Aptamer-based simultaneous spatial mRNA / protein assay

Abstract

The present disclosure relates generally to methods for preparing spatial proteome and / or transcriptome sequencing libraries from biological samples, which in some embodiments are useful for determining genetic profiles, aiding in the diagnosis of subjects having or at risk of having a disorder, and improving treatment of subjects.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 477,096, filed December 23, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to methods for preparing spatial proteogenomic sequencing libraries. Summary of the Invention

[0003] There is a need for techniques to map both the spatial transcriptome and the spatial proteome in the same tissue slice. Colocalization of protein and mRNA signals provides a better understanding of how mRNA and protein expression are co-regulated. Certain diseases (e.g., but not limited to, Alzheimer's disease) are characterized by abnormal protein deposits, and understanding how gene expression is altered near these protein deposits may elucidate the mechanisms underlying these disorders. Aptamers targeting cell membrane or nuclear membrane proteins can also be used to define cell and nuclear boundaries, simplifying cell segmentation of spatial transcriptome data.

[0004] Commercially available ex situ spatial assays capable of detecting both proteins and mRNA are limited to detecting only a few proteins using immunofluorescence. Future products capable of detecting many more proteins are based on oligo-conjugated antibodies. However, aptamers can also be used to detect proteins in situ and have several advantages over antibodies. Aptamers are oligonucleotides (e.g., DNA or RNA oligonucleotides) that can specifically bind to proteins. Due to their smaller size compared to antibodies, aptamers can diffuse more easily into tissues. Aptamers are more stable and less sensitive to temperature and pH changes. Aptamers can also be manufactured more reproducibly and on a larger scale compared to antibodies.

[0005] Aptamers can be modified with sequences that allow capture on barcoded surfaces. However, designing a spatial mRNA / protein simultaneous assay presents several challenges. 1. The addition of long sequences to aptamers to enable capture may affect protein binding by aptamers. 2. Aptamers are difficult to remove from their target proteins. 3. Free aptamers that are not bound to proteins must be prevented from binding to the barcoded surface. 4. Proteins are much more abundant than mRNA molecules, and protein levels span a wide dynamic range.

[0006] In various aspects, the present disclosure provides methods for using aptamers in simultaneous spatial mRNA / protein assays that address these challenges.

[0007] In some aspects, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) a surface, comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3′ end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier. (b) contacting a plurality of aptamers on the surface with a biological sample, wherein the contacting results in association of individual aptamers in the plurality of aptamers with individual proteins in the biological sample, each aptamer in the plurality of aptamers comprising (i) a target nucleotide sequence, (ii) an aptamer barcode nucleotide sequence, and (iii) a cleavage site; (c) removing aptamers in the plurality of aptamers that did not associate with a protein in the biological sample; and (d) cleaving the plurality of aptamers to release (i) the target nucleotide sequence and (ii) the aptamer barcode nucleotide sequence, thereby resulting in association of the target nucleotide sequence with a capture nucleotide sequence, thereby preparing a spatial proteome sequencing library. In some embodiments, the UMI comprises the spatial barcode nucleotide sequence, and is included in the aptamer but not in the capture nucleotide sequence. In some embodiments, the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence. In some embodiments, the blocker nucleic acid is removed from the capture oligonucleotide after step (c). In various embodiments, the plurality of aptamers are cleaved via ultraviolet radiation, enzymes, or chemical cleavage. In some embodiments, the methods of the present disclosure further comprise (e) extending the capture nucleotide sequence to create copies of individual aptamers, thereby creating extended capture oligonucleotides.In some embodiments, the methods of the present disclosure further comprise (f) adding a template switch oligonucleotide (TSO) to the 3' end of the extended capture oligonucleotide.

[0008] In some aspects, the present disclosure provides a method of preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3′ end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, the UMI comprising a spatial barcode nucleotide sequence; and (b) contacting a plurality of aptamers with the biological sample on the surface, wherein the contacting comprises: The method includes contacting an individual aptamer in the plurality of aptamers with an individual protein in the biological sample, wherein each aptamer in the plurality of aptamers comprises (i) a target nucleotide sequence, (ii) an aptamer barcode nucleotide sequence, and (iii) a truncated adapter nucleotide sequence; (c) removing aptamers in the plurality of aptamers that did not associate with a protein in the biological sample; and (d) eluting the individual aptamers from the individual proteins, thereby resulting in association of the target nucleotide sequence with a capture nucleotide sequence, thereby preparing a spatial proteome sequencing library. In some embodiments, the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence. In further embodiments, the blocker nucleic acid is removed from the capture oligonucleotide after step (c). In some embodiments, the method of the present disclosure further comprises (e) extending the capture nucleotide sequence to create copies of the individual aptamers, thereby creating extended capture oligonucleotides. In some embodiments, the methods of the present disclosure further comprise (f) hybridizing and extending the truncated adapter nucleotide sequence to the full-length adapter nucleotide sequence primer to synthesize a second strand.

[0009] In some aspects, the disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3′ end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; and (b) contacting a plurality of aptamers with the biological sample on the surface, wherein the contacting results in association of each aptamer complex in the plurality of aptamer complexes with each protein in the biological sample, and each aptamer complex in the plurality of aptamer complexes comprises: (1) (i) a capture nucleotide sequence at a 3′ end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence. and (ii) an aptamer-specific nucleotide sequence; and (2) an oligonucleotide hybridized to the aptamer prior to the contacting, the oligonucleotide comprising (i) a target nucleotide sequence, (ii) a sequence complementary to the aptamer-specific nucleotide sequence, and (iii) an aptamer barcode nucleotide sequence. After association of each aptamer complex in the plurality of aptamer complexes with each protein in the biological sample, the oligonucleotide is released from the aptamer, thereby resulting in association of the target nucleotide sequence of the released oligonucleotide with a capture nucleotide sequence of one capture oligonucleotide of the plurality of capture oligonucleotides, thereby preparing a spatial proteome sequencing library. In some embodiments, the aptamer-specific nucleotide sequence is about 5 to about 20 nucleotides in length. In further embodiments, the aptamer-specific nucleotide sequence is about 10 nucleotides in length. In some embodiments, the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence. In a further embodiment, the blocker nucleic acid is removed from the capture oligonucleotide after contacting.In some embodiments, association of an individual aptamer complex in the plurality of aptamer complexes with an individual protein in the biological sample results in release of the oligonucleotide from the aptamer. In some embodiments, after association of an individual aptamer complex in the plurality of aptamer complexes with an individual protein in the biological sample, conditions are changed, thereby resulting in release of the oligonucleotide from the aptamer. In further embodiments, the conditions are temperature, pH, or salt concentration. In yet further embodiments, after association of an individual aptamer complex in the plurality of aptamer complexes with an individual protein in the biological sample, formamide is added, thereby resulting in release of the oligonucleotide from the aptamer. In various embodiments, the blocker oligonucleotide is removed from the capture oligonucleotide by exonuclease digestion. In further embodiments, the exonuclease digestion is performed using T7 exonuclease or lambda exonuclease. In some embodiments, the aptamer comprises a detectable moiety. In some embodiments, the detectable moiety is a fluorescent moiety.

[0010] In a further aspect, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3′ end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, the UMI comprising a spatial barcode nucleotide sequence; and (b) contacting a plurality of aptamers with the biological sample on the surface. (c) removing aptamers from the plurality of aptamers that did not associate with proteins in the biological sample; and (d) eluting the individual aptamers from the individual proteins, thereby resulting in hybridization of the target nucleotide sequence with the capture nucleotide sequence, thereby preparing a spatial proteome sequencing library. In some embodiments, the plurality of capture oligonucleotides comprise a cleavable site at their 5' ends. In some embodiments, step (d) further comprises contacting at least one aptamer from the plurality of aptamers with a blocker nucleic acid, thereby forming a blocked aptamer, wherein the blocker nucleic acid is complementary to the target nucleotide sequence, and the blocked aptamer is unable to associate with the capture nucleotide sequence. In some embodiments, the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence. In some embodiments, the blocker nucleic acid is removed from the capture oligonucleotide after step (c). In some embodiments, the elution in step (d) comprises digesting proteins in the biological sample or competing with excess aptamers. In some embodiments, the method further comprises (e) extending the capture nucleotide sequence to create copies of individual aptamers, thereby creating extended capture oligonucleotides.In some embodiments, step (e) further comprises hybridizing a plurality of aptamer-barcoded oligonucleotides to an extended capture oligonucleotide and extending the extended capture oligonucleotide, thereby creating a plurality of barcoded capture oligonucleotides, each of the aptamer-barcoded oligonucleotides comprising at least a portion of an individual aptamer sequence. In some embodiments, the plurality of aptamer-barcoded oligonucleotides comprises a plurality of aptamer blocker nucleic acids, each of the aptamer blocker nucleic acids comprising at least a portion of an individual aptamer sequence.

