95 results about "Ribozyme" patented technology

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 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 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 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.

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.

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.

The invention discloses a method for site-specific modification of the cyclization efficiency of an anabaena ribozyme PIE carrier based on a cryoelectron microscope structure. The RNA (Ribonucleic Acid) of the Anabaena mutant comprises at least one of an RNA (Ribonucleic Acid) of an Anabaena intron G37 mutant and an RNA of an Anabaena PIE POLR2A mutant. The invention also provides an application of the mutant in research of increasing the yield of circRNA. The method has the beneficial effects that cryo-EM is utilized to analyze a tertiary structure of Anabaena ribozyme in a cyclization reaction process, a complete molecular structure mechanism for forming circRNA is researched through a cryoelectron microscope technology, the Anabaena mutant RNA with improved cyclization efficiency as well as the preparation method and application thereof are provided accordingly, the provided mutant RNA can increase the yield of circRNA by 1-2 times, and the yield of circRNA is increased by 1-2 times. The mutant is obviously superior to a wild type and other site mutants.

This invention discloses a bisphosphonate-nucleic acid nanoparticle, its preparation method, and its applications. The preparation method involves adding divalent metal ions to a rolling circle amplification (RoBA) reaction solution, followed by the addition of bisphosphonate, to perform a RoBA reaction to obtain the bisphosphonate-nucleic acid nanoparticles. The RoBA reaction solution contains a circular DNA template. The preparation method of this invention has the advantages of high speed, precise synthesis, and simplicity, realizing multiple reactions—RoBA, ion coordination, crystallization, and co-assembly of DNA and bisphosphonate—in the same reaction system, greatly simplifying the reaction process and improving reaction efficiency. The obtained bisphosphonate-nucleic acid nanoparticles exhibit dual-pathway therapeutic effects and excellent ribozyme stability, showing promising application prospects.

The invention discloses a paper-based sensor device based on a deoxyribozyme probe and application, and belongs to the technical field of analysis and detection. The paper-based sensor based on the deoxyribozyme probe by using the fluorescence signal is constructed, complex treatment on a sample is not needed, only a bacterial lysis buffer needs to be added into the sample for lysis, the deoxyribozyme is enabled to cut the deoxyribozyme probe by the generated escherichia coli CIM specificity, so that a sequence fragment containing an FAM group modified on the deoxyribozyme probe falls off, and the detection result is accurate. A complementary sequence modified at a detection line is used for capturing, a fluorescence signal is read to realize the detection purpose, and the whole reaction process can be completed within 30 minutes. The method integrates the processes of bacterial lysis, reaction sampling, bacterial detection and the like, avoids the complex and time-consuming operation process of a traditional method, has the analysis performance of rapidness, simplicity, rapidness, low cost and the like, and has potential application value in analysis and detection of various complex liquid bacteria in the clinical medicine field.

The present disclosure provides compositions and methods for inserting a heterologous payload sequence into a target site in the genome of a host cell. The compositions and methods use a non-LTR reverse transcriptase reverse transcriptase protein that binds to a template RNA comprising a payload sequence that encodes the protein or regulates the RNA. The template RNA may comprise a modified uridine that cannot be cleaved by a ribozyme. The incorporation of the modified uridine increases the integration and expression efficiency of the payload sequence and reduces cytotoxicity.

The invention relates to negative functional modulators of at least one variant of the non-erythrogenic erythropoietin (V-EPO) and pharmaceutical compositions or kits containing them. Such functional negative modulators of V-EPO may be a mono- or multi-specific antibody anti-EV3, anti-EV4, anti-EV1-4, anti-EV1-5, anti-EV1-1, or EV2-1, or anti-Epo receptor (EpoR) anti-EPHB4, anti-CSF2RB, an antisense oligonucleotide, DNA decoy, RNA decoy, a ribozyme, an antagomiR, a shRNA, LNA or siRNA.Several uses of these functional modulators are described, which have been advantageously employed as a medicament and for the treatment of an oncological pathology, a proliferative pathology, chronic inflammatory diseases on an autoimmune and non-autoimmune basis, of neurodegenerative diseases, and in the treatment of patients undergoing organ or tissue transplantation.The invention also describes variants of EPO for use in the diagnosis and in the treatment of an oncological pathology, a proliferative pathology, chronic inflammatory diseases on an autoimmune and non-autoimmune basis, of neurodegenerative diseases, and in the treatment of patients undergoing an organ or tissue transplantation and as a diagnostic agent.According to another aspect, a monoclonal antibody to at least one of the variants of the erythropoietin is described.According to yet another aspect, the use of at least one alternative splicing variant of non-erythrogenic EPO and the measurement thereof at tissue and / or systemic level is described, as well as the study of the methylation status of the promoters of the genes involved in the EPO signalling pathway (by way of example EPO, EPOR, EPHB4, CSF2RB), as diagnostic, prognostic and predictive markers of an oncological pathology, a proliferative pathology, neurodegenerative or inflammatory pathology.

