MODULAR APTAMAR-REGULATED RIBOZYMES
First Claim
1. An aptamer-regulated ribozyme, comprising(a) a cis-acting hammerhead ribozyme comprising a catalytic core and stem I, stem II and stem III duplex regions extending therefrom, said stem I having a loop I single-stranded loop region opposite to said catalytic core, and said stem II having a loop II single-stranded loop region opposite to said catalytic core;
- (b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop I or loop II through said first end; and
(c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to a ligand,wherein, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme undergoes self-cleavage of a backbone phosphodiester bond at a rate dependent upon the presence or absence of said ligand.
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Abstract
An extensible RNA-based framework for engineering ligand-controlled gene regulatory systems, called ribozyme switches, that exhibit tunable regulation, design modularity, and target specificity is provided. These switch platforms typically contain a sensor domain, comprised of an aptamer sequence, and an actuator domain, comprised of a hammerhead ribozyme sequence. A variety of modes of standardized information transmission between these domains can be employed, and this application demonstrates a mechanism that allows for the reliable and modular assembly of functioning synthetic hammerhead ribozyme switches and regulation of ribozyme activity in response to various effectors. In some embodiments aptamer-regulated cis-acting hammerhead ribozymes are provided.
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Citations
60 Claims
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1. An aptamer-regulated ribozyme, comprising
(a) a cis-acting hammerhead ribozyme comprising a catalytic core and stem I, stem II and stem III duplex regions extending therefrom, said stem I having a loop I single-stranded loop region opposite to said catalytic core, and said stem II having a loop II single-stranded loop region opposite to said catalytic core; -
(b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop I or loop II through said first end; and (c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to a ligand, wherein, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme undergoes self-cleavage of a backbone phosphodiester bond at a rate dependent upon the presence or absence of said ligand. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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21. An aptamer-regulated ribozyme for cleaving a target nucleic acid, comprising
(a) a trans-acting hammerhead ribozyme comprising catalytic core, a 5′ - targeting arm which hybridizes to a 3′
sequence of said target nucleic acid, a 3′
targeting arm which hybridizes to a 5′
sequence of said target nucleic acid, and a stem duplex region extending from said catalytic core with a single-stranded loop region opposite said catalytic core;(b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop through said first end; and (c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to a ligand, wherein, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme cleaves a target nucleic acid at a rate dependent upon the presence or absence of said ligand. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 48, 49, 60)
- targeting arm which hybridizes to a 3′
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35. A cell comprising:
- a metabolic pathway of one or more reactions that are regulated at least in part by a target gene; and
one or more trans-acting hammerhead ribozymes that act as control elements on said metabolic pathway by regulating expression of said target gene, each trans-acting nucleic acid including(a) a catalytic core, a 5′
targeting arm which hybridizes to a 3′
sequence of said target gene, a 3′
targeting arm which hybridizes to a 5′
sequence of said target gene, and a stem duplex region extending from said catalytic core with a single-stranded loop region opposite said catalytic core;(b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop through said first end; and (c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to a ligand selected from an enzyme co-factor, a reactant, a substrate or a product of a reaction in said metabolic pathway; wherein, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme cleaves mRNA transcribed from said target gene at a rate dependent upon the presence or absence of said ligand. - View Dependent Claims (36)
- a metabolic pathway of one or more reactions that are regulated at least in part by a target gene; and
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37. A cell comprising a metabolic pathway of one or more reactions that are regulated at least in part by a target gene, said target gene engineered to include, upon expression as an mRNA, one or more cis-acting ribozymes that act to control expression of said target gene, each cis-acting ribozyme including
(a) a cis-acting hammerhead ribozyme comprising a catalytic core and stem I, stem II and stem III duplex regions extending therefrom, said stem I having a loop I single-stranded loop region opposite to said catalytic core, and said stem II having a loop II single-stranded loop region opposite to said catalytic core; -