[0011] In some embodiments, the method further comprises contacting a second plurality of aptamers with the biological sample on the surface, wherein the contacting results in association of individual aptamers in the second plurality of aptamers with individual proteins in the biological sample, and each aptamer in the second plurality of aptamers comprises a detectable moiety. In some embodiments, the detectable moiety is a fluorophore. In some embodiments, each aptamer in the second plurality of aptamers comprises a target nucleotide sequence and a truncated adapter nucleotide sequence. In some embodiments, each aptamer in the second plurality of aptamers further comprises an aptamer barcode nucleotide sequence. In some embodiments, after contacting the second plurality of aptamers with the biological sample on the surface, the method further comprises imaging the biological sample, thereby obtaining an image of the biological sample. In some embodiments, the method does not comprise contacting the biological sample with a hematoxylin and eosin (H&E) staining reagent. In some embodiments, at least one aptamer in the second plurality of aptamers is specific for a cell membrane-associated protein. In some embodiments, at least one aptamer in the second plurality of aptamers is specific for a nuclear membrane-associated protein. In some embodiments, at least one aptamer in the second plurality of aptamers is specific for a cell membrane-associated protein, at least one aptamer in the second plurality of aptamers is specific for a nuclear membrane-associated protein, and the at least one aptamer specific for the nuclear membrane-associated protein comprises a different detectable moiety than the at least one aptamer specific for the cell membrane-associated protein. In some embodiments, the at least one aptamer specific for the nuclear membrane-associated protein comprises a different aptamer barcode nucleotide sequence than the at least one aptamer specific for the cell membrane-associated protein. In some embodiments, the cell membrane-associated protein is E-cadherin, N-cadherin, or Na + / K + In some embodiments, the nuclear membrane-associated protein is a nuclear pore complex protein.

[0012] In any of the aspects of the present disclosure, the biological sample is from a mammal. In a further aspect, the biological sample is from a human.

[0013] In further aspects, the present disclosure provides methods for identifying a disorder in a subject having or at risk of having the disorder, comprising: i) generating a spatial proteome and / or transcriptome library from a biological sample from the subject according to the methods of the present disclosure; ii) comparing the proteome and / or genetic information from the sample proteome and / or transcriptome library with a control proteome and / or transcriptome library; and iii) identifying genetic variations in the sample proteome and / or transcriptome library that are associated with the disease. In some aspects, the disorder is a neurodegenerative disorder. In a further aspect, the disorder is Alzheimer's disease. [Brief explanation of the drawings]

[0014] [Figure 1] A spatial proteomics workflow is shown using an aptamer tagged with a polyA sequence at the 3' end and a truncated B15 adaptor at the 5' end. [Figure 2] We show how the methods provided herein can be modified to be compatible with simultaneous spatial protein / mRNA assays. [Figure 3] 1 shows an exemplary variation of the workflow of the present disclosure that uses an aptamer containing a cleavable tag. [Figure 4] 1 shows an exemplary variation of the disclosed workflow that uses a tagged aptamer bound to single-stranded DNA, which is released from the tagged aptamer upon protein binding. [Figure 5] 1 illustrates an exemplary method provided by the present disclosure that addresses the problem of accommodating proteins with large dynamic ranges and copy numbers. [Figure 6A]Figure 6A shows an exemplary variation of the disclosed workflow using modified aptamers containing unique barcodes and adapter sequences, where a blocker oligonucleotide complementary to the aptamer adapter sequence is included to accommodate a large dynamic range of protein abundance. Figure 6A is a representative illustration of a cellular environment containing a mixture of proteins (labeled "1," "2," and "X" for mRNA-binding proteins) and mRNA. [Figure 6B] Figure 6B shows an exemplary variation of the disclosed workflow using modified aptamers containing unique barcodes and adapter sequences, where a blocker oligonucleotide complementary to the aptamer adapter sequence is included to accommodate a large dynamic range of protein abundance. Figure 6B shows the step of permeabilizing cells to allow the modified aptamers to enter the cells. A wash step (not shown) removes unbound aptamers. [Figure 6C] Figure 6C shows an exemplary variation of the disclosed workflow using modified aptamers containing unique barcodes and adapter sequences, where a blocker oligonucleotide complementary to the aptamer adapter sequence is included to accommodate a large dynamic range of protein abundance. Figure 6D shows immobilized surface primers hybridized to the blocking oligonucleotide. These surface primers can have the same or different capture sequences. [Figure 6D] Figure 6D shows an exemplary variation of the disclosed workflow using modified aptamers containing unique barcodes and adapter sequences, where a blocker oligonucleotide complementary to the aptamer adapter sequence is included to accommodate a large dynamic range of protein abundance. Figure 6C shows the step of deblocking the surface primer to expose a free 3' end. [Figure 6E]Figure 6E shows an exemplary variation of the disclosed workflow using modified aptamers containing unique barcodes and adapter sequences, where a blocker oligonucleotide complementary to the aptamer adapter sequence is included to accommodate a large dynamic range of protein abundance. Figure 6F shows the steps of digesting the protein to release the mRNA and aptamer, and optionally adding a dynamic range compression (DRC) blocker oligonucleotide specific for the abundant protein aptamer. [Figure 6F] Figure 6F shows an exemplary variation of the disclosed workflow using a modified aptamer containing a unique barcode and adapter sequence, with a blocker oligonucleotide complementary to the aptamer adapter sequence included to accommodate a large dynamic range of protein abundance. Figure 6F illustrates the capture of released mRNA and aptamer barcodes onto an immobilized surface primer, followed by reverse transcription and polymerase extension to generate covalently attached complements of the mRNA and aptamer barcode. In some embodiments, the mRNA is captured on a surface after permeabilization in the presence of an aptamer to minimize loss of cellular mRNA in the process. In some embodiments, the mRNA capture sequence is unblocked and the aptamer capture sequence is blocked. [Figure 7A] An exemplary variation of the disclosed workflow, which uses a DRC blocker to maintain abundant SOMAmers in solution, is shown. Figure 7A shows cell permeabilization, aptamer binding, and protein digestion followed by reverse transcription and polymerase extension of the released mRNA and SOMAmer onto an immobilized surface primer. STUBBY corresponds to the universal adapter sequence at the 3' end of the aptamer (i.e., SOMAmer). The addition of a DRC blocker (i.e., a blocker oligo containing a complement to the STUBBY sequence and a complement to the SOMAmer sequence) to the SOMAmer. [Figure 7B]Figure 7B shows an exemplary variation of the disclosed workflow that uses a DRC blocker to maintain abundant SOMAmers in solution. Following reverse transcription and polymerase extension, unbound mRNA and SOMAmers are washed away, leaving behind immobilized extended surface primers containing the complement of the mRNA's cDNA or STUBBY and SOMAmer sequences. [Figure 7C] Figure 7C shows an exemplary variation of the disclosed workflow that uses a DRC blocker to maintain abundant SOMAmers in solution. Figure 7C illustrates the addition of a SOMAmer barcode to an extended surface primer containing the complement of the SOMAmer sequence, and an oligo containing the SOMAmer-SEQ sequence, the barcode sequence, and the B-15′ sequencing primer sequence is hybridized to the extended surface primer and copied. A "dummy" oligo, lacking the barcode sequence and sequencing primer sequence, can be included to act as a DRC blocker for highly abundant SOMAmers. [Figure 7D] Figure 7D shows an exemplary variation of the disclosed workflow that uses DRC blockers to maintain abundant SOMAmers in solution. Figure 7D shows a library preparation process in which a cleavage step to release extended surface primers containing SOMAmer barcodes and B-15 adapter sequences is followed by the addition of sequencing adapters using PCR. Sequencing the barcodes of SOMAmers provides an alternative method for identifying SOMAmer sequences without directly sequencing them. [Figure 8A] Figure 8A shows an exemplary variation of the disclosed workflow using fluorescently labeled aptamers targeting cell membranes, e.g., for cell segmentation analysis and image alignment processes. Figure 8A shows the steps of attaching the fluorescently labeled aptamers to proteins, flushing away any unbound aptamers, and then imaging the tissue to visualize the bound aptamers. Immobilized surface primers on a solid support are blocked, e.g., with complementary blocking oligonucleotides. [Figure 8B]Figure 8B shows an exemplary variation of the disclosed workflow using fluorescently labeled aptamers targeted to the cell membrane, e.g., for cell segmentation analysis and image alignment processes. Figure 8B shows the steps of removing the blocking oligo from the surface primer using, e.g., T7 or lambda exonuclease, eluting the protein-bound aptamer using excess unlabeled aptamer, capturing the eluted aptamer, extending the surface primer to copy the aptamer to the surface, and performing second strand synthesis using a full B15 adapter sequence oligo hybridized to a truncated B15 adapter. [Figure 9A] Various examples of cell membrane-targeting aptamers and nuclear membrane-targeting aptamers are shown. Figure 9A shows aptamers targeting multiple types of cell membranes that contain a common fluorophore and barcode, for example, to enable membrane detection across different tissue types. Each aptamer can contain a cell membrane barcode and a truncated B15 adapter sequence. [Figure 9B] Figure 9B shows various examples of aptamers targeting the cell membrane and nuclear membrane. Figure 9B shows an exemplary aptamer for co-detection of cell membrane and nuclear membrane proteins using differentially labeled aptamers (e.g., aptamers with different fluorophores and barcode sequences). DETAILED DESCRIPTION OF THE INVENTION