The present invention provides compositions, systems and methods for expressing a protein of interest or a fusion protein using ribozyme mediated cis-cleavage and trans-cleavage or cis-ligation of RNA molecules.

The invention relates to the technical field of biology, in particular to a screening method of a high-throughput RNA delivery system based on a Barcode marker, and relates to a functional RNA molecule containing a specific barcode sequence, a delivery efficiency evaluation system and a detection method. Wherein the 5'end and the 3 'end of the functional RNA are respectively provided with ribozyme sequences and form a suture fragment which can be recognized by intracellular RtcB ligase through complementary pairing of partial bases, and quantitative analysis on RNA delivery efficiency and in-vivo distribution characteristics is realized by detecting a specific sequence containing a bar code after suture; the optimized screening method can specifically track the intracellular RNA delivery process of the delivery vector, the sensitivity and accuracy of in-vitro cell and in-vivo multi-organ targeted delivery vector detection are remarkably improved, and the high-throughput detection technology can accelerate the research and development process of the RNA delivery vector and has important application value in the fields of gene therapy and vaccine development.

The invention discloses a glyphosate fluorescence detection method based on RNase H cycle amplified magnetic bead-aptamer-Cas9 / deoxyribozyme, belongs to the technical field of environmental monitoring and biological detection, and can realize high-sensitivity and rapid quantitative detection of glyphosate residues in soil, water bodies and agricultural products. According to the method, the glyphosate is combined with an aptamer to release complementary DNA (cDNA) combined with the aptamer, then signal cascade amplification is realized through RNase H-mediated cDNA cyclic utilization, the anti-interference advantage brought by magnetic bead separation is combined, and the known characteristics of Cas9 endonuclease and DNAzyme in the aspects of nucleic acid recognition and cutting are used as signal amplification and transduction means, so that the detection sensitivity of the glyphosate is improved, and the detection sensitivity of the glyphosate is improved. Therefore, trace detection of glyphosate is realized. According to the invention, sensitive detection of other targets can be realized by replacing nucleic acid aptamers capable of specifically recognizing other targets and correspondingly adjusting the sequence of cDNA to maintain base pairing.

The invention belongs to the technical field of agricultural biological protection, and discloses a targeted screen tube-vector insect lysozyme-nano magnesium compound system and application thereof in prevention and control of areca yellows, the compound system comprises an in-plant treatment module and a propagation blocking module, the in-plant treatment module comprises a screen tube targeted delivery unit, and the propagation blocking module comprises a propagation blocking unit. The sieve tube targeted delivery unit is formed by combining betel nut sieve tube navigation peptide ArePEP1 and a lysozyme-nano magnesium complex through a non-covalent bond; the propagation blocking module comprises an antifeedant micro-capsule activated by salivary enzyme of a vector insect, and phytoplasma deoxyribozyme PdeA and an insect antifeedant are encapsulated in the antifeedant micro-capsule; the target sequence of the phytoplasma deoxyribozyme PdeA is 5 '-GGAAACAG-3'; the in-plant treatment module and the propagation blocking module are jointly loaded on the same delivery vehicle. According to the areca yellows prevention and control compound system and the application method, pathogens in plants can be synchronously inhibited, insect propagation can be blocked, and high-altitude accurate delivery can be realized.

The invention discloses a droplet microfluidic detection system for detecting pathogenic bacteria based on a deoxyribozyme probe, which belongs to the technical field of microbiological detection, and mainly comprises an automatic sample introduction device, a microfluidic droplet generation chip, a capillary tube-laser induced fluorescence detector and a signal acquisition and analysis device, the pathogen detection process comprises the following steps: respectively and automatically injecting an actual water sample, a deoxyribozyme probe, liquid drop oil, a pathogen lysis solution and a reaction buffer solution into the microfluidic liquid drop generation chip by the automatic sample injection device, stably generating liquid drops, and detecting the liquid drops through the capillary tube-laser induced fluorescence detector, and the signal acquisition and analysis device acquires and analyzes the fluorescence signal. The method has the advantages of high detection flux, high stability, high sensitivity, rapidness and the like, and can be applied to the field of environmental microbiological detection.

The application discloses a double-stranded nucleic acid detection composition based on lambda exonuclease and deoxyribozyme and application thereof, and belongs to the technical field of biological technology, and aims to efficiently and sensitively detect double-stranded nucleic acid. The double-stranded nucleic acid detection composition based on lambda exonuclease and deoxyribozyme comprises a double-stranded nucleic acid detection probe, lambda exonuclease and a reporter molecule, the double-stranded nucleic acid detection probe comprises one long single-stranded DNA named recognition chain R and n short single-stranded DNAs named Dz; the recognition chain R comprises a target binding domain and n Dz binding domains arranged in sequence from 5' to 3' in order; the 5' end of the recognition chain R is modified with a functional group; the Dz is a deoxyribozyme capable of cleaving the reporter molecule when the Dz is single-stranded, and the 5' end of the Dz is modified with sulfur. The application has high sensitivity and specificity, and the detection limit can be as low as a femtomole.

