Methods for spatial analysis using proximity ligation

Abstract

The present disclosure provides methods and compositions for detecting and spatially locating analyte interactions and gene expression in a biological sample. For example, provided herein are methods of determining a location of at least one analyte in a biological sample using analyte-binding moieties, proximity ligation, and an array including capture probes.

1 . A method of determining a location or abundance of an interaction between a first analyte and a second analyte in a biological sample, the method comprising:
 (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain;   (b) attaching a first analyte-binding moiety to the first analyte, wherein the first analyte-binding moiety is bound to a first oligonucleotide comprising a first barcode that is unique to the interaction between the first analyte and the first analyte-binding moiety;   (c) attaching a second analyte-binding moiety to the second analyte, wherein the second analyte-binding moiety is bound to a second oligonucleotide comprising a second barcode that is unique to the interaction between the second analyte and the second analyte-binding moiety;   (d) hybridizing a third oligonucleotide to the first oligonucleotide and to the second oligonucleotide;   (e) ligating the first oligonucleotide and the second oligonucleotide, thereby generating a ligation product comprising a capture probe capture domain sequence; and   (f) hybridizing the capture probe capture domain sequence to the capture domain; and   (g) determining (i) all or a portion of the sequence of the spatial barcode or a complement thereof; (ii) all or a portion of the sequence of the first barcode or a complement thereof; (iii) all or a portion of the sequence of the second barcode or a complement thereof; and using the determined sequences of (i), (ii), and (iii) to identify the location or abundance of the interaction between the first analyte and the second analyte in the biological sample.   
     
     
         2 . The method of  claim 1 , wherein the first oligonucleotide further comprises a first functional sequence, the capture probe capture domain sequence, or both. 
     
     
         3 . The method of  claim 2 , wherein the second oligonucleotide further comprises a second functional sequence, the capture probe capture domain sequence, or both. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 3 , wherein the third oligonucleotide comprises:
 (i) a functional sequence, the capture probe capture domain sequence, or both; or   (ii) a sequence complementary to the first barcode and a sequence complementary to the second barcode.   
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , further comprising dissociating the third oligonucleotide from the first oligonucleotide and the second oligonucleotide between steps (e) and (f). 
     
     
         9 . A method of determining a location or abundance of an interaction between a first analyte and a second analyte in a biological sample, the method comprising:
 (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain;   (b) attaching a first analyte-binding moiety to the first analyte, wherein the first analyte-binding moiety is bound to a first oligonucleotide comprising a first barcode that is unique to the interaction between the first analyte and the first analyte-binding moiety and a first bridge sequence;   (c) attaching a second analyte-binding moiety to the second analyte, wherein the second analyte-binding moiety is bound to a second oligonucleotide comprising a second barcode that is unique to the interaction between the second analyte and the second analyte-binding moiety and a second bridge sequence;   (d) hybridizing the first bridge sequence to the second bridge sequence, thereby creating a hybridized proximity oligonucleotide comprising a capture probe capture domain sequence;   (e) hybridizing the capture probe capture domain sequence to the capture domain;   (f) determining (i) all or a portion of the sequence of the first barcode or a complement thereof; (ii) all or a portion of the sequence of the second barcode or a complement thereof, and (iii) all or a part of the sequence of the spatial barcode or a complement thereof, and using the determined sequence of (i), (ii), and (iii) to identify the location or abundance of the interaction between the first analyte and the second analyte in the biological sample.   
     
     
         10 - 14 . (canceled) 
     
     
         15 . The method of  claim 1 , further comprising extending (i) the first oligonucleotide using the second oligonucleotide as a template, and (ii) the second oligonucleotide using the first oligonucleotide as a template. 
     
     
         16 - 18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the first analyte-binding moiety is a first protein and wherein the second analyte-binding moiety is a second protein. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . The method of  claim 1 , wherein the first analyte-binding moiety is associated with the first oligonucleotide via a first linker, wherein the first linker is a cleavable linker, wherein the first cleavable linker is a photocleavable linker, UV-cleavable linker, or an enzyme-cleavable linker. 
     
     
         24 - 28 . (canceled) 
     
     
         29 . The method of  claim 1 , wherein the second analyte-binding moiety is associated with the second oligonucleotide via a second linker, wherein the second linker is a second cleavable linker, wherein the second cleavable linker is a photocleavable linker, UV-cleavable linker, or an enzyme-cleavable linker. 
     
     
         30 - 34 . (canceled) 
     
     
         35 . The method of  claim 1 , wherein the first analyte is selected from the group consisting of: lipids, carbohydrates, peptides, post-translational modifications, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 1 , wherein the second analyte is selected from the group consisting of: lipids, carbohydrates, peptides, post-translational modifications, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments. 
     
     
         38 . (canceled) 
     
     
         39 . The method of  claim 1 , wherein the ligating step uses enzymatic ligation or chemical ligation to create a ligation product. 
     
     
         40 . (canceled) 
     
     
         41 . The method of  claim 1 , further comprising a releasing step comprising removing (i) the first oligonucleotide from the first analyte-binding moiety and (ii) the second oligonucleotide from the second analyte-binding moiety. 
     
     
         42 . (canceled) 
     
     
         43 . The method of  claim 1 , wherein the determining step comprises amplifying all or part of the ligation product bound to the capture domain, thereby generating an amplifying product. 
     
     
         44 . The method of  claim 43 , wherein the amplifying product comprises (i) all or part of sequence of the ligation product or the hybridized proximity oligonucleotide bound to the capture domain, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof. 
     
     
         45 - 46 . (canceled) 
     
     
         47 . The method of  claim 1 , wherein the first analyte is a host analyte and the second analyte is viral analyte or the first analyte is a viral analyte and the second analyte is a host analyte. 
     
     
         48 . The method of  claim 47 , wherein the host analyte is selected from the group consisting of: a host protein, a cell surface moiety, a cell motor protein, a nuclear pore protein, and a RNA polymerase. 
     
     
         49 . The method of  claim 47 , wherein the viral analyte selected from the group consisting of: is a viral protein, a viral capsid, a glycoprotein, an envelope protein, a viral coat protein, or an integrase protein. 
     
     
         50 - 52 . (canceled) 
     
     
         53 . A kit comprising:
 (a) an array comprising a plurality of capture probes;   (b) a first analyte-binding moiety and a second analyte-binding moiety, wherein the first analyte-binding moiety is bound to a first oligonucleotide, wherein the first oligonucleotide comprises: a first barcode, wherein the second analyte-binding moiety is bound to a second oligonucleotide; wherein the second oligonucleotide comprises: (i) a capture probe capture domain sequence, and (ii) a second barcode;   (c) a third oligonucleotide, wherein the third nucleotide comprises a sequence that is substantially complementary to the first oligonucleotide, and a sequence that is substantially complementary to the second oligonucleotide;   (d) a plurality of enzymes comprising a ligase and a polymerase; and   (e) instructions for performing the method of  claim 1 .   
     
     
         54 - 55 . (canceled)