[0015] The emerging field of spatial proteogenomics is being driven by the development of new technologies that enable the mapping of single-cell organisms to their spatial locations within tissue sections. One method for spatially mapping single-cell transcriptomes (called the ex situ approach) involves the use of a surface coated with barcoded oligonucleotides, where the spatial location of each barcode is known. The barcoded oligonucleotides are localized to individual features, where all oligonucleotides in the same feature carry the same spatial barcode. Different implementations of this surface include bead arrays, spotted arrays, clustered flow cells, or clustered particles arranged on the surface. These oligonucleotides also contain oligo(dT) capture sequences that bind to mRNA and act as primers for reverse transcription. A tissue section is then placed on this surface, and polyA mRNA molecules within the tissue are diffused into the features and captured on the surface. The captured RNA is reverse transcribed into cDNA, linking the spatial barcode to the cDNA sequence. Following this, libraries are prepared and sequenced using a standard (e.g., Illumina) sequencer. During analysis, the spatial barcode is used to map the physical location of the molecule from which the read originates.

[0016] term As used in this specification and enumerated paragraphs herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0017] "About" and "approximately" generally refer to an acceptable degree of error for the quantity measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20-25 percent (%) of the stated value or range of values, e.g., within 20 percent, 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent.

[0018] As used herein, the terms "includes," "including," "includes," "including," "contains," "containing," "have," "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product defined by a process, or composition of matter that includes or contains an element or list of elements may include not only those elements, but also other elements not expressly listed in or inherent in such process, method, product defined by a process, or composition of matter. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting.

[0019] As used herein, a "surface" may refer to a portion of a substrate or support structure that is accessible for contact with a reagent, bead, or analyte. A surface may be substantially flat or planar. Alternatively, a surface may be rounded or contoured. Exemplary contours that may be included on a surface include wells (e.g., microwells or nanowells), depressions, posts, ridges, channels, and the like. Exemplary materials that can be used as a substrate or support structure include glass, such as modified or functionalized glass; plastics, such as acrylic, polystyrene, or copolymers of styrene with another material, polypropylene, polyethylene, polybutylene, polyurethane, or TEFLON; polysaccharides or cross-linked polysaccharides, such as agarose or Sepharose; nylon; nitrocellulose; resins; silica or silica-based materials, including silicon and modified silicon, carbon fiber; metals; inorganic glasses; fiber optic bundles, or various other polymers. A single material or a mixture of several different materials can form a surface useful in the present invention. In some examples, the surface includes a well (e.g., a microwell or nanowell). In some embodiments, the surface comprises a well in an array of wells (e.g., microwells or nanowells) on glass, silicon, plastic, or other suitable solid support with a patterned, covalently linked gel, such as poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM, see, e.g., U.S. Patent Application Publication No. 2014 / 0079923 A1, incorporated herein by reference). In some examples, the support structure can comprise one or more layers. Non-limiting examples of surfaces include bead arrays, spot arrays, clustered particles disposed on the surface of a chip, films, multiwell plates, and flow cells.

[0020] In certain aspects, the "surface" and / or "substrate" disclosed herein can further comprise islands or clusters of immobilized capture agents or capture oligonucleotides. Islands or clusters can be generated on the surface of a substrate (e.g., a flow cell) by using bridge amplification. In such cases, the substrate comprises multiple immobilized capture oligonucleotides on the surface of the substrate, which bind to complementary adapter regions present on adjacent primers or oligonucleotides to form bridge-like structures. These bridge-like structures are then extended using a polymerase enzyme to generate double-stranded molecules, which are then denatured, leaving single-stranded capture oligonucleotides tethered to the substrate. After multiple repetitions of the aforementioned process, islands or clusters of immobilized capture oligonucleotides are created. Examples of the aforementioned processes that can be used with the methods and compositions disclosed herein can be found in WO 2022 / 015913 (A1), which is incorporated herein by reference in its entirety. In certain aspects, adjacent primers or oligonucleotides are attached to the substrate (e.g., a flow cell) by selectively cleavable linkers. Each island or cluster can be approximately circular or elliptical in shape. Each island or cluster can have an average diameter of 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, 1050 nm, 1100 nm, 1200 nm, or a range including or between any two of the foregoing diameters. In further embodiments, the surface of the substrate (e.g., a flow cell) has a surface area of ​​1 mm 2The present invention also encompasses 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 million clusters per substrate, or any range between or including any two of the foregoing numbers. In certain embodiments, a "substrate" disclosed herein comprises islands or clusters of immobilized capture oligonucleotides comprising an adapter sequence, a spatial address sequence, an optional sequence primer site, and a capture moiety for a targeted analyte. In yet further embodiments, each cluster or island on a substrate (e.g., a flow cell) comprises a capture oligonucleotide with a unique spatial address sequence, thereby allowing the x,y location of each cluster or island to be identified. In such cases, the x,y location of each cluster or island can be determined by decoding the spatial address sequence. Methods for decoding spatial address sequences include, but are not limited to, the hybridization-based or sequencing-based methods disclosed herein.

[0021] As used herein, the term "interstitial region" refers to an area within a substrate or on a surface that separates other areas of the substrate or surface. For example, a gap region can separate one feature of an array from another feature of the array. Two regions separated from each other can be distinct and lack contact with each other. In another example, a gap region can separate a first portion of a feature from a second portion of a feature. The separation provided by the gap region can be partial or complete. A gap region typically has a surface material that is different from the surface material of the features on the surface. For example, an array feature can have an amount or concentration of capture agent or capture oligonucleotide that exceeds the amount or concentration present in the gap region. In some embodiments, a capture agent or primer may not be present in the gap region.

[0022] In some embodiments, the substrate comprises an array of wells or depressions in its surface, which can be fabricated as commonly known in the art using a variety of techniques, including but not limited to photolithography, stamping techniques, molding techniques, and microetching techniques. As will be understood in the art, the technique used will depend on the composition and shape of the array substrate.

[0023] Exemplary flow cells include, but are not limited to, those used in nucleic acid sequencing devices such as the flow cells for the Genome Analyzer®, MiSeq®, NextSeq®, or HiSeq® platforms marketed by Illumina, Inc. (San Diego, Calif.), or the SOLiD™ or Ion Torrent™ sequencing platforms marketed by Life Technologies (Carlsbad, Calif.). Exemplary flow cells and methods for their manufacture and use are also described, for example, in International Publication No. WO 2014 / 142841 (A1); U.S. Patent Application Publication No. 2010 / 0111768 (A1); and U.S. Patent No. 8,951,781, each of which is incorporated herein by reference. The flow cell may be a "non-patterned flow cell," in which the surface of the flow cell includes randomly or semi-randomly arranged features (e.g., areas containing clusters or islands of oligonucleotides). Alternatively, the flow cell may be a "patterned flow cell," in which the flow cell comprises features (e.g., nanowells) at fixed locations across the surface of the flow cell. The features of a "patterned flow cell" may further comprise immobilized oligonucleotides, or clusters or islands of immobilized oligonucleotides. A "patterned flow cell" may be an "ordered substrate," in that the features of the patterned flow cell have assigned or readily determinable x,y spatial addresses.

[0024] "Complementary" means that an oligonucleotide contains a sequence of nucleotides that can form a double-stranded structure by base pairing with another oligonucleotide or portion thereof. "Complementary" means that the oligonucleotide has at least 85%, 90%, 95%, 98%, 99%, or 100% overall sequence identity to the complementary sequence.