(b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop I or loop II through said first end; and (c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to a ligand selected from an enzyme co-factor, a reactant, a substrate or a product of a reaction in said metabolic pathway; wherein, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme undergoes self-cleavage of a backbone phosphodiester bond at a rate dependent upon the presence or absence of said ligand so as to render expression of target gene dependent on said ligand. - View Dependent Claims (38)
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39. A method for rendering expression of a target gene in a cell dependent on the presence or absence of a ligand, comprising introducing into the cell a trans-acting ribozyme comprising:
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(a) a catalytic core, a 5′
targeting arm which hybridizes to a 3′
sequence of said target gene, a 3′
targeting arm which hybridizes to a 5′
sequence of said target gene, and a stem duplex region extending from said catalytic core with a single-stranded loop region opposite said catalytic core;(b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop through said first end; and (c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to a ligand; wherein, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme cleaves mRNA transcribed by said target gene at a rate dependent upon the presence or absence of said ligand. - View Dependent Claims (40, 41)
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42. A method for rendering expression of a target gene in a cell dependent on the presence or absence of a ligand, comprising introducing into the cell an expression construct including a coding sequence that for the target gene, which when transcribed to an mRNA transcript, also includes one or more cis-acting ribozymes in said mRNA that regulate expression of the target gene, said cis-acting ribozyme comprising:
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(a) a catalytic core and stem I, stem II and stem III duplex regions extending therefrom, said stem I having a loop I single-stranded loop region opposite said catalytic core, and said stem II having a loop II single-stranded loop region opposite said catalytic core; (b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop I or loop II through said first end; and (c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to a ligand, wherein, binding of said ligand to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme undergoes self-cleavage of a backbone phosphodiester bond at a rate dependent upon the presence or absence of said ligand so as to render expression of target gene dependent on said ligand. - View Dependent Claims (43, 44, 45)
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46. A method of determining the amount of an analyte in a cell which expresses a reporter gene, comprising:
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introducing into the cell a trans-acting ribozyme comprising (a) a catalytic core, a 5′
targeting arm which hybridizes to a 3′
sequence of said reporter gene, a 3′
targeting arm which hybridizes to a 5′
sequence of said reporter gene, and a stem duplex region extending from said catalytic core with a single-stranded loop region opposite said catalytic core;(b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop through said first end; and (c) an aptamer coupled to said information transmission domain through said second end, said aptamer selectively binds to said analyte; wherein, binding of said analyte to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme cleaves mRNA transcribed from said reporter gene at a rate dependent upon the presence or absence of said analyte; (ii) measuring the amount of expression of said reporter gene; and (iii) correlating the amount of expression of said reporter gene with the amount of analyte, thereby determining the amount of the analyte in the cell.
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47. A method of determining the amount of an analyte in a cell which expresses a reporter gene, comprising
(i) introducing into the cell an expression construct including a coding sequence that for the reporter gene, which when transcribed to an mRNA transcript, also includes one or more cis-acting ribozymes in said mRNA that regulate expression of the reporter gene, said cis-acting ribozyme comprising: -
(a) a catalytic core and stem I, stem II and stem III duplex regions extending therefrom, said stem I having a loop I single-stranded loop region opposite said catalytic core, and said stem II having a loop II single-stranded loop region opposite said catalytic core; and (b) an information transmission domain having a first and second end, which information transmission domain is directly coupled to said loop I or loop II through said first end; and (c) an aptamer coupled to said information transmission domain through said second end, said aptamer binds to an analyte, wherein, binding of said analyte to said aptamer causes a change in the interaction of said information transmission domain with one or more of said loop, said stem or said catalytic core, such that said ribozyme undergoes self-cleavage of a backbone phosphodiester bond at a rate dependent upon the presence or absence of said analyte so as to render expression of reporter gene dependent on said analyte; (ii) measuring the amount of expression of said reporter gene; and (iii) correlating the amount of expression of said reporter gene with the amount of analyte, thereby determining the amount of the ligand in the cell.
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Specification