160 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 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 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 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.

The technology described herein is directed to Aptamer and Ribozyme Equilibrium Shifting (ARES) regions, including ON-switches and OFF-switches, which can be harnessed to regulate the stability of RNA molecules. Also described herein are compositions comprising such RNA molecules and methods of using them to regulate translation of cargo polypeptides.

The present invention relates to a recombinant nucleic acid molecule for preparing a circular RNA comprising, from the 5'end to the 3 'end: a) an internal guide sequence (IGS), b) a ribozyme, c) a first portion of an extended anticodon arm (eACA) sequence, d) a gene of interest, and e) a second portion of the eACA sequence. One nucleotide in the second part of the eACA sequence and one nucleotide in the IGS form a swinging base pair. Recombinant nucleic acid molecules, circular RNAs, and methods of making and using the circular RNAs are provided.

The present invention discloses a capillary monolithic column modified with a deoxyribozyme nanoflower, a preparation method thereof, and an application thereof in separating chiral molecules, belonging to the field of pharmaceutical analysis. The present invention synthesizes a deoxyribozyme-based nanoflower chiral selector, the nucleotide sequence of the single-stranded DNA template of the deoxyribozyme being shown in SEQ ID NO.1, and the synthesized deoxyribozyme nanoflower having good pH and temperature stability. The prepared deoxyribozyme nanoflower is bonded to a graphene oxide-functionalized capillary silica monolithic column, and chiral molecules are separated under electrophoretic conditions, achieving baseline separation of 2'-deoxyadenosine, atenolol, propranolol, tyrosine, and nafopam enantiomers.

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.

Provided are methods and compositions for screening / identifying / characterizing nucleic acid delivery vehicles (e.g., liquid nanoparticle (LNP) formulations). Provided is a library of nucleic acid delivery vehicles with different chemical compositions, where the members of the library are barcoded by virtue of including a barcoded RNA molecule (in some cases a barcoded ligation-ready RNA molecule). The barcoded RNA molecule includes a first ribozyme, a second ribozyme, and a barcode sequence between the first and second ribozymes. Once a barcoded RNA molecule is successfully delivered into a cell, the first and second ribozymes cleave the barcoded RNA molecule to produce a 5'-OH end and a 2',3'-cyclic phosphate end, which ends can be ligated to one another by an intracellular RNA ligase, producing a circular RNA (cRNA) that includes the barcode sequence. Detecting barcode sequences from cRNAs therefore identifies the chemical composition of successful delivery vehicles.

This invention relates to recombinant nucleic acid molecules and their use in producing circular RNA. The recombinant nucleic acid molecules comprise in the 5' to 3' direction: a) an internal guide sequence (IGS), b) a ribozyme, c) a first portion of an extended anticodon arm (eACA) sequence, d) a gene of interest, and e) a second portion of the eACA sequence. The 3' nucleotide in the second portion of the eACA sequence forms a canonical base pair with a nucleotide in the IGS or does not form a base pair with a nucleotide in the IGS. Recombinant nucleic acid molecules, methods for producing circular RNA and circular RNA thus produced are provided.

The application discloses a kind of photoactivation manganese ion bridging type deoxyribozyme probe preparation and its application in Cistanche inhibiting acetylcholinesterase activity.The probe is pSD@MnO2 Composite structure, which is composed of manganese dioxide nanosheet and functional nucleic acid assembly adsorbed on its surface, the assembly includes S chain labeled with fluorescence / quenching group and containing deoxyribozyme cleavage site, and pD chain partially hybridized with S chain and containing photocleavage site.Its preparation includes nucleic acid probe assembly, MnO2 Nanosheet complexing and purification steps.When applied, AChE catalytic product decomposes MnO2, releases Mn 2+ And nucleic acid chain; after centrifugation, pD chain is cut by ultraviolet light activation trigger, and released DNAzyme is cut under the assistance of Mn 2+ It produces amplified fluorescence signal; by comparing signal change before and after adding inhibitor, inhibition activity can be quantitatively evaluated.The application combines photocontrol activation with enzyme cascade amplification, realizes high sensitivity and operation synchronism of detection process, and is especially suitable for high-throughput rapid screening of AChE inhibitor in Cistanche and other natural products.

To provide compositions and methods for the treatment of leukodystrophy.SOLUTION: Provided is use of administering an effective amount of a compound that down-modulates the expression of both wild-type and mutant TUBB4-A in the manufacture of a pharmaceutical composition for the treatment or prevention of hypomyelination and atrophy of the basal ganglia (H-ABC) leukodystrophy, the pharmaceutical composition ameliorating symptoms of H-ABC, and the compound being selected from short hairpin RNA (shRNA), short interfering RNA (siRNA), antisense RNA, antisense DNA, chimeric antisense DNA / RNA, microRNA, and a ribozyme that is sufficiently complementary to either the gene or mRNA encoding TUBB4A.SELECTED DRAWING: Figure 1-1

Polynucleotides, vectors, systems of vectors or polynucleotides, cells, and methods for expressing AAV Rep proteins are provided. In certain aspects, among the provided embodiments are polynucleotides, vectors, and systems of vectors or polynucleotides, and cells including the same, that include a polynucleotide that includes a ribozyme. In certain aspects, these polynucleotides, vectors, systems of vectors or polynucleotides, cells, and methods may be used to produce higher levels of small Rep transcripts as compared to large Rep transcripts. In certain aspects, these polynucleotides, vector systems, cells, and methods may be used to produce higher levels of small Rep proteins as compared to large Rep proteins. In certain aspects, these polynucleotides, vectors, vector systems, cells, and methods may be used to produce recombinant AAV (rAAV). Increased expression of small Rep as compared to large Rep is useful in many aspects, such as, increasing total virions and increasing packaged virions during the production of rAAV.