[0025] In any of the embodiments of the present disclosure, the methods described herein include a sequencing procedure, such as, but not limited to, a sequencing-by-synthesis (SBS) technique or nanopore sequencing. Briefly, SBS can be initiated by contacting a barcode with one or more labeled nucleotides, a DNA polymerase, or the like. These features allow a primer to be extended using the sequence containing the barcode as a template, incorporating a labeled nucleotide that can be detected. Optionally, the labeled nucleotide can further include a reversible termination feature that terminates further primer extension once the nucleotide is added to the primer. For example, a nucleotide analog with a reversible terminator moiety can be added to the primer so that further extension does not occur until a deblocking agent is delivered to remove the moiety. Thus, in embodiments using reversible termination, a deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washing can be performed between the various delivery steps. The cycle is then repeated n times to extend the primer with n nucleotides, thereby allowing a sequence of length n to be detected. Exemplary SBS procedures, fluidics systems, and detection platforms that can be readily adapted for use with libraries produced by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, WO 91 / 06678, WO 07 / 123744, U.S. Pat. Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, or 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082 A1, each of which is incorporated herein by reference.

[0026] As used herein, a "primer" is a nucleic acid molecule capable of hybridizing to a target sequence, such as an adapter attached to a library fragment. In some embodiments, amplification primers can serve as the starting point for template amplification and cluster generation. In another example, a synthesized nucleic acid (template) strand may contain a site to which a primer (e.g., a sequencing primer) can hybridize to prime the synthesis of a new strand complementary to the synthesized nucleic acid strand. Any primer can contain any combination of nucleotides or their analogs. In some examples, primers are single-stranded oligonucleotides or polynucleotides. Primers can be any number of bases in length and can contain a variety of non-naturally occurring nucleotides. In various embodiments, sequencing primers are short strands ranging from 5-60 bases, 10-60 bases, 10-20 bases, 10-30 bases, 10-40 bases, 10-50 bases, or 20-40 bases. One skilled in the art can adjust these factors to provide optimal hybridization and signal generation for a given hybridization procedure. A primer allows for the addition of nucleotide residues to it or for oligonucleotide or polynucleotide synthesis therefrom under suitable conditions. In embodiments, the primer is a DNA primer, i.e., a primer consisting of or consisting predominantly of deoxyribonucleotide residues. The primer is designed to have a sequence that is the complement of the region of the template / target DNA to which the primer hybridizes. Addition of a nucleotide residue to the 3' end of the primer by forming a phosphodiester bond results in a DNA extension product. Addition of a nucleotide residue to the 3' end of the DNA extension product by forming a phosphodiester bond results in a further DNA extension product. In another embodiment, the primer is an RNA primer. In embodiments, the primer is hybridized to a target polynucleotide.A "primer" is complementary to a polynucleotide template and complexes with the template by hydrogen bonding or hybridization to provide a primer / template complex for initiation of synthesis by a polymerase, which is extended in the process of DNA synthesis by the addition of covalently bound bases linked at its 3' end that are complementary to the template.

[0027] As used herein, the term "unique molecular identifier" or "UMI" refers to a molecular tag that can be attached to a nucleic acid, either randomly, non-randomly, or semi-randomly. When incorporated into a nucleic acid, the unique molecular identifier (UMI) can be used to correct for subsequent amplification bias by directly counting the UMI when sequenced after amplification. The UMI can be attached to similar nucleic acids, such as adapters, making each nucleic acid unique. In some embodiments, the UMI comprises a spatial barcode.

[0028] As used herein, a "semi-random" nucleotide sequence comprises or consists of a partially predetermined nucleotide sequence combined with random nucleotide sequences.

[0029] As used herein, the term "adapter" generally refers to any linear nucleic acid molecule that can be added to an oligonucleotide of the present disclosure. In some embodiments, the adapter is copied onto a library molecule using templated polymerase synthesis. In some embodiments, the adapter comprises two reversibly complementary oligonucleotides that form a double-stranded structure. In some embodiments, the adapter comprises two oligonucleotides that are complementary in one portion and mismatched in another portion, forming a Y-shaped or fork-shaped adapter that is double-stranded in the complementary portion and has two floppy overhangs in the mismatched portion. In some embodiments, the adapter is a template switch oligonucleotide (TSO) adapter.

[0030] The term "template switch oligonucleotide" refers to an oligonucleotide template onto which polymerase activity is switched from an initial template (e.g., a single-stranded nucleic acid provided by a sample of the present invention). In one embodiment of the present invention, the template switch oligonucleotide is a DNA / RNA hybrid oligonucleotide used by a template-dependent DNA or RNA polymerase (preferably RT, preferably MMLV RT) to continue reverse transcription (i.e., template-independent) after the enzyme (preferably MMLV RT) reaches the 5' end of the template nucleic acid and adds nucleotides to the 3' end of the cDNA or cRNA strand synthesized by its terminal transferase activity. The 3' end of the TSO hybridizes to the nucleotides added by the terminal transferase activity of the template-dependent DNA or RNA polymerase, effectively extending the 5' end of the template DNA or RNA such that the template-dependent DNA or RNA polymerase (preferably RT, more preferably MMLV RT) also reverse transcribes the remaining 5' portion of the TSO, which contains the defined sequence to be added to the 5' end of the template nucleic acid. The TSO may contain one or more modified or non-naturally occurring nucleotides (or analogs thereof). For example, the template-switching oligonucleotide may contain one or more nucleotide analogs (e.g., LNA, FANA, 2'-O-methylribonucleotides, 2'-fluororibonucleotides, etc.), ligation modifications (e.g., phosphorothioates, 3'-3' and 5'-5' reverse ligation), 5' and / or 3' terminal modifications (e.g., 5' and / or 3' amino, biotin, DIG, phosphate, thiol, dye, quencher, etc.), one or more fluorescently labeled nucleotides, or any other feature that provides a desired function to the template-switching oligonucleotide.

[0031] The terms "P5" and "P7" may be used when referring to exemplary adapters. The terms "P5'" (P5 primer) and "P7'" (P7 primer) refer to the complements of P5 and P7, respectively. It will be understood that any suitable adapter can be used in the methods presented herein, and the use of P5 and P7 is exemplary only. The use of adapters such as P5 and P7 or their complements on flow cells is known in the art, as exemplified by the disclosures of WO 2007 / 010251, WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941, WO 1998 / 044151, and WO 2000 / 018957, which are incorporated by reference in their entireties. For example, any suitable forward amplification primer, whether immobilized or in solution, can be useful in the methods provided herein for hybridization to and amplification of complementary sequences and sequences. Similarly, any suitable reverse amplification primer, whether immobilized or in solution, can be useful in the methods provided herein for hybridization to and amplification of complementary sequences and sequences. Those skilled in the art will understand how to design and use suitable primer sequences for capture and / or amplification of nucleic acids as provided herein.

[0032] As used herein, the term "barcode" is intended to mean a series of nucleotides in an oligonucleotide that can be used to identify an oligonucleotide, a spatial address on a surface (i.e., a "spatial barcode" or "spatially addressable sequence"), a feature of the oligonucleotide, and / or an operation performed on the oligonucleotide. A barcode may be a naturally occurring nucleotide sequence or a nucleotide sequence that does not naturally occur in the organism from which the barcoded nucleic acid is obtained. In aspects, a barcode is unique in a pool of barcodes that differ from each other in sequence, or is uniquely associated with a particular sample polynucleotide in a pool of sample polynucleotides. In aspects, all barcodes in a pool of adapters are unique such that sequencing reads containing a barcode can be identified as originating from a single sample polynucleotide molecule based solely on the barcode. In other embodiments, individual barcode sequences may be used more than once, but the adapters comprising the overlapping barcodes are associated with different sequences and / or different combinations of barcoded adapters, such that sequence reads can still be uniquely distinguished as originating from a single sample polynucleotide molecule based on the barcode and flanking sequence information (e.g., the sample polynucleotide sequence and / or one or more flanking barcodes). In embodiments, the barcodes are about or at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, or more nucleotides in length. In embodiments, the barcodes are less than 20, 15, 10, 9, 8, 7, 6, or 5 nucleotides in length. In embodiments, the barcodes are about 10 to about 50 nucleotides in length, e.g., about 15 to about 40 or about 20 to about 30 nucleotides in length. In a pool of different barcodes, the barcodes can have the same or different lengths. Generally, barcodes are of sufficient length and contain sufficiently different sequences to allow for the discrimination of sequencing reads originating from the same sample polynucleotide molecule.In embodiments, each barcode in the plurality of barcodes differs from every other barcode in the plurality by at least three nucleotide positions, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions. In some embodiments, a sufficiently degenerate barcode may be known as random. In some embodiments, a barcode may comprise a nucleic acid sequence from within a pool of known sequences. In some embodiments, a barcode may be predefined.

