Methods for determining a location of a biological analyte in a biological sample

Abstract

Provided herein are methods of determining a location of a biological analyte in a biological sample.

What is claimed is: 
     
         1 . A method for determining a location of a nucleic acid in a biological sample, the method comprising:
 (a) contacting the biological sample with an array comprising a plurality of capture probes, wherein a capture probe comprises (i) a spatial barcode and (ii) a capture domain that specifically binds the nucleic acid;   (b) binding the nucleic acid to the capture domain of the capture probe from the biological sample;   (c) amplifying the nucleic acid bound to the capture probe to generate an amplicon, wherein the amplicon comprises the spatial barcode from the capture probe; and   (d) determining the location of the nucleic acid within the biological sample by hybridizing a plurality of labelled probes to the amplicon, thereby determining the location of the nucleic acid within the biological sample.   
     
     
         2 . The method of  claim 1 , wherein the array comprises one or more features. 
     
     
         3 . The method of  claim 1 , wherein the biological sample is permeabilized under conditions sufficient to allow the nucleic acid to specifically bind the capture probe. 
     
     
         4 . The method of  claim 1 , wherein the capture probe further comprises one or more of a cleavage domain, a functional domain, a unique molecular identifier, and combinations thereof. 
     
     
         5 . The method of  claim 1 , wherein step (c) comprises amplifying the nucleic acid bound to the capture probe in situ using rolling circle amplification, bridge amplification, isothermal amplification, linear PCR, or exponential PCR. 
     
     
         6 . The method of  claim 1 , wherein the plurality of labelled probes in step (d) comprises a plurality of fluorescently-labelled probes. 
     
     
         7 . The method of  claim 1 , wherein analyzing the amplicon comprises RNA sequential probing of targets (RNA SPOTs), sequential fluorescent in situ hybridization (SeqFISH), single-molecule fluorescent in situ hybridization (smFISH), multiplexed error-robust fluorescence in situ hybridization (MERFISH), in situ sequencing, targeted in situ sequencing, fluorescent in situ sequencing (FISSEQ), or spatially-resolved transcript amplicon readout mapping (STARmap). 
     
     
         8 . The method of  claim 1 , further comprising contacting a polymer material to the biological sample, and activating the polymer material to form a hydrogel-embedded biological sample. 
     
     
         9 . The method of  claim 8 , wherein the polymer material comprises acrylamide. 
     
     
         10 . The method of  claim 8 , wherein the hydrogel-embedded biological sample is further subjected to a chemical permeabilization condition, an enzymatic permeabilization condition, or both, wherein the chemical permeabilization condition comprises contacting the biological sample with an alkaline solution and the enzymatic permeabilization condition comprises contacting the biological sample with an acidic solution comprising a protease. 
     
     
         11 . The method of  claim 10 , further comprising clearing the hydrogel-embedded biological sample, wherein the clearing substantially removes a plurality of cellular components from the biological sample. 
     
     
         12 . The method of  claim 1 , wherein the nucleic acid is RNA or DNA. 
     
     
         13 . The method of  claim 1 , wherein the capture domain comprises a poly(T) sequence. 
     
     
         14 . The method of  claim 1 , wherein the method further comprises generating a nucleic acid sequence complementary to the sequence of the nucleic acid bound to the capture probe, using the capture probe as a primer. 
     
     
         15 . The method of  claim 1 , wherein analyzing the amplicon comprises sequencing the amplicon, thereby determining (i) all or a portion of a sequence of the spatial barcode or complement thereof, and (ii) all or a portion of a sequence of the nucleic acid or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the nucleic acid in the biological sample. 
     
     
         16 . A method for determining a location of an analyte in a biological sample, the method comprising:
 (a) contacting the biological sample with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a capture domain that specifically binds the analyte, thereby generating a captured analyte;   (b) contacting the captured analyte with a plurality of detectable probes, wherein one or more detectable probes bind to with the captured analyte;   (c) detecting the one or more detectable probes bound to the captured analyte; and   (d) determining a location of the one or more detectable probes bound to the captured analyte, thereby identifying the location of the analyte in the biological sample.   
     
     
         17 . The method of  claim 16 , wherein the one or more detectable probes comprise: (i) a sequence that specifically binds the captured analyte, wherein the captured analyte interacts with at least two or more detectable probes; and (ii) one or more readout sequences that interact with a detectable label, wherein the detectable label is a nucleic acid comprising a fluorophore. 
     
     
         18 . The method of  claim 17 , wherein two or more readout sequences interact with different detectable labels. 
     
     
         19 . The method of  claim 1 , wherein the capture probe comprises a spatial barcode. 
     
     
         20 . The method of  claim 17 , wherein a plurality of detectable labels are contacted with the biological sample, wherein the plurality of detectable labels interact with a detectable probe bound to the captured analyte. 
     
     
         21 . The method of  claim 17 , further comprising imaging the plurality of detectable labels bound to the one or more readout sequences of the detectable probe to identify the location of the captured analyte. 
     
     
         22 . The method of  claim 21 , further comprising generating a signature, wherein the signature comprises assigned values from fluorescence of the plurality of detectable labels after imaging the plurality of detectable labels bound to the one or more readout sequences of the detectable probe. 
     
     
         23 . The method of  claim 16 , wherein the detecting step (c) further comprises:
 1) contacting the captured analyte with a plurality of detectable probes;   2) labeling the detectable probes with a plurality of detectable labels;   3) imaging the plurality of detectable labels;   4) recording the location of the plurality of detectable labels;   5) removing the plurality of detectable labels; and   6) repeating steps (2)-(5) for one or more rounds, wherein after each recording step, a signature is generated from fluorescence of the plurality of detectable labels, the signature comprising n number of assigned values uniquely representing a specific analyte.   
     
     
         24 . The method of  claim 23 , wherein steps (2)-(5) are repeated 4 or more times. 
     
     
         25 . The method of  claim 23 , wherein the signature is based on combinatorial labeling of the detectable label(s) and is matched to the specific analyte, thereby identifying the location of the specific analyte in the biological sample. 
     
     
         26 . The method of  claim 23 , wherein the plurality of detectable labels are removable from the detectable probes by at least one of light, a chemical molecule, and an enzyme. 
     
     
         27 . The method of  claim 16 , further comprising, prior to step (b), amplifying the captured analyte in situ using isothermal amplification, rolling circle amplification, or bridge amplification. 
     
     
         28 . The method of  claim 16 , wherein the detectable probe is contacted with the captured analyte after the biological sample is removed from the array. 
     
     
         29 . The method of  claim 16 , wherein the analyte comprises at least one of RNA, DNA, a protein, a small molecule, and a metabolite. 
     
     
         30 . The method of  claim 16 , wherein the biological sample comprises a tissue, a tissue section, an organ, an organism, or a cell culture sample.