Molecular sensors activated by disinhibition
AssigneeKaloBios, Inc.
InventorsRobert F. Balint, Jen-Horng Her
Life of this patent
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Abstract
The current invention provides methods and systems for detecting the presence of a target molecule either in vitro or in vivo. The systems of the invention comprise interacting components, a reporter and a low-affinity inhibitor of the reporter, each of which is fused to a member of a binding pair. A target molecule that interferes with binding of the binding pair members can therefore be identified by detecting activation of the reporter molecule.
Claims48
A system for detecting a target molecule that interferes with the binding interaction between members of a binding pair, the system comprising:
- i) a reporter molecule linked to a first binding pair member, and ii) a low-affinity inhibitor of the reporter molecule linked to a second binding pair member;
- wherein, when the first and second binding pair members interact, the reporter molecule is inhibited;
- and further;
- wherein binding of the target molecule to at least one binding pair member prevents the inhibitor from binding to the reporter molecule, thereby activating the reporter molecule.
A method of detecting a target molecule in a sample, the method comprising:
- contacting a sample that is being tested for the presence of the target molecule with:
- i) a reporter molecule linked to a binding pair member, and ii) a low-affinity inhibitor of the reporter molecule linked to a second binding pair member,
- wherein binding of the target molecule to at least one binding pair member prevents the inhibitor from binding to the reporter molecule, thereby activating the reporter molecule.
A method of identifying a target molecule in a cell population, the method comprising:
- introducing into the population of cells expression vector(s) comprising a nucleic acid sequence encoding a first binding pair member linked to a reporter molecule and further comprising a nucleic acid sequence encoding a second binding pair member linked to an inhibitor of the reporter molecule;
- wherein the reporter molecule is inhibited when the binding pair members interact;
- culturing the population of cells under conditions in which the first binding pair member linked to the reporter and the second binding pair member linked to the inhibitor are expressed in the presence of a candidate target molecule, wherein a target molecule that binds to at least one binding pair member prevents the inhibitor from binding to the reporter molecule, thereby activating the reporter molecule;
- and
- selecting a cell in which the reporter molecule is active.
A system for detecting a target molecule that interferes with a binding interaction between members of a binding pair, the system comprising two components:
- (i) a reporter molecule linked to a first binding pair member,
- (ii) an inhibitor of the reporter molecule linked to a second binding pair member;
- wherein component (i) further comprises a mask that has low affinity for the reporter molecule, which mask binds to the reporter molecule and prevents the inhibitor from binding to the reporter molecule when there is no binding pair interaction, or
- component (ii) further comprises a mask that has low affinity for the inhibitor, which mask binds to the inhibitor and prevents the inhibitor from binding to the reporter molecule when there is no binding pair interaction,wherein interaction of the binding pair inactivates the reporter by displacement of the mask from the reporter or inhibitor;
- and further,
- wherein binding of the target molecule to the first or second binding pair member prevents the interaction between the first and the second binding pair member, thereby preventing displacement of the mask and binding of the inhibitor to the reporter molecule.
Dependencies are inferred from the claim text.