[0033] As used herein, a "biological sample" can include one or more biological or chemical substances, such as nucleic acids, oligonucleotides, proteins, cells, tissues, organisms, and / or biologically active chemical compounds, such as analogs or mimetics of the aforementioned species. In some cases, a biological sample may include whole blood, lymph, serum, plasma, sweat, tears, saliva, sputum, cerebrospinal fluid, amniotic fluid, semen, vaginal discharge, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudate, exudate, cystic fluid, bile, urine, gastric juice, intestinal fluid, fecal sample, fluid containing single or multiple cells, fluid containing cellular organelles, tissue fluid, biological fluid, viruses including viral pathogens, fluid containing multicellular organisms, biological swabs, and biological washes. In further examples, the sample may be derived from an organ comprising, for example, an organ of the musculoskeletal system, such as muscle, bone, tendon, or ligament; an organ of the digestive system, such as the salivary gland, pharynx, esophagus, stomach, small intestine, large intestine, liver, gallbladder, or pancreas; an organ of the respiratory system, such as the larynx, trachea, bronchi, lungs, or diaphragm; an organ of the urinary system, such as the kidney, ureter, bladder, or urethra; a reproductive organ, such as the ovaries, fallopian tubes, uterus, vagina, placenta, testes, epididymis, vas deferens, seminal vesicles, prostate, penis, or scrotum; an organ of the endocrine system, such as the pituitary gland, pineal gland, thyroid gland, parathyroid gland, or adrenal gland; an organ of the circulatory system, such as the heart, arteries, veins, or capillaries; an organ of the lymphatic system, such as the lymphatic vessels, lymph nodes, bone marrow, thymus, or spleen; an organ of the central nervous system, such as the brain, brainstem, cerebellum, spinal cord, cranial nerves, or spinal nerves; a sensory organ, such as the eye, ear, nose, or tongue; or an organ of the integument, such as the skin, subcutaneous tissue, or mammary gland. In various embodiments, the tissue may be derived from a multicellular organism. In some embodiments, a tissue section may be contacted with the surface, for example, by placing the tissue on the surface. The tissue may be freshly excised from the organism, or the tissue may have been previously preserved, for example, by freezing (e.g., fresh frozen tissue), embedding in a material such as paraffin (e.g., formalin fixed paraffin embedded (FFPE) sample), formalin fixation, infiltration, dehydration, etc. Optionally, the tissue section may be attached to the surface using, for example, the techniques and compositions described in U.S. Pat. No. 11,390,912, the entire contents of which are incorporated herein by reference.In some embodiments, tissue can be permeabilized, allowing cells in the tissue to lyse when the tissue contacts the surface. Any of a variety of treatments can be used, such as those described above for lysing cells. Target proteins and / or nucleic acids released from the permeabilized tissue can be captured by capture oligonucleotides on the surface. The thickness of the tissue or other biological sample contacted with the surface in the methods described herein can be any suitable thickness desired. In representative embodiments, the thickness is at least 0.1 μm, 0.25 μm, 0.5 μm, 0.75 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, or greater. Alternatively, or in addition, the thickness of the biological sample contacted with the surface can be 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 0.25 μm, 0.1 μm, or less.

[0034] As used herein, the term "permeability" refers to a property of a material that allows a substance to pass through it. "Permeability" can be used to describe a biological sample, such as a cell or nucleus, that allows an analyte in the biological sample to leave the biological sample. "Permeabilization" is, for example, an action taken to cause a biological sample (e.g., a cell) to release its analyte. In some examples, permeabilization of a biological sample is achieved by affecting (e.g., damaging) the integrity of a biological sample membrane (e.g., a cell membrane or a nuclear membrane), for example, by applying a protease or other enzyme that can disrupt the membrane and allow the analyte to diffuse out of the biological sample. In some aspects, permeabilizing a biological sample does not release biomolecules (e.g., proteins and / or nucleic acids) contained within the sample.

[0035] As used herein, a "capture oligonucleotide" generally refers to an oligonucleotide comprising a nucleotide sequence capable of hybridizing to or otherwise associating with an aptamer or other oligonucleotide as described herein (e.g., mRNA, a single-stranded oligonucleotide released from an aptamer complex after association of the aptamer complex with a protein, a probe that binds to an mRNA target). Nucleotide sequences capable of hybridizing to or otherwise associating with an aptamer or other oligonucleotide include, but are not limited to, a universal sequence (e.g., a poly-T sequence) or a target-specific sequence. Capture oligonucleotides may contain additional elements, including, but not limited to, a unique molecular identifier (UMI), a spatial barcode, a primer sequence for amplification (e.g., A14-ME), a sequence used to generate a barcoded feature (e.g., a P7 sequence used in clustering and an SBS12 sequence used as a sequencing primer binding site), or a combination thereof.

[0036] As used herein, "universal sequence" refers to a nucleotide sequence that is common among multiple capture oligonucleotides.The common nucleotide sequence can be, for example, a sequence that is complementary to the same adapter sequence.A universal capture oligonucleotide can be used to examine multiple different oligonucleotides without necessarily distinguishing between different species, while a target-specific capture sequence can be used to distinguish between different species.A non-limiting example of a universal sequence is a poly-T nucleotide sequence.

[0037] As used herein, "hybridize" is intended to mean the non-covalent association of a first oligonucleotide to a second oligonucleotide along the length of the polymer to form a double-stranded "duplex." For example, two DNA oligonucleotide strands may associate through complementary base pairing. The strength of association between a first and second oligonucleotide increases with the complementarity between the nucleotide sequences within the oligonucleotides. The strength of hybridization between oligonucleotides is determined by the melting temperature (T), at which 50% of the oligonucleotide strands in the duplex have dissociated from each other. m Oligonucleotides that are "partially" hybridized to one another mean that they have sequences that are complementary to one another, but that such sequences hybridize to one another along only a portion of their length, forming a partial duplex. Oligonucleotides that are "unable" to hybridize include those that are physically separated from one another, with an insufficient number of bases that can contact one another to hybridize. For example, hybridization can be performed at temperatures ranging from 15°C to 95°C. In some embodiments, hybridization is performed at temperatures of about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, or about 95°C. In other embodiments, the stringency of hybridization can be further altered by the addition or removal of components of the buffer solution.

[0038] As used herein, the term "plurality" is intended to mean a population of two or more members, which may all be the same or the two or more members may be different. Pluralities can range in size from small, medium, large, to very large. A small-sized plurality can range, for example, from a few members to tens of members. A medium-sized plurality can range, for example, from tens of members to about 100 or hundreds of members. A large plurality can range, for example, from about hundreds of members to about 1,000 members, thousands of members, and tens of thousands of members. A very large plurality can range, for example, from tens of thousands of members to about hundreds of thousands, millions, tens of millions, or hundreds of millions or more members. Thus, pluralities can range in size from 2 to well over 100 million members, as well as all sizes measured by number of members and ranges larger than the exemplary ranges listed above. Therefore, the definition of this term is intended to include all integer values ​​greater than 2. The upper limit of the plurality can be set, for example, by the theoretical limit of oligonucleotides (e.g., capture oligonucleotides) on a surface.

[0039] In some embodiments, the nucleic acid comprises a label. As used herein, the term "label" is used according to its clear and ordinary meaning to refer to a molecule that can directly or indirectly produce or result in a detectable signal by itself or upon interaction with another molecule. Non-limiting examples of detectable labels include fluorescent dyes, biotin, digoxin, haptens, and epitopes. Generally, a dye is a molecule, compound, or substance that can provide an optically detectable signal, such as a colorimetric, luminescent, bioluminescent, chemiluminescent, phosphorescent, or fluorescent signal. In embodiments, the label is a dye. In embodiments, the dye is a fluorescent dye. Non-limiting examples of dyes, some of which are commercially available, include CF dyes (Biotium, Inc.), Alexa Fluor dyes (Thermo Fisher), DyLight dyes (Thermo Fisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes (Anaspec, Inc.). In embodiments, specific nucleotide types are associated with specific labels, such that identifying the label identifies the nucleotide with which it is associated. In embodiments, the label is luciferin, which reacts with luciferase to produce a detectable signal in response to one or more bases being incorporated into an elongated complementary strand, such as in pyrosequencing. In embodiments, the nucleotide comprises a label (e.g., a dye). In embodiments, the label is not associated with any particular nucleotide, but detection of the label identifies whether one or more nucleotides of known identity have been added during the extension step (e.g., in pyrosequencing). Examples of detectable agents (i.e., labels) include imaging agents, including fluorescent and luminescent substances, molecules, or compositions, including, but not limited to, various organic or inorganic small molecules commonly referred to as "dyes," "labels," or "indicators." Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. In embodiments, the detectable moiety is a fluorescent molecule (eg, an acridine dye, a cyanine dye, a fluorine dye, an oxazine dye, a phenanthridine dye, or a rhodamine dye).In embodiments, the detectable moiety is a fluorescent molecule (e.g., an acridine dye, a cyanine dye, a fluorine dye, an oxazine dye, a phenanthridine dye, or a rhodamine dye). As used herein, the term "cyanine" or "cyanine moiety" refers to a detectable moiety containing two nitrogen groups separated by a polymethine linkage. In embodiments, the cyanine moiety has three methine structures (i.e., cyanine 3 or Cy3). In embodiments, the cyanine moiety has five methine structures (i.e., cyanine 5 or Cy5). In embodiments, the cyanine moiety has seven methine structures (i.e., cyanine 7 or Cy7).

