Method of generating branched and multi-chain nucleic acid switches for ligand detection
First Claim
1. A method to generate a multichain nucleic acid switch adapted to switch from a first conformation to a second conformation upon ligand binding, said switch comprising:
- a probe strand P comprising a ligand binding domain;
a switching framework comprising a cover strand (C);
a tether comprising a base-pair stem that holds P and C together while the switch changes between the first and second conformations, wherein P and C are on different nucleic acid chains; and
a signaling apparatus comprising a combination of signaling entities, said method comprising the following steps;
(i) altering two nucleic acid sequences (a) and (b) to introduce mismatches between P and C;
(ii) measuring the signal for the two nucleic acid sequences (a) and (b), thereby determining an equilibrium constant, K1 for the switch from the first conformation to the second conformation for each of the two sequences (a) and (b);
(iii) adjusting the equilibrium constant, K1 between 0.1-0.01 based on K1 for sequences (a) and (b); and
(iv) generating the multichain nucleic acid switch having the equilibrium constant, K1 between 0.1-0.01.
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Abstract
Embodiments of the invention relate to a branched or multichain nucleic acid switch adapted to switch from a first conformation to a second conformation upon ligand binding. The switch includes a probe strand, P, which includes the ligand binding domain; a switching framework which includes a cover strand (C), and a tether that holds P and C together and a signaling apparatus. Some embodiments include a toggle strand (T) where now the tether holds P, C, T, and the signaling apparatus together. As the switch changes between the first and second conformations; the signaling apparatus reports the state of the switch. The signaling entity is typically a lumiphore and a quencher located along the switching framework. Nucleic acid switches have applications in real time assays for diverse agents including infectious agents, environmental toxins, and terrorist agents, as well as screening methods for such agents. Further applications are found for nanoelectronics, nanofabrication and nanomachines.
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Citations
18 Claims
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1. A method to generate a multichain nucleic acid switch adapted to switch from a first conformation to a second conformation upon ligand binding, said switch comprising:
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a probe strand P comprising a ligand binding domain; a switching framework comprising a cover strand (C); a tether comprising a base-pair stem that holds P and C together while the switch changes between the first and second conformations, wherein P and C are on different nucleic acid chains; and a signaling apparatus comprising a combination of signaling entities, said method comprising the following steps; (i) altering two nucleic acid sequences (a) and (b) to introduce mismatches between P and C; (ii) measuring the signal for the two nucleic acid sequences (a) and (b), thereby determining an equilibrium constant, K1 for the switch from the first conformation to the second conformation for each of the two sequences (a) and (b); (iii) adjusting the equilibrium constant, K1 between 0.1-0.01 based on K1 for sequences (a) and (b); and (iv) generating the multichain nucleic acid switch having the equilibrium constant, K1 between 0.1-0.01. - View Dependent Claims (2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13)
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7. A method to generate a branched, unimolecular nucleic acid switch adapted to switch from a first conformation to a second conformation upon ligand binding, said switch comprising:
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a probe strand P comprising a ligand binding domain; a switching framework comprising a cover strand (C); a toggle strand (T); a tether at a single vertex that holds P, C and T together while the switch changes between the first and second conformations; and a signaling apparatus comprising a combination of signaling entities said method comprising the following steps; (i) altering sequences of C and/or T to introduce mismatches between C and T in a sequence (a) which favors the first conformation and a sequence (b) which favors the second conformation; (ii) measuring the signal for sequences (a) and (b), thereby determining an equilibrium constant, K1 for each of the two sequences (a) and (b); (iii) adjusting the equilibrium constant, K1, based upon the free energy difference between the K1 for each of the two sequences (a) and (b); and (iv) generating the branched, unimolecular nucleic acid switch having the adjusted equilibrium constant, K1. - View Dependent Claims (14, 15, 16, 17, 18)
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Specification