The invention discloses screening of a Cronobacter sakazakii aptamer and development of a functional nucleic acid double-recognition element biosensor. The screening comprises the following steps: (1) determining a sequence of the Cronobacter sakazakii aptamer; (2) optimizing reaction conditions of the cronobacter sakazakii biosensor; and (3) carrying out visual detection on the cronobacter sakazakii. The detection principle of the sensor is as follows: an aptamer is coupled to a carboxyl magnetic bead to serve as a capture element, enrichment of target bacterial cells is realized through magnetic separation, and a large amount of dissolved contents provide a target with a higher order of magnitude for ribozyme cutting by splitting the target bacterial cells. Short-chain nucleic acid generated after ribozyme cutting is used as a primer of RCA amplification reaction, generation of a product with a large number of G-quadruplex sequences can be triggered, and finally, TMB is catalyzed to generate chromogenic reaction by utilizing peroxidase-like activity of a G-quadruplex / hemin compound, so that visual output of signals can be realized, and ultra-sensitive detection of cronobacter sakazakii is completed.

This invention belongs to the medical field and provides a calcium carbonate manganese nanoprobe for in situ imaging detection of vascular epidermal growth factor, its preparation method, and its application. In this strategy, a DNA strand (P1 and P2) containing a protein recognition antibody, a protein recognition sequence, and a deoxyribonuclease, along with a molecular beacon DNA strand (H1), is stirred in a DMEM cell culture medium containing calcium chloride and manganese chloride to prepare the calcium carbonate manganese nanoprobe. In acidic tumor cells, this nanoprobe releases P1, P2, H1, and manganese ions. P1 and P2 can simultaneously recognize proteins, forming a complete deoxyribonuclease center, which catalyzes the cleavage of H1 under the action of the cofactor manganese ions, generating a fluorescent signal. This strategy achieves specific in situ dynamic imaging of low-abundance proteins in vivo and has great potential in biomedical research.

The invention discloses deoxyribozyme targeting enolase 1 and application of deoxyribozyme in preparation of a gene medicine for treating rheumatoid arthritis, and belongs to the technical field of biology. On the basis of a mouse ENO1 mRNA secondary structure, 15 deoxyribozyme is designed and screened, Dz8 has the strongest ENO1 mRNA cutting capacity, and the expression level of the gene can be effectively reduced; meanwhile, the Dz8 has Mg-dependent efficient ENO1 mRNA cleavage activity and time-dependent multi-turnover kinetic characteristics, ENO1 expression can be remarkably inhibited at the gene and protein levels, then inflammatory factor secretion is reduced, the glycolysis level is regulated and controlled, and the Dz8 has important application prospects in the field of rheumatoid arthritis gene therapy. The invention provides a new target and a treatment strategy for rheumatoid arthritis gene therapy, and Dz8 is expected to become a potential tool for rheumatoid arthritis gene therapy.

This invention provides a Csm6-based deoxyribonuclease sensor, characterized by comprising the following components: (1) a deoxyribonuclease enzyme chain and a deoxyribonuclease substrate chain, wherein the nucleotide sequence of the deoxyribonuclease substrate chain is shown in SEQ ID No. 1 or SEQ ID No. 2 of the sequence listing, and the nucleotide sequence of the deoxyribonuclease enzyme chain is shown in SEQ ID No. 10 of the sequence listing; (2) a Csm6 protease; and (3) a single-stranded RNA reporter probe modified with fluorescent and quenching groups, wherein the sequence of the single-stranded RNA reporter probe modified with fluorescent and quenching groups is shown in SEQ ID No. 16 of the sequence listing. This invention also provides an application of this deoxyribonuclease sensor for lead detection. The deoxyribonuclease sensor provided by this invention can improve the sensitivity and selectivity of existing biosensors in lead ion detection.

The invention belongs to the technical field of RNA stability detection, and particularly relates to a method for detecting tRNA structure stability based on ribozyme. The invention discovers that when a non-modified site of the tRNA is taken as a cutting target, the tRNA with a more compact space structure can hinder pairing of VMC10A20p to the tRNA and hinder cutting of the VMC10A20p to the tRNA target. By comparing cleavage product levels of tRNA targets of different biological samples, the relative stability of the space structure of the tRNA target is detected. Therefore, the invention provides a method for detecting the tRNA structure, upstream and downstream arms of VMC10A20p are complementarily paired with upstream and downstream of a non-modified site of to-be-detected tRNA, a cleavage sequence is designed for the to-be-detected tRNA site, the to-be-detected tRNA is cleaved, and the structural stability of the tRNA is judged based on a cleavage result.