[0040] Oligonucleotides Oligonucleotides are polymers composed of nucleotides. The oligonucleotides (e.g., aptamers) of the present disclosure can be of any length, and in various embodiments, include DNA oligonucleotides, RNA oligonucleotides, their analogs, or combinations thereof. In any embodiment described herein, the oligonucleotides are single-stranded, double-stranded, or partially double-stranded.

[0041] Nucleotides may include naturally occurring nucleotides and their functional analogs. Examples of functional analogs are those that are capable of hybridizing to nucleic acids in a sequence-specific manner or that can be used as templates for replicating a specific nucleotide sequence. Naturally occurring nucleotides generally have a backbone containing phosphodiester bonds. Analog structures can have alternative backbone linkages, including any of a variety known in the art. Naturally occurring nucleotides generally have a deoxyribose sugar (e.g., found in DNA) or a ribose sugar (e.g., found in RNA). Analog structures can have alternative sugar moieties, including any of a variety known in the art. Nucleotides can contain natural or unnatural bases. Natural DNA can contain one or more of adenine, thymine, cytosine, and / or guanine, while natural RNA can contain one or more of adenine, uracil, cytosine, and / or guanine. Any unnatural base can be used, such as locked nucleic acids (LNA) and bridged nucleic acids (BNA). Examples of modified nucleotides include inosine, xanthate, hypoxanthate, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine. These include tosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, and the like.As is known in the art, certain nucleotide analogs cannot become incorporated into polynucleotides, e.g., nucleotide analogs such as adenosine 5'-phosphosulfate. A nucleotide may contain any suitable number of phosphates, e.g., 3, 4, 5, 6, or more than 6 phosphates.

[0042] Oligonucleotides contemplated by the present disclosure also include those having at least one modified internucleoside linkage. In some embodiments, the oligonucleotide is entirely or partially a peptide nucleic acid. Other modified internucleoside linkages include at least one phosphorothioate linkage. Still other modified oligonucleotides include those containing one or more universal bases. A "universal base" refers to a molecule that can replace any one of A, C, G, T, and U in a nucleic acid by forming a hydrogen bond without significant structural destabilization. Examples of universal bases include, but are not limited to, 5'-nitroindole-2'-deoxyriboside, 3-nitropyrrole, inosine, and hypoxanthine.

[0043] In various embodiments, the oligonucleotides of the present disclosure, or modified forms thereof, are generally from about 5 to about 150 nucleotides in length. In further embodiments, the oligonucleotides of the present disclosure are from about 5 to about 125 nucleotides in length, from about 5 to about 100 nucleotides in length, from about 5 to about 90 nucleotides in length, from about 5 to about 50 nucleotides in length, from about 5 to about 45 nucleotides in length, from about 5 to about 40 nucleotides in length, from about 5 to about 35 nucleotides in length, from about 5 to about 30 nucleotides in length, from about 5 to about 25 nucleotides in length, from about 5 to about 20 nucleotides in length, from about 5 to about 15 nucleotides in length, from about 5 to about 10 nucleotides in length, from about 10 to about 150 nucleotides in length, from about 10 to about 125 nucleotides in length, or from about 5 to about 150 nucleotides in length. nucleotides in length, about 10 to about 100 nucleotides in length, about 10 to about 90 nucleotides in length, about 10 to about 50 nucleotides in length, about 10 to about 45 nucleotides in length, about 10 to about 40 nucleotides in length, about 10 to about 35 nucleotides in length, about 10 to about 30 nucleotides in length, about 10 to about 25 nucleotides in length, about 10 to about 20 nucleotides in length, about 10 to about 15 nucleotides in length, and all oligonucleotides of lengths intermediate to the specifically disclosed sizes to the extent that the oligonucleotide achieves the desired result.Thus, in various aspects, the oligonucleotides of the present disclosure can be any of the following: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 9, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 , 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or more nucleotides in length, or is at least that nucleotide in length.In further aspects, the oligonucleotides of the present disclosure are 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 3, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156 7, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, and less than 23, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or more nucleotides in length.

[0044] As used herein, the terms "poly T" or "poly A," when used in reference to a nucleic acid sequence, are intended to mean a string of two or more thiamine (T) or adenine (A) bases, respectively. The poly T or poly A may contain at least about 2, 5, 8, 10, 12, 15, 18, 20, or more T or A bases, respectively. Alternatively, or in addition, the poly T or poly A may contain up to about 30, 20, 18, 15, 12, 10, 8, 5, or 2 T or A bases, respectively. In some embodiments, the present disclosure contemplates the use of a "poly T V N" sequence, which is a poly T sequence followed by a V (any base other than T) and an N. The poly T V N sequence is used in some embodiments to bias reverse transcription toward the bases of the poly A tail on an mRNA molecule.

[0045] As used herein, the term "immobilized," when used with respect to oligonucleotides, is intended to mean direct or indirect attachment to a surface via covalent or non-covalent bonds. While covalent attachment may be used in certain embodiments, all that is required is that the oligonucleotide remain immobilized or attached to the surface under the conditions under which the surface is intended to be used, e.g., in applications requiring nucleic acid capture, amplification, and / or sequencing. Oligonucleotides used as capture oligonucleotides can be immobilized such that the 3' end is available for enzymatic extension and at least a portion of the sequence is capable of hybridizing to a complementary sequence. Immobilization can occur via hybridization to a surface-attached oligonucleotide, in which case the immobilized oligonucleotide or polynucleotide can be in a 3' to 5' orientation. Alternatively, immobilization of the oligonucleotide can involve the use of a selectively cleavable linker. Examples of selectively cleavable linkers include, but are not limited to, biotin-based molecules (e.g., desthiobiotin molecules (ddBio)), PCR linkers, and recognition sites for rare-cutter enzymes. Typically, the selectively cleavable linker can be cleaved by heating, competitive binding, pH change, chemical cleavage, enzymatic cleavage, and / or photocleavage. Cleavage of the selectively cleavable linker results in release of the nucleic acid or portion thereof from the substrate or substrate feature.

[0046] Certain embodiments employ an inert substrate or matrix (e.g., glass slide, polymer beads, etc.) that has been functionalized by the application of a layer or coating of an intermediate material containing reactive groups that allow for covalent attachment of, for example, biomolecules such as polynucleotides. Examples of such substrates include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, particularly the polyacrylamide hydrogels described in International Publication No. WO 2005 / 065814 and U.S. Patent Application Publication No. 2008 / 0280773, the contents of which are incorporated herein by reference in their entireties. In such embodiments, biomolecules (e.g., polynucleotides) can be covalently attached directly to the intermediate material (e.g., hydrogel), although the intermediate material may itself be noncovalently attached to the substrate or matrix (e.g., glass substrate). The term "covalent attachment to a substrate" should be interpreted accordingly to encompass this type of arrangement.

[0047] Exemplary covalent linkages include, for example, those resulting from the use of click chemistry techniques. Exemplary non-covalent linkages include, but are not limited to, non-specific interactions (e.g., hydrogen bonds, ionic bonds, van der Waals interactions, etc.) or specific interactions (e.g., affinity interactions, receptor-ligand interactions, antibody-epitope interactions, avidin-biotin interactions, streptavidin-biotin interactions, lectin-carbohydrate interactions, etc.). Exemplary linkages are described in U.S. Patent Nos. 6,737,236, 7,259,258, 7,375,234, and 7,427,678, and U.S. Patent Publication No. 2011 / 0059865(A1), each of which is incorporated herein by reference.

