Fluorogenic sensors for detecting antigens
AssigneePresident and Fellows of Harvard College
InventorsErkin Kuru, George M. Church, Janathan Rittichier, Isaac Han, Helena de Puig Guixe
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Abstract
Provided herein are fluorogenic sensors which can be used to detect targets (e.g., antigens). In general, the fluorogenic sensors provided herein comprise a protein (e.g., antibody, nanobody, mini-protein) and a fluorogenic small molecule conjugated to the target-binding domain (e.g., antigen-binding domain) of the protein. Upon binding of the protein to said target (e.g., antigen), the fluorogenic small molecule may increase or decrease in fluorescence or exhibit a change in fluorescence lifetime (i.e., “turn on”), thereby indicating the presence of the target (e.g., antigen).
Claims121
A fluorogenic sensor for detecting a target comprising:
- a nanobody;
- and
- a fluorogenic small molecule conjugated at or around a target-binding domain of the nanobody.
A fluorogenic sensor for detecting a target comprising:
- a mini-protein;
- and
- a fluorogenic small molecule conjugated to the mini-protein.
The fluorogenic sensor of any one of the preceding claims, wherein the the nanobody or the mini-protein comprises an unnatural amino acid comprising the fluorogenic small molecule;
- optionally wherein a target-binding domain of the nanobody or the mini-protein comprises an unnatural amino acid comprising the fluorogenic small molecule.
The fluorogenic sensor of any one of the preceding claims, wherein the amino acid sequence comprises at least one amino acid substituted by an unnatural amino acid comprising the fluorogenic small molecule.
The fluorogenic sensor of any one of the preceding claims, wherein the fluorogenic small molecule is 7-nitrobenz-2-Oxa-1,3-Diazol-4-yl (NBD).
The fluorogenic sensor of any one of the preceding claims, wherein the fluorogenic small molecule conjugated to the nanobody or the mini-protein results from conjugating a compound selected from those in FIG. 2A and FIG. 2B.
The fluorogenic sensor of any one of the preceding claims, wherein the fluorescence of the fluorogenic small molecule increases upon binding of the nanobody or the mini-protein to the target.
The fluorogenic sensor of any one of the preceding claims, wherein the fluorescence lifetime of the fluorogenic small molecule changes upon binding of the nanobody or the mini-protein to the target.
The fluorogenic sensor of any one of the preceding claims further comprising a second fluorogenic small molecule conjugated to the N-terminus of the nanobody.
The fluorogenic sensor of 98 or 99, wherein the second fluorogenic small molecule is tetramethylrhodamine (TMR).
A method of detecting a target, the method comprising:
- (i) contacting a target with a fluorogenic sensor of any one of the preceding claims;
- and
- (ii.a) measuring or observing the fluorescence of the fluorogenic sensor, or (ii.b) measuring or observing a change in the fluorescence lifetime of the fluorogenic sensor.
A method of detecting an antigen, the method comprising:
- (i) contacting an antigen with a fluorogenic sensor of any one of the preceding claims;
- and
- (ii.a) measuring or observing the fluorescence of the fluorogenic sensor, or (ii.b) measuring or observing a change in the fluorescence lifetime of the fluorogenic sensor.
A compound of the following formula:
- or a salt, stereoisomer, or tautomer thereof, wherein:
- A is a reactive moiety comprising one of the following formulae:
- RH is halogen or a leaving group;
- each instance of X is independently —N(RN)2, —ORO, or —SRS;
- each instance of R′ is independently hydrogen, halogen, —CN, —NO2, —N3, —N(RN)2, —ORO, —SRS, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted acyl, optionally substituted sulfinyl, or optionally substituted sulfonyl;
- each instance of m is independently 0, 1, 2, 3, or 4;
- and
- each instance of RN, RO, and RS is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted acyl.
A compound of the following formula:
- or a salt or tautomer thereof, wherein:
- A is a reactive moiety;
- each instance of X is independently —N(RN)2, —ORO, or —SRS;
- n is 0 or an integer from 1-20, inclusive.
- each instance of R′ is independently hydrogen, halogen, —CN, —NO2, —N3, —N(RN)2, —ORO, —SRS, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted acyl, optionally substituted sulfinyl, or optionally substituted sulfonyl;
- each instance of m is independently 0, 1, 2, 3, or 4;
- each instance of RN, RO, and RS is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted acyl.
A compound of the following formula:
- or a salt, stereoisomer, or tautomer thereof;
- wherein:
- L is a bond or a linker;
- A is a reactive moiety;
- each instance of R′ and X is independently hydrogen, halogen, —CN, —NO2, —N3, —N(RN)2, —ORO, —SRS, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted acyl, optionally substituted sulfinyl, or optionally substituted sulfonyl;
- and
- each instance of RN, RO, and RS is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or optionally substituted acyl.
A compound of the following formula:
- or a salt, stereoisomer, or tautomer thereof;
- wherein the compound is further optionally substituted.
A kit comprising a fluorogenic sensor or compound of any one of the preceding claims, and optionally instructions for use.
Dependencies are inferred from the claim text.