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US 2017/0253918 A1Published application
Combining protein barcoding with expansion microscopy for in-situ, spatially-resolved proteomics
AssigneeExpansion Technologies
InventorRichie E. KOHMAN
Life of this patent
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Abstract
This invention relates to imaging, such as by expansion microscopy, labelling, and analyzing biological samples, such as cells and tissues, as well as reagents and kits for doing so.
Claims30
A method of labeling a biological sample, the method comprising:
- contacting the sample with at least one binding composition under conditions where it selectively recognizes a target biomolecule, wherein the binding composition comprises a double-stranded nucleic acid having a first strand comprising a first sequence and a second strand comprising a second sequence that is complementary to the first sequence, wherein the first strand is operably linked to an affinity tag for the target biomolecule and is operably linked to a first detectable label, and wherein the second strand is operably linked to a polyelectrolyte gel binding moiety;
- contacting the sample with a solution comprising monomers of a polyelectrolyte gel;
- by free radical polymerization, polymerizing the monomers to form the polyelectrolyte gel and covalently conjugate the polyelectrolyte gel binding moiety to the polyelectrolyte gel;
- proteolytically digesting the sample;
- and
- dialyzing the sample in water to expand the polyelectrolyte gel.
3 more elements
A method of labeling a biological sample, the method comprising:
- contacting the sample with at least one binding composition under conditions where it selectively recognizes a target biomolecule, wherein the binding composition comprises a double-stranded nucleic acid having a first strand comprising a first sequence and a second strand comprising a second sequence that is complementary to the first sequence, wherein the first strand is operably linked to an affinity tag for the target biomolecule and is operably linked to a polyelectrolyte gel binding moiety, and wherein the second stand is operably linked to a first detectable label;
- contacting the sample with a solution comprising monomers of a polyelectrolyte gel;
- by free radical polymerization, polymerizing the monomers to form the polyelectrolyte gel and covalently conjugate the polyelectrolyte gel binding moiety to the polyelectrolyte gel;
- proteolytically digesting the sample;
- and
- dialyzing the sample in water to expand the polyelectrolyte gel.
3 more elements
A method of labeling a biological sample, the method comprising:
- contacting the sample with at least one binding composition under conditions where it selectively recognizes a target biomolecule, wherein the binding composition comprises a single-stranded nucleic acid comprising a first sequence, wherein the single-stranded nucleic acid is operably linked to (i) an affinity tag for the target biomolecule, and (ii) a polyelectrolyte gel binding moiety, and wherein the affinity tag is operably linked to a first detectable label;
- contacting the sample with a solution comprising monomers for the polyelectrolyte gel;
- by free radical polymerization, polymerizing the monomers to form the polyelectrolyte gel and covalently conjugate the polyelectrolyte gel binding moiety to the polyelectrolyte gel;
- proteolytically digesting the sample;
- and
- dialyzing the sample in water to expand the polyelectrolyte gel.
3 more elements
A method of labeling a biological sample, the method comprising:
- contacting the sample with at least one binding composition under conditions where it selectively recognizes a target biomolecule, wherein the binding composition comprises a double-stranded nucleic acid having a first strand comprising a first sequence and a second strand comprising a second sequence that is complementary to the first sequence, wherein the first strand is operably linked to an affinity tag for the target biomolecule, and wherein the second strand is operably linked to a polyelectrolyte gel binding moiety;
- contacting the sample with a solution comprising monomers of a polyelectrolyte gel;
- by free radical polymerization, polymerizing the monomers to form the polyelectrolyte gel and covalently conjugate the polyelectrolyte gel binding moiety to the polyelectrolyte gel;
- proteolytically digesting the sample;
- dialyzing the sample in water to expand the polyelectrolyte gel;
- removing the second strand;
- and
- hybridizing a nucleic acid probe operably linked to a detectable label and comprising a probe sequence that is complementary to the first sequence.
5 more elements
A method of labeling a biological sample, the method comprising:
- contacting the sample with at least one binding composition under conditions where it selectively recognizes a target biomolecule, wherein the binding composition comprises a single-stranded nucleic acid comprising a first sequence, wherein the single-stranded nucleic acid is operably linked to (i) an affinity tag for the target biomolecule, and (ii) a polyelectrolyte gel binding moiety;
- contacting the sample with a solution comprising monomers of a polyelectrolyte gel;
- by free radical polymerization, polymerizing the monomers to form the polyelectrolyte gel and covalently conjugate the polyelectrolyte gel binding moiety to the polyelectrolyte gel;
- proteolytically digesting the sample;
- dialyzing the sample in water to expand the polyelectrolyte gel;
- and
- hybridizing a nucleic acid probe operably linked to a detectable label and comprising a probe sequence that is complementary to the first sequence.
4 more elements
A method of analyzing a biological sample, the method comprising:
- (a) contacting the sample with a set of adapters that selectively recognize a set of target biomolecules under conditions where the adapters selectively recognize the target biomolecules, wherein each adapter comprises a double stranded nucleic acid comprising a first strand operably linked to an affinity tag specific for one of the target biomolecules and having a sequence specific for that target biomolecule, and a second strand comprising a sequence specific for that target biomolecule and operably linked to a polyelectrolyte gel binding moiety;
- (b) contacting the sample with a solution comprising monomers of a polyelectrolyte gel;
- (c) by free radical polymerization, polymerizing the monomers to form the polyelectrolyte gel and covalently conjugate the polyelectrolyte gel binding moiety to the polyelectrolyte gel;
- (d) proteolytically digesting the sample;
- (e) dialyzing the sample in water to expand the polyelectrolyte gel;
- and
- (f) for each target biomolecule, hybridizing a nucleic acid probe operably linked to a fluorescent label and comprising a probe sequence complementary to the sequence specific for that target biomolecule, and detecting the fluorescent label.
4 more elements
A method of analyzing a biological sample, the method comprising:
- (a) contacting the sample with a set of adapters that selectively recognize a set of target biomolecules under conditions where the adapters selectively recognize the target biomolecules, wherein each adapter comprises a single-stranded nucleic acid operably linked to a polyelectrolyte gel binding moiety and to an affinity tag for one of the target biomolecules and having a sequence specific for that target biomolecule;
- (b) contacting the sample with a solution comprising monomers of a polyelectrolyte gel;
- (c) by free radical polymerization, polymerizing the monomers to form the polyelectrolyte gel and covalently conjugate the polyelectrolyte gel binding moiety to the polyelectrolyte gel;
- (d) proteolytically digesting the sample;
- (e) dialyzing the sample in water to expand the polyelectrolyte gel;
- and
- (f) for each target biomolecule, hybridizing a nucleic acid probe operably linked to a fluorescent label and comprising a probe sequence complementary to the sequence specific for that target biomolecule, and detecting the fluorescent label.
4 more elements
Dependencies are inferred from the claim text.