[0048] As used herein, the term "extending," when used with respect to a nucleic acid, is intended to mean the addition of at least one nucleotide or oligonucleotide to a nucleic acid. In certain embodiments, one or more nucleotides can be added to the 3'-end of a nucleic acid, for example, via polymerase catalysis (e.g., DNA polymerase, RNA polymerase, or reverse transcriptase). Chemical or enzymatic methods can be used to add one or more nucleotides to the 3'- or 5'-end of a nucleic acid. One or more oligonucleotides can be added to the 3'- or 5'-end of a nucleic acid, for example, by chemical or enzymatic (e.g., ligase-catalyzed) methods. Nucleic acids can be extended in a template-directed manner, whereby the extension product is complementary to a template nucleic acid hybridized to the nucleic acid being extended.

[0049] As used herein, the terms "DNA polymerase" and "nucleic acid polymerase" are used according to their plain ordinary meaning and refer to an enzyme capable of synthesizing a nucleic acid molecule from nucleotides (e.g., deoxyribonucleotides). Typically, a DNA polymerase adds nucleotides to the 3' end of a DNA strand one nucleotide at a time. In embodiments, the DNA polymerase is Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol β DNA polymerase, Pol μ DNA polymerase, Pol λ DNA polymerase, Pol σ DNA polymerase, Pol α DNA polymerase, Pol δ DNA polymerase, Pol ε DNA polymerase, Pol η DNA polymerase, Pol ι DNA polymerase, Pol κ DNA polymerase, Pol ζ DNA polymerase, Pol γ DNA polymerase, Pol θ DNA polymerase, Pol υ DNA polymerase, or a thermophilic nucleic acid polymerase (e.g., Therminator γ, 9°N polymerase (exo-), Therminator II, Therminator III, or Therminator IX). In embodiments, the DNA polymerase is a modified archaeal DNA polymerase. In some embodiments, the polymerase is a reverse transcriptase. For example, the polymerase catalyzes the addition of the next correct nucleotide to the 3'-OH group of the primer via a phosphodiester bond, thereby chemically incorporating the nucleotide into the primer. Optionally, the polymerase used in the provided method is a processive polymerase. Optionally, the polymerase used in the provided method is a partitioning polymerase.

[0050] As used herein, the term "exonuclease activity" is used according to its ordinary meaning in the art and refers to the removal of nucleotides from nucleic acids by a DNA polymerase. For example, during polymerization, a nucleotide is added to the 3'-end of a primer strand. Occasionally, a DNA polymerase incorporates an incorrect nucleotide at the 3'-OH end of the primer strand, where the incorrect nucleotide is unable to form a hydrogen bond to the corresponding base in the template strand. Such an incorrectly added nucleotide is removed from the primer as a result of the 3' to 5' exonuclease activity of the DNA polymerase. In some embodiments, exonuclease activity may be referred to as "proofreading." When referring to 3' to 5' exonuclease activity, it is understood that the DNA polymerase breaks the phosphodiester bond at the 3' end of the polynucleotide chain to facilitate a hydrolysis reaction that excises the nucleotide. In embodiments, 3' to 5' exonuclease activity refers to the sequential removal of nucleotides in single-stranded DNA in the 3' to 5' direction, releasing deoxyribonucleoside 5'-monophosphates one after the other. Methods for quantifying exonuclease activity are known in the art; see, for example, Southworth et al., PNAS Vol. 93, 8281-8285 (1996). In embodiments, 5' to 3' exonuclease activity refers to the sequential removal of nucleotides in double-stranded DNA in the 5' to 3' direction. In embodiments, the 5' to 3' exonuclease is lambda exonuclease. For example, lambda exonuclease catalyzes the removal of 5' mononucleotides from duplex DNA with a preference for 5' phosphorylated double-stranded DNA. In other embodiments, the 5' to 3' exonuclease is E. coli DNA polymerase I.

[0051] As used herein, the term "cleavable linker" or "cleavable moiety" refers to a divalent or monovalent moiety, respectively, that can be separated into separate entities (e.g., by elimination, fission, scission, hydrolysis, or rupture of a stable bond within the moiety). A cleavable linker is cleavable (e.g., specifically cleavable) in response to an external stimulus (e.g., an enzyme, a nucleophilic / basic reagent, a reducing agent, light irradiation, an electrophilic / acidic reagent, an organometallic and metallic reagent, or an oxidizing reagent). A chemically cleavable linker refers to a linker that can be cleaved in response to the presence of a chemical (e.g., an acid, a base, an oxidizing agent, a reducing agent, Pd(0), tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride, tris(3-hydroxypropyl)phosphine), sodium dithionite (Na2S2O4), or hydrazine (NH4)). A chemically cleavable linker can be cleaved non-enzymatically. In embodiments, the cleavable linker is cleaved by contacting the cleavable linker with a cleaving agent. In embodiments, the cleaving agent is a phosphine-containing reagent (e.g., TCEP or THPP), sodium dithionite (Na2S2O4), a weak acid, hydrazine (NH4), Pd(0), or light irradiation (e.g., ultraviolet radiation). In embodiments, cleaving includes removing. A "cleavable site" or "cleavable linkage" in the context of a polynucleotide is a site that allows for controlled cleavage of a polynucleotide chain (e.g., a linker, a primer, or a polynucleotide) by chemical, enzymatic, or photochemical means known in the art and described herein. A cleavable site can refer to a nucleotide linkage between two other nucleotides in a nucleotide chain (i.e., an internucleoside linkage). In embodiments, the cleavable linkage can be located anywhere within one or more nucleic acid molecules, including at or near the terminal end of one or more nucleic acid molecules (e.g., the 3' end of an oligonucleotide) or within an internal portion of one or more nucleic acid molecules. In embodiments, conditions suitable for separating the cleavable linkage include modifying pH and / or temperature. In embodiments, the cleavable site can include at least one acid-labile linkage. For example, the acid-labile linkage can include a phosphoramidate linkage.In embodiments, the phosphoramidate linkage may be hydrolyzable under acidic conditions, including mild acidic conditions such as trifluoroacetic acid and a suitable temperature (e.g., 30°C), or other conditions known in the art, e.g., Matthias Mag, et al., Tetrahedron Letters, Volume 33, Issue 48, 1992, 7319-7322. In embodiments, the cleavable site may comprise at least one photolabile internucleoside linkage (e.g., an o-nitrobenzyl linkage as described in Walker et al., J. Am. Chem. Soc. 1988, 110, 21, 7170-7177), e.g., an o-nitrobenzyloxymethyl or p-nitrobenzyloxymethyl group. In embodiments, the cleavage site comprises at least one uracil nucleobase. In embodiments, uracil nucleobases can be cleaved with uracil DNA glycosylase (UDG) or formamidopyrimidine DNA glycosylase Fpg. In embodiments, the cleavable linkage comprises a sequence-specific nicking site having a nucleotide sequence that is recognized and nicked by a nicking endonuclease enzyme or uracil DNA glycosylase. In embodiments, the cleavable site can be cleaved at or near the modified nucleotide or bond by an enzyme or chemical reagent collectively referred to herein and in the claims as a "cleavage agent." Examples of cleavage agents include DNA repair enzymes, glycosylases, DNA cleavage endonucleases, or ribonucleases. For example, cleavage at dUTP can be achieved using uracil DNA glycosylase and endonuclease VIII (USER™, NEB, Ipswich, Mass.), as described in U.S. Pat. No. 7,435,572. In aspects, when the modified nucleotide is a ribonucleotide, the cleavable site is capable of being cleaved with an endoribonuclease.In embodiments, cleaving the extension product comprises contacting the cleavable site with a cleavage agent, wherein the cleavage agent comprises a reducing agent, sodium periodate, RNase, formamidopyrimidine DNA glycosylase (Fpg), an endonuclease, a restriction enzyme, or uracil DNA glycosylase (UDG). In embodiments, the cleavage agent is an endonuclease enzyme, such as nuclease P1, AP endonuclease, T7 endonuclease, T4 endonuclease IV, Bal31 endonuclease, endonuclease I (Endo I), micrococcal nuclease, endonuclease II (Endo VI, Exo III), nuclease BAL-31, or mung bean nuclease. In embodiments, the cleavage agent comprises a restriction endonuclease, including, for example, a Type IIS restriction endonuclease. In embodiments, the cleavage agent is an exonuclease (e.g., RecBCD), restriction nuclease, endoribonuclease, exoribonuclease, or RNase (e.g., RNase I, II, or III). In embodiments, the cleavage agent is a restriction enzyme. In embodiments, the cleavage agent comprises a glycosylase and one or more suitable endonucleases. In embodiments, cleavage is performed under alkaline (e.g., pH greater than 8) buffer conditions at 40°C to 80°C (e.g., 65°C).

