Loop-Mediated Synthetic Riboregulators
AssigneeArizona Board of Regents on behalf of Arizona State University
InventorsAlexander Green, Duo Ma
Life of this patent
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Abstract
Provided herein are synthetic nucleic acid molecules known as loop-mediated riboregulators that have single-nucleotide polymorphism (SNP) sensitivity and ultralow OFF state signal levels. Loop-mediated riboregulators can activate or repress gene expression in response to trigger RNAs bearing completely arbitrary sequences. Also provided herein are methods of using such synthetic nucleic acid molecules for detecting the presence or absence of a particular target RNA in, for example, a biological sample.
Claims12
A method of detecting presence of an endogenous RNA in a cell, the method comprising:
- introducing into the cell a loop-mediated riboregulator comprising a synthetic nucleic acid molecule, the synthetic nucleic acid molecule comprising:
- (a) a stem-loop structure, the stem-loop structure comprising:
- a first stem-forming domain;
- a loop-forming domain after the final 3′ nucleotide of the first stem-forming domain;
- and
- a second stem-forming domain after the final 3′ nucleotide of the loop domain;
- the second stem-forming domain comprising a ribosomal binding site (RBS), a translational start codon, and a 6 nucleotide clamp;
- wherein the loop domain and a portion of the first stem-forming domain form an endogenous RNA docking site that is complementary to the endogenous RNA;
- and
- (b) a coding sequence located downstream of the start codon;
- wherein the second stem-forming domain is substantially complementary to the first stem-forming domain;
- wherein the coding sequence encodes a reporter protein;
- wherein the 6 nucleotide clamp in the second stem-forming domain corresponds to the first 6 nucleotides of the coding sequence;
- and
- wherein the coding sequence does not comprise a linker;
- culturing the cell under conditions that allow translation of the reporter protein in the presence of the endogenous RNA but not in the absence of the endogenous RNA;
- and
- detecting the reporter protein as an indicator of the endogenous RNA in the cell.
A method for altering expression of a target gene product in a cell, the method comprising
- introducing into the cell a sequence encoding a loop-mediated riboregulator comprising a synthetic nucleic acid molecule, the synthetic nucleic acid molecule comprising:
- (a) a stem-loop structure, the stem-loop structure comprising:
- a first stem-forming domain;
- a loop-forming domain after the final 3′ nucleotide of the first stem-forming domain;
- and
- a second stem-forming domain after the final 3′ nucleotide of the loop domain;
- the second stem-forming domain comprising a ribosomal binding site (RBS), a translational start codon, and a 6 nucleotide clamp;
- wherein the loop domain and a portion of the first stem-forming domain form a trigger RNA docking site that is complementary to a trigger RNA;
- and
- (b) a coding sequence located downstream of the start codon;
- wherein the second stem-forming domain is substantially complementary to the first stem-forming domain;
- wherein the coding sequence encodes the target gene product;
- wherein the 6 nucleotide clamp in the second stem-forming domain corresponds to the first 6 nucleotides of the coding sequence;
- wherein the coding sequence does not comprise a linker;
- and
- wherein, in the presence of the trigger RNA in the cell, expression of the target gene product is altered.
Dependencies are inferred from the claim text.