35 results about "Riboswitch" patented technology

The present invention provides constructs comprising modified riboswitches to regulate expression of a transgene within a subject. Methods of treating a disease, specifically an eye disease, are also contemplated.

The present disclosure provides methods for genetically modifying lymphocytes and methods for performing adoptive cellular therapy that include transducing T cells and / or NK cells. The methods can include inhibitory RNA molecule(s) and / or engineered signaling polypeptides that can include a lymphoproliferative element, and / or a chimeric antigen receptor (CAR), for example a microenvironment restricted biologic CAR (MRB-CAR). Additional elements of such engineered signaling polypeptides are provided herein, such as those that drive proliferation and regulatory elements therefor, as well as replication incompetent recombinant retroviral particles and packaging cell lines and methods of making the same. Numerous elements and methods for regulating transduced and / or genetically modified T cells and / or NK cells are provided, such as, for example, those including riboswitches, MRB-CARS, recognition domains, and / or pH-modulating agents.

Naturally occurring RNA pseudoknots fold into many topologies, yet their formation is poorly understood. Herein, by using high-resolution single-molecule force spectroscopy, the folding pathways of the H-type pseudoknot found in the preQ1-riboswitch in B. subtilis were investigated By holding a single riboswitch RNA molecule in the optical-trap, the structural rearrangements as the end-to-end distance change along the pulling direction, x at a force, F were followed. The data reveal a multistate folding, wherein the intermediate hairpin undergoes a unidirectional conformational switching in the presence of ligand to form the pseudoknot receptor. Specifically-designed mutant RNAs resisted the switching mechanism and resulted in a significantly reduced pseudoknot population (4.5%) compared to the wild-type (100%). The free-energy landscape highlighted two kinetic barriers (ΔG‡) that interrupt the folding pathway. By coupling the exothermic ligand-binding reaction (ΔGbinding=−16 kT) to the folding events, the nascent transcript ensures successful barrier crossing, thus favoring the pseudoknot conformation.

The present disclosure relates to oligonucleotide aptamers that bind to certain small molecules including thiamine analogs and derivatives and methods of generating aptamers that bind to the small molecules. Also contemplated are riboswitches and polynucleotide cassettes comprising the aptamers disclosed herein. Further provided are methods of using said aptamers, riboswitches, and / or polynucleotide for the regulation of target genes, including therapeutic genes. Also provided herein are small molecules that are modulators of target gene expression where the target gene contains a riboswitch comprising an aptamer described herein.

The present invention provides methods and materials that can be used to simply and rapidly determine the three-dimensional structures of RNA hairpin loops and their complexes with inhibitors. The scaffold RNA, a YdaO-type c-di-AMP riboswitch from Thermoanaerobacter pseudethanolicus, readily forms crystals with large cavities having a diameter exceeding. The hairpin of interest can be engineered into the P2 stem of this RNA such that the hairpin is accommodated within the cavity. The fused RNA is then crystallized and the structure can be determined using X-ray or electron crystallography. Embodiments of the present invention can be used to identify compounds that bind to the hairpin loop so as to elicit, for example, therapeutic and other biological activities.

The present invention discloses a method for screening a riboswitch that specifically recognizes doxycycline, as well as its rational modification and application, relating to the field of gene expression regulation. The invention provides a doxycycline-specific riboswitch and establishes a high-throughput screening method based on flow cytometry. Computer-assisted prediction of riboswitch binding sites and calculation of mutation sites are used to obtain a doxycycline riboswitch with a further enhanced activation factor. Dose-response curves show that the riboswitch exhibits a linear correlation at 40-100 μg / L. The constructed whole-cell sensor is low-cost, highly stable, and simple to operate, making it suitable for rapid on-site detection.

Disclosed is an aptamer capable of binding to luteolin and other naringenin derived flavonoids, wherein the aptamer is: an RNA aptamer having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2; or a DNA aptamer having a nucleotide sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4; wherein for SEQ ID NO: 1 and 2, N is one nucleotide selected from the group consisting of A, G, C and U, and wherein for SEQ ID NO: 3 and 4, N is one nucleotide selected from the group consisting of A, G, C and T. Also disclosed is a riboswitch and a DNA expression cassette comprising the aptamer, a composition thereof, and a microbe containing the riboswitch or DNA expression cassette. Further disclosed is a method of detecting luteolin and other naringenin derived flavonoids produced in a microbe using the aptamer or riboswitch as disclosed herein.

