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US 11,236,326 B2Granted
Methods and compositions for assessing protein function
AssigneeUniversity of Oregon
InventorJeffrey McKnight
Life of this patent
Estimating the term…
Abstract
Compositions and methods for assessing protein function are provided. A modified SpyCatcher protein that can include a tag is provided. Modified SpyCatcher proteins linked to a protein of interest, such as a nuclease are also provided. The methods include contacting a SpyCatcher protein and a SpyTagged protein to form a complex that may further include a protein of interest, one or more nucleic acids, and/or a nuclease. The methods can be used to purify a protein of interest or identify or target a protein binding site in a nucleic acid.
Claims17
A method of mapping protein binding to a nucleic acid, comprising:
- (a) contacting a modified SpyCatcher polypeptide covalently linked to a tag with a protein of interest covalently linked to a SpyTag polypeptide or a SpyTag-sequence-specific nickel-assisted cleavage tag (SpyTag-SNAC tag) polypeptide and a population of nucleic acids under conditions sufficient for the protein of interest to bind to the nucleic acids and the SpyCatcher polypeptide and the SpyTag polypeptide or SpyTag-SNAC tag polypeptide to bind to form a SpyCatcher-SpyTag-protein of interest-nucleic acid complex or a SpyCatcher-SpyTag-SNAC tag-protein of interest-nucleic acid complex, wherein the modified SpyCatcher polypeptide comprises the amino acid sequence of SEQ ID NO:
- 7;
- (b) contacting the SpyCatcher-SpyTag-protein of interest-nucleic acid complex or SpyCatcher-SpyTag-SNAC tag-protein of interest-nucleic acid complex with a binding partner for the tag covalently linked to the SpyCatcher polypeptide under conditions sufficient for the binding partner to bind to the tag;
- (c) contacting the complex with a nuclease under conditions sufficient for the nuclease to cleave nucleic acid flanking the complex;
- (d) performing one or more washes of the complex;
- (e) disassociating the SpyCatcher-SpyTag-protein of interest or SpyCatcher-SpyTag-SNAC tag-protein of interest from the complex, thereby producing dissociated nucleic acid;
- and
- (f) isolating the dissociated nucleic acid.
5 more elements
A method of mapping protein binding to a nucleic acid, comprising:
- (a) contacting a SpyCatcher polypeptide covalently linked to a nuclease with a protein of interest covalently linked to a SpyTag polypeptide or a SpyTag-sequence-specific nickel-assisted cleavage tag (SpyTag-SNAC tag) polypeptide and a population of nucleic acids under conditions sufficient for the protein of interest to bind to the nucleic acids and the SpyCatcher polypeptide and the SpyTag polypeptide or SpyTag-SNAC tag polypeptide to bind to form a SpyCatcher-SpyTag-protein of interest-nucleic acid complex or a SpyCatcher-SpyTag-SNAC tag-protein of interest-nucleic acid complex;
- (b) incubating the SpyCatcher-SpyTag-protein of interest-nucleic acid complex or the SpyCatcher-SpyTag-SNAC tag-protein of interest-nucleic acid complex under conditions sufficient to activate the nuclease;
- (c) performing one or more washes of the complex;
- (d) dissociating the SpyCatcher-SpyTag-protein of interest or the SpyCatcher-SpyTag-SNAC tag-protein of interest from the complex, thereby producing dissociated nucleic acid;
- and
- (e) isolating the dissociated nucleic acid.
3 more elements
A method of mapping protein binding to a nucleic acid, comprising:
- (a) contacting a modified SpyCatcher polypeptide covalently linked to a Tn5 transposase with a protein of interest covalently linked to a SpyTag polypeptide or a SpyTag-sequence-specific nickel-assisted cleavage tag (SpyTag-SNAC tag) polypeptide and a population of nucleic acids under conditions sufficient for the protein of interest to bind to the nucleic acids and the SpyCatcher polypeptide and the SpyTag polypeptide or SpyTag-SNAC tag polypeptide to bind to form a SpyCatcher-SpyTag-protein of interest-nucleic acid complex or a SpyCatcher-SpyTag-SNAC tag-protein of interest-nucleic acid complex, wherein the modified SpyCatcher polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:
- 7, and wherein the modified SpyCatcher polypeptide comprises a cysteine at amino acid position 50 of SEQ ID NO:
- 7;
- (b) incubating the SpyCatcher-SpyTag-protein of interest-nucleic acid complex or the SpyCatcher-SpyTag-SNAC tag-protein of interest-nucleic acid complex under conditions sufficient to activate the Tn5 transposase;
- (c) performing one or more washes of the complex;
- (d) dissociating the SpyCatcher-SpyTag-protein of interest or the SpyCatcher-SpyTag-SNAC tag-protein of interest from the complex, thereby producing dissociated nucleic acid;
- and
- (e) isolating the dissociated nucleic acid.
5 more elements
A method of localizing a protein to a nucleic acid, comprising:
- contacting a SpyCatcher polypeptide covalently linked to a dCas9 nuclease with:
- (i) a protein of interest covalently linked to a SpyCatcher tag polypeptide comprising a SpyTag polypeptide;
- (ii) a guide RNA specific for a target nucleic acid;
- and
- (iii) a population of nucleic acids,
- under conditions sufficient for the SpyCatcher polypeptide and the SpyCatcher tag polypeptide and nucleic acids to form a SpyCatcher-SpyCatcher tag-protein of interest-gRNA-nucleic acid complex.
3 more elements
Dependencies are inferred from the claim text.