemical solid-phase synthesis method, an enzymatic method and a chemical labeling method. However, the methods have the limitations of poor site specificity, low efficiency, complex manipulation and the like in the use process. The engineering ribozyme for catalyzing RNA biotinylation is obtained by taking 12: 0 biotin coenzyme A as a biotin donor through an in-vitro screening technology. The ribozyme provided by the invention can efficiently and specifically carry out biotin labeling on target RNA, the reaction process is simple, and the labeling activity is up to 90% or above.

The present disclosure relates to a recombinant nucleic acid molecule and an application thereof in preparation of a circular RNA, and in particular, to a recombinant nucleic acid molecule for preparing a circular RNA, a recombinant expression vector, a circular RNA, a composition, a method for preparing a circular RNA, a method for expressing a target polypeptide in a cell, a method for screening a target coding region sequence, a system for screening a target coding region sequence, and a method for screening a ribozyme recognition site sequence. The recombinant nucleic acid molecule provided by the present disclosure provides a Clean PIE system with a novel structure for preparing a circRNA in vitro, which can avoid introducing additional exon sequences into the circular RNA, improve the sequence accuracy of circular RNA molecules, reduce changes in a secondary structure of the circular RNA, and further reduce the immunogenicity of the circular RNA, and has a good application prospects in the fields of nucleic acid vaccines, expression of therapeutic proteins, gene therapy, and the like.

The invention provides a method for producing circular RNA (Ribonucleic Acid) by utilizing cyclic substitution (CP) II type intron ribozyme and application of the circular RNA. Specifically, the inventor provides a recombinant polynucleotide for preparing the circular RNA, and a precursor for generating the circular RNA is constructed by using a natural or modified CP II intron and can be used for cyclization of a target RNA sequence. According to the annular RNA precursor constructed by the CP II intron, the catalysis process does not depend on protein participation, GTP and high temperature conditions are not needed, the generated annular RNA sequence is accurate, no extra sequence exists, and the cyclization efficiency is high. The polynucleotide precursor constructed on the basis of the natural or modified CP II intron has an application prospect in the field of circular RNA production.

A toehold exchange riboregulator gate includes: a gate input toehold domain; a ribosome binding sequence; a start codon; a c spacer domain positioned between the ribosome binding sequence and the start codon; a protein coding sequence; and a self-cleaving ribozyme sequence that produces a double-stranded RNA gate suitable for strand exchange and produces the gate input toehold domain.

The invention belongs to the technical field of biological detection, and particularly relates to an escherichia coli non-amplification type rapid detection method based on a deoxyribozyme probe. The method comprises the following steps: detecting Escherichia coli by adopting a fluorescent chip, mixing Escherichia coli lysate and RFD-EC recognition molecules for reaction, dripping into the fluorescent chip for reaction, and detecting the fluorescence intensity after the reaction; the fluorescent chip takes paper as a substrate and is coated with d-DNA, the nucleotide sequence of the d-DNA is shown as SEQ ID NO.3, and the 5'end of the d-DNA is marked with biotin. Fluorescent DNA molecules are enriched through a fluorescent chip micro-fluidic technology, and detection can be completed within 0.5-1 hour without enrichment culture in combination with multi-channel fluorescence detection. The detection lower limit of the method is as low as 100 CFU, and the method has the advantages of rapidness, sensitivity and low cost, is suitable for on-site rapid detection of Escherichia coli in clinical samples and environmental samples, and has important application value in the fields of pollution control and the like.