Disclosed are a method for screening a riboswitch capable of specifically recognizing doxycycline as well as rational engineering and application of the riboswitch, and belongs to the field of gene expression regulation. The present disclosure provides a doxycycline specific riboswitch, and establishes a high-throughput screening method based on flow cytometry, and obtains the doxycycline riboswitch with further improved activation fold by way of computer-aided prediction of binding sites of the riboswitch and calculation of mutation sites. According to a dose-response curve, the riboswitch shows linear correlation at 40-100 μg / L, and meanwhile, a whole-cell sensor constructed is low in price, high in stability and simple to operate, and can be used for on-site rapid detection.

Provided herein are membrane encapsulated cell-free systems comprising transcriptionally regulated biosensors for detecting analytes, and methods of their use. In embodiments, the biosensor is a riboswitch for detection of fluoride in a sample.

The invention provides a method for screening an aptamer, the screening of the aptamer is realized on the basis of a random nucleic acid library formed by 16 random basic groups and fixed sequences at intervals, and Capture-SELEX one-round screening of the aptamer is combined with high-throughput sequencing. Wherein the sequence of the random nucleic acid library for screening is as shown in SEQ ID NO: 1; the sequence of the etomidate aptamer screened by the invention is as shown in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; the etomidate accounting aptamer disclosed by the invention can be applied to identification, analysis and detection of etomidate, and an aptamer biosensor is constructed by responding to the expression level of etomidate regulation downstream genes in the riboswitch designed by the sequence.

The invention discloses a riboflavin biosensor construction method based on an RNA element, and belongs to the field of synthetic biology. On the basis of an FMN riboswitch and a foothold switch, an expression platform of a wild type FMN riboswitch is artificially modified, a sensitive riboswitch biosensor is constructed, and efficient detection of the riboflavin concentration is achieved; the fluorescence value of a riboswitch of the detection strain constructed by the method under the riboflavin concentration of 1 mmol / L is 4.3 times of the fluorescence value under the riboflavin concentration of 0 mmol / L.

The invention discloses the preparation and application of a 5-aminolevulinic acid biosensor, relates to the technical field of biosensors, and comprises the following steps: step one: design and synthesize an RNA aptamer sequence with high affinity and specificity for 5-ALA; step two: combine the RNA aptamer sequence with the expression regulation region of a fluorescent reporter gene to construct a riboswitch structure; step three: clone the riboswitch structure into an expression vector, transform Escherichia coli or yeast; step four: screen and optimize the engineering strains for efficient expression; step five: immobilize the engineering strains on a biocompatible material to prepare a biosensor chip. In the present invention, an RNA aptamer capable of specifically recognizing 5-ALA is designed and combined with the expression regulation of a fluorescent reporter gene to achieve rapid and sensitive detection of 5-ALA.

The present disclosure provides an expression cassette comprising from 5' to 3': (i) promoter, (ii) an upstream exon, (iii) an upstream intron, (iv) an alternatively spliced exon comprising a translation initiation sequence, (v) a downstream intron, (vi) a downstream exon, and (vii) a heterologous nucleic acid sequence. In some aspects, the upstream exon, the upstream intron, the alternatively spliced exon, the downstream intron, and the downstream exon, each comprise a portion of a gene selected from the group consisting of a Transient Receptor Potential Cation Channel Subfamily V Member 3 (TRPV3) gene, a Serine / Threonine Kinase 31 (STK31) gene, an Ubiquitin Specific Peptidase 25 (USP25) gene, a IK gene, a Testis Expressed 14, Intercellular Bridge Forming Factor (TEX14) gene, a General Transcription Factor IIB (GTF2B) gene, and a LY6 / PLAUR Domain Containing 3 (LYPD3) gene.

The present disclosure relates to riboswitches and polynucleotide cassettes that modulate the expression of chimeric antigen receptors (CARs) in response to small molecule ligands, wherein the polynucleotide cassettes comprise the riboswitches and aptamers disclosed herein. Also provided are methods for generating a population of T cells wherein the T cells comprise a CAR transgene comprising an expression construct that inductively expresses a CAR in response to a small molecule inducer that binds to a riboswitch aptamer, which is part of the expression construct. Also provided are methods for treating cancer by administering to a patient in need thereof: (i) a population of T cells comprising an induced CAR comprising the riboswitch described herein; and (ii) a small molecule inducer (aptamer ligand) disclosed herein.

