Covalent aptamers
AssigneeUniversity of Pittsburgh—Of the Commonwealth System of Higher Education
InventorsAlexander DEITERS, Yaniv TIVON, Mary CACACE, Savannah ALBRIGHT
Life of this patent
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Abstract
Disclosed are chemically modified aptamers comprising an electrophilic group and a handle and methods for using said aptamers to deliver the handle to a target protein, crosslink the aptamer to the target protein, as well as methods of using said aptamers to detect proteins and to treat viral infections or cancer.
Claims68
A modified aptamer-handle conjugate comprising an aptamer, an electrophilic leaving group and a handle.
A modified aptamer comprising an electrophilic leaving group for crosslinking to a target.
A method of delivering biotin, a chemiluminescent marker, a fluorescent marker, radiomarker, dye, quantum dot, enzyme, enzyme substrate, catalyst, small molecule ligand, PROTAC® (E3 ligase ligand), or LYTAC® (cation-independent mannose-6-phosphate receptor (CI-M6PR) ligand) a or to a target cell comprising conjugating a labeled electrophile to an aptamer creating a modified aptamer or an aptamer-handle complex;
- and contacting the target cell with said aptamer-handle complex.
A method of labeling one or more protein targets on or in a cell comprising conjugating a labeled electrophile to an aptamer creating a modified aptamer or an aptamer-handle complex;
- and contacting the cell with the aptamer electrophile complex.
A modified aptamer-handle conjugate of Formula I
- wherein:
- A1 comprises an aptamer;
- L1 and L2 are independently selected from a bond or a linker;
- E1 is a cleavable electrophilic moiety;
- H1 is a handle;
- m1 is at least 1;
- and
- n1 is at least 1.
A modified aptamer of Formula II
- wherein:
- A1 comprises an aptamer;
- L1 is selected from a bond or a linker;
- E2 is an electrophilic moiety;
- and
- m2 is at least 1.
A modified aptamer of Formula III
- wherein:
- A1 comprises an aptamer;
- L1 is selected from a bond or a linker;
- C1 is a catalytic moiety;
- m3 is at least 1;
- and
- m4 is at least 1.
A method for identifying aptamers suitable as ligands for a target from a candidate mixture of modified aptamers, the method comprising:
- a) preparing a candidate mixture of modified aptamers, wherein each modified aptamer may independently be selected from a modified aptamer of Formula II-a
- wherein:
- A1 comprises an aptamer;
- L1A is a linker comprising at least one cleavable moiety;
- E2 is an electrophilic moiety;
- and
- m2 is at least 1;
- b) contacting the candidate mixture of modified aptamers with the target, wherein modified aptamers capable of forming a covalent bond with the target provide a subpopulation of covalently bound aptamers;
- c) partitioning the subpopulation of covalently bound aptamers from a remainder of the candidate mixture of modified aptamers;
- d) contacting the subpopulation of covalently bound aptamers with a reagent capable of reacting with the at least one cleavable moiety, wherein the at least one cleavable moiety is cleaved to provide a subpopulation of modified aptamers;
- and
- e) amplifying the subpopulation of modified aptamers such that aptamers suitable as ligands for the target may be identified.
A method for identifying aptamers suitable as ligands for a target from a candidate mixture of modified aptamers, the method comprising:
- a) preparing a candidate mixture of modified aptamers, wherein each modified aptamer may independently be selected from a modified aptamer of Formula II
- wherein:
- A1 comprises an aptamer;
- L1 is a bond or a linker;
- E2 is an electrophilic moiety;
- and
- m2 is at least 1;
- b) contacting the candidate mixture of modified aptamers with the target, wherein modified aptamers capable of forming a covalent bond with the target provide a subpopulation of covalently bound aptamers;
- c) partitioning the subpopulation of covalently bound aptamers from a remainder of the candidate mixture of modified aptamers;
- d) contacting the subpopulation of covalently bound aptamers with a reagent capable of cleaving the target bound to the aptamer;
- and
- e) amplifying the subpopulation of modified aptamers such that aptamers suitable as ligands for the target may be identified.
A method of detecting the presence or quantifying the amount of a target protein comprising contacting the target protein with a modified aptamer-handle conjugate comprising an aptamer, an electrophilic leaving group, and a handle;
- wherein the handle comprises or can be modified with a detectable label.
Dependencies are inferred from the claim text.