The invention discloses a bacillus amyloliquefaciens riboswitch regulation and control system and application thereof. The regulation and control system is composed of a P43 promoter, a theophylline aptamer TC, RBS, RepA-tag and a required regulation and control gene. A theophylline ribose switch regulation element is integrated on the upstream of a bacillus amyloliquefaciens regulation gene, and the expression quantity of the gene is precisely regulated through theophylline induced expression. Furthermore, a theophylline ribose switch regulation element is integrated to the upstream of the genes hemX and mtnN to obtain the dual-regulation engineering bacterium HZ-B. Under induction of 2mM theophylline, the heme yield and the 5-adenosylmethionine (SAM) yield of the dual-regulation engineering bacterium HZ-B are remarkably increased and are respectively increased by 33.1 times and 0.5 times compared with those of an original strain HZ-12.

The invention provides a platform and methods of using the platform for the regulation of the expression of a target gene using exposure to an aptamer ligand (for example, a small molecule). The platform features a polynucleotide gene regulation cassette that is placed in the target gene and includes a synthetic riboswitch positioned in the context of a 5′ intron-alternative exon-3′ intron. The riboswitch comprises an effector region and a sensor region (e.g., an aptamer that binds a small molecule ligand) such that the alternative exon is spliced into the target gene mRNA when the ligand is not present thereby preventing expression of the target gene. When the ligand is present, the alternative exon is not spliced into the target gene mRNA thereby providing expression of the target gene.

Disclosed herein are dual-specificity (DS) aptamers involving modular designed RNA that connect two aptamer motifs with a linker domain. In cells, they induce proximity between O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) and a designated protein, and increase or decrease O-GlcNAcylation on the substrate. These RNA aptamers have short half-lives in cells and their binding can be controlled by riboswitches.

The invention provides a platform and methods of using the platform for the regulation of the expression of a target gene using exposure to an aptamer ligand (for example, a small molecule). The platform features a polynucleotide gene regulation cassette that is placed in the target gene and includes a synthetic riboswitch positioned in the context of a 5′ intron-alternative exon-3′ intron. The riboswitch comprises an effector region and a sensor region (e.g., an aptamer that binds a small molecule ligand) such that the alternative exon is spliced into the target gene mRNA when the ligand is not present thereby preventing expression of the target gene. When the ligand is present, the alternative exon is not spliced into the target gene mRNA thereby providing expression of the target gene.

This disclosure provides a small molecule of formula (I) that binds to an aptamer. Also intended are riboswitches and polynucleotide cassettes for regulating the expression of a target gene in response to the small molecule, the polynucleotide cassette comprising the aptamer disclosed herein. The small molecule disclosed herein, bound to the aptamer disclosed herein, is a modulator of target gene expression, and the target gene comprises a riboswitch containing the aptamer described herein. JPEG2026524820000284.jpg40164

The present disclosure provides riboswitches to modulate gene expression or vector stability in response to the presence or absence of target nucleic acids. Also provided are vectors that include one or more of such riboswitches, for example, a plurality of the riboswitches combined into a logic gate. Such vectors are useful as programmable genetic medicines, permitting expression of desired cargos only in the precise cell types or disease states that have recognized molecular signatures. The present disclosure also provides related DNA vectors, host cells, manufacturing processes, and uses of the vectors in diagnostic and therapeutic methods.

The invention provides eukaryotic cell-based screening methods for identifying an aptamer that specifically binds a ligand or a ligand that specifically binds an aptamer, using a polynucleotide cassette to regulate the expression of a reporter gene, wherein the polynucleotide cassette contains a riboswitch within a 5' intron-alternative exon-3' intron frame. The riboswitch comprises an effector region and an aptamer, such that when the aptamer binds a ligand, reporter gene expression is induced.

Disclosed is an aptamer capable of binding to Cannabielsoin (CBE), wherein the aptamer is: an RNA aptamer having a nucleotide sequence of: CGCGGGCAGUGNNNUGCANNNNNNNNUGUGNNNUAUUAGC (SEQ ID NO: 1); or a DNA aptamer having a nucleotide sequence of: CGCGGGCAGTGNNNTGCANNNNNNNNTGTGNNNTATTAGC (SEQ ID NO: 2), wherein for SEQ ID NO: 1, N is one nucleotide selected from the group consisting of A, G, U and C, and wherein for SEQ ID NO: 2, N is one nucleotide selected from the group consisting of A, G, C and T. Also disclosed is a riboswitch and a DNA expression cassette comprising the aptamer, a composition thereof, and a microbe containing the riboswitch or DNA expression cassette. Further disclosed is a method of detecting CBE produced in a microbe using the aptamer or riboswitch as disclosed herein.

Disclosed are methods and systems for detecting fluoride as a target molecule in a test sample which utilize a cell-protein synthesis (CFPS) reaction and an engineered fluoride-sensing riboswitch. The components used in the disclosed methods and systems may be dried or lyophilized and may be present or immobilized on a paper substrate.

The present disclosure provides small molecules of formula (I) that bind to aptamers. Also contemplated are riboswitches and polynucleotide cassettes that modulate the expression of a target gene in response to the small molecules, wherein the polynucleotide cassettes comprise the aptamers disclosed herein. The small molecules disclosed herein bound to the aptamers disclosed herein are modulators of expression of a target gene, wherein the target gene contains a riboswitch comprising the aptamers described herein.

The invention provides polynucleotide constructs for the regulation of gene expression by aptamer-based modulation of U1 snRNP-mediated suppression of polyadenylation and methods of using the constructs to regulate gene expression in response to the presence or absence of a ligand that binds the aptamer. The polynucleotide construct contains a U1 binding site in the context of a riboswitch comprising an effector region and an aptamer such that when the aptamer binds a ligand, target gene expression occurs.

The present disclosure relates to compounds that bind to target RNA molecules, such as TPP riboswitches, compositions comprising the compounds, and methods of making and using the compounds. Compared to compounds that bind only to a single RNA binding site, the compounds contain two structurally distinct fragments that allow binding to the target RNA at two different binding sites, thereby generating a higher affinity 5-binding ligand.

The invention provides polynucleotide constructs for the regulation of gene expression by aptamer-based modulation of self-cleaving ribozymes and methods of using the constructs to regulate gene expression in response to the presence or absence of a ligand that binds the aptamer. The invention further provides methods for making and using riboswitches that decrease target gene expression in response to an aptamer ligand as well as riboswitches that increase target gene expression in response to an aptamer ligand.

The invention provides eukaryotic cell-based screening methods to identify an aptamer that specifically binds a ligand, or a ligand that specifically binds an aptamer, using a polynucleotide cassette for the regulation of the expression of a reporter gene where the polynucleotide cassette contains a riboswitch in the context of a 5′ intron-alternative exon-3′ intron. The riboswitch comprises an effector region and an aptamer such that when the aptamer binds a ligand, reporter gene expression occurs.

The application discloses a riboswitch element responding to vitamin B12 and construction and application of a vitamin B12 biosensor based on the riboswitch element, and belongs to the fields of genetic engineering technology, molecular detection and biosensing. The biosensor comprises a riboswitch element responding to vitamin B12 and a selectable marker gene. An effective relationship between vitamin B12 concentration in a bacterium and expression of the marker gene is established by using the sensor. The biosensor can be used for selecting a high-yield vitamin B12 strain in a relatively fast and easy way, and has an important application prospect in detection and screening of vitamin B12-producing microorganisms.

The invention discloses a microbial whole-cell sensor based on a riboswitch as well as a construction method and application thereof, and belongs to the technical field of biosensors. The invention provides a microbial whole-cell sensor based on a riboswitch, escherichia coli is modified by synthetic biology, so that the escherichia coli responds to mercury ions and expresses red fluorescent protein through the riboswitch, protein signals with different intensities are further generated, and the effect of detecting the mercury ions is achieved; according to the microbial whole-cell sensor based on the riboswitch, mercury ion signals can be quickly responded through the designed artificial riboswitch; through combination with transcription factor MerR, chaperonin ProQ and BL21 Star (DE3) strains, the probability of false positive results can be effectively reduced, the anti-interference capability is improved, and the detection specificity, sensitivity and accuracy of the whole-cell sensor are greatly improved.