[0052] Aptamers The method of the present disclosure involves the use of aptamers, which are oligonucleotides that can specifically bind to proteins. Generally, the present disclosure provides a method in which aptamers specifically bind to proteins in biological samples. After the aptamer binds to the protein, the aptamer (or a portion thereof), or the oligonucleotide hybridized to the aptamer, is released from the protein and captured on a surface via a capture oligonucleotide. The aptamer sequence can then be copied, amplified, and sequenced to determine (i) the identity of the aptamer, and therefore the identity of the protein, and (ii) the spatial information of the protein in the biological sample.

[0053] As used herein, "aptamer" and "SOMAmer" are used interchangeably to refer to a non-naturally occurring nucleic acid that has a desired action on a target molecule. The desired action includes, but is not limited to, binding the target, catalytically altering the target, reacting with the target in a manner that modifies or alters the target or its functional activity, covalently attaching to the target, and facilitating a reaction between the target and another molecule. In one aspect, the action is specific binding affinity for a target molecule, where such a target molecule is a three-dimensional chemical structure other than a polynucleotide that binds to a nucleic acid ligand through a mechanism independent of Watson / Crick base pairing or triple helix formation, and wherein the aptamer is not a nucleic acid with a physiological function known to be bound by the target molecule. Aptamers for a given target include nucleic acids identified from a candidate mixture of nucleic acids by a method comprising: (a) contacting a candidate mixture with the target, wherein nucleic acids with increased affinity for the target relative to other nucleic acids in the candidate mixture can be partitioned from the remainder of the candidate mixture; (b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to obtain a ligand-enriched mixture of nucleic acids, thereby identifying aptamers for the target molecule, where the aptamers are ligands of the target. While it is recognized that affinity interactions are a matter of degree, in this context, the "specific binding affinity" of an aptamer for its target means that the aptamer binds to the target with a degree of affinity that is significantly greater than the aptamer binds to other non-target components in the mixture or sample. Aptamers can contain any suitable number of nucleotides. "Aptamer" refers to a set of two or more such molecules. Different aptamers can have either the same or different numbers of nucleotides. Aptamers can be DNA or RNA, can be single-stranded, double-stranded, or contain double- or triple-stranded regions. Aptamers can be designed with any combination of desired base-modified nucleotides.

[0054] As used herein, a "SOMAmer" or slow off-rate modified aptamer refers to an aptamer (including aptamers containing at least one nucleotide with a hydrophobic modification) that has an off-rate (t) of ≥ 30 minutes. In some embodiments, SOMAmers are generated using the improved SELEX method described in U.S. Patent No. 7,947,447, entitled "Method for Generating Aptamers with Improved Off-Rates," which is incorporated herein by reference.

[0055] Aptamers can be identified using any known method, including the SELEX process. See, e.g., U.S. Patent No. 5,475,096, entitled "Nucleic Acid Ligands." Once identified, aptamers can be prepared or synthesized according to any known method, including chemical and enzymatic synthesis.

[0056] As used herein, the terms "aptamer-target affinity complex," "aptamer affinity complex," or "aptamer complex" refer to a non-covalent complex formed by the interaction of an aptamer with its target molecule. An "aptamer-target affinity complex," "aptamer affinity complex," or "aptamer complex" refers to a set of two or more such complexes. Aptamer-target affinity complexes, aptamer affinity complexes, or aptamer complexes generally can be reversed or dissociated by changes in environmental conditions, such as increasing temperature, increasing salt concentration, or adding denaturing agents.

[0057] In some embodiments, a non-covalent complex of an aptamer and its target is provided, wherein the aptamer has a Kd for the target of about 100 nM or less, the dissociation rate of the aptamer from the target (given by the half-life of the complex; t1 / 2) is about 30 minutes or more, and / or one, some, or all of the pyrimidines in the nucleic acid sequence of the aptamer are modified at the 5-position of the base.

[0058] As used herein, a "non-specific complex" refers to a non-covalent association between two or more molecules other than an aptamer and its target molecule. Non-specific complexes are not selected based on affinity interactions between their constituent molecules, but rather represent interactions between classes of molecules, and the associated molecules in a non-specific complex exhibit, on average, much lower affinities for each other and have correspondingly higher dissociation rates than an aptamer and its target molecule. Non-specific complexes include complexes formed between an aptamer and a non-target molecule, an aptamer and another aptamer, a competitor and a non-target molecule, a competitor and a target molecule, an aptamer and a competitor, and a target molecule and a non-target molecule, as well as higher-order aggregates of an aptamer, a target molecule, a non-target molecule, a surface, and a competitor.

[0059] As used herein, "target molecule," "analyte," and "target" are used interchangeably to refer to any molecule of interest to which an aptamer can bind with high affinity and specificity and which may be present in a test sample. A "molecule of interest" includes any minor alteration of the particular molecule, e.g., in the case of a protein, minor alterations in the amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling moiety, that does not substantially alter the identity of the molecule. Exemplary target molecules include proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affibodies, antibody mimetics, viruses, pathogens, toxic substances, substrates, metabolites, transition-state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, and any fragments or portions of any of the foregoing. Aptamers can be identified for virtually any chemical or biological molecule of any size, and therefore virtually any chemical or biological molecule of any size can be a suitable target. The target can also be modified to enhance the likelihood or strength of the interaction between the target and the aptamer. The target can also be modified to include a tag as defined above. In an exemplary embodiment, the target molecule is a protein. For methods in which the SELEX target is a peptide, see U.S. Patent No. 6,376,190, entitled "Modified SELEX Processes Without Purified Protein."

[0060] In various embodiments, the aptamers of the present disclosure comprise one or more elements, including, but not limited to, a protein-binding nucleotide sequence, a complementary target nucleotide sequence, an adapter nucleotide sequence or portion thereof, an aptamer barcode nucleotide sequence, a cleavage site, an aptamer-specific nucleotide sequence, or a combination thereof. In some embodiments, the aptamers of the present disclosure comprise a capture nucleotide sequence, and an oligonucleotide hybridized to the aptamer comprises an at least partially complementary target nucleotide sequence. As used herein, a "capture nucleotide sequence" is a sequence that can hybridize to a target nucleotide sequence on an aptamer or on an oligonucleotide hybridized to the aptamer. In some embodiments, the capture nucleotide sequence is a homopolymeric nucleotide sequence (e.g., a poly-T sequence). As used herein, a "target nucleotide sequence" is a sequence that can hybridize to a capture nucleotide sequence (e.g., a capture nucleotide sequence present on a capture oligonucleotide). In some embodiments, the target nucleotide sequence is a homopolymeric nucleotide sequence (e.g., a poly-A sequence).

[0061] In some aspects, the aptamer comprises a protein-binding nucleotide sequence, a target nucleotide sequence, and an adapter nucleotide sequence or portion thereof. In further aspects, the aptamer comprises a protein-binding nucleotide sequence, a target nucleotide sequence, and an aptamer barcode nucleotide sequence. In yet additional examples, the present disclosure provides an aptamer complex comprising: (1) an aptamer comprising (i) a capture nucleotide sequence and (ii) an aptamer-specific nucleotide sequence; and (2) an oligonucleotide hybridized to the aptamer prior to contacting the aptamer complex with a protein, wherein the oligonucleotide comprises (i) the target nucleotide sequence, (ii) a sequence complementary to the aptamer-specific nucleotide sequence, and (iii) the aptamer barcode nucleotide sequence.

[0062] As described below, aptamers can range from about 20 to about 100 nucleotides in length. In some embodiments, aptamers are about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 nucleotides in length. In some embodiments, aptamers include RNA bases. In some embodiments, aptamers include DNA bases. In some embodiments, aptamers include both RNA and DNA bases.

[0063] method In some embodiments of the present disclosure, a plurality of aptamers are provided that associate with one or more proteins in a biological sample (e.g., a tissue slice) and result in the capture of the aptamers, portions of the aptamers, and / or oligonucleotides hybridized to the aptamers on a surface. In some embodiments, nucleic acids (e.g., mRNA) in the biological sample (e.g., a tissue slice) are transferred to an array and captured thereon. In some embodiments of the present disclosure, simultaneous assays are performed that provide spatial information about proteins and nucleic acids from a biological sample (e.g., a tissue slice).

[0064] For example, in some embodiments, a biological sample (e.g., a tissue section) is placed in contact with a surface, and proteins from the biological sample are allowed to associate with aptamers. In various embodiments, the aptamers are then released from the proteins, captured on the surface (e.g., by hybridization to a capture oligonucleotide), and tagged with a spatial address (barcode). In some embodiments, the aptamers comprise aptamer barcodes. In some embodiments, oligonucleotides hybridized to the aptamers are released after association of the aptamers with the proteins, and the oligonucleotides are captured on the surface and tagged with a spatial address (barcode). The spatially tagged oligonucleotides are released from the array and analyzed, for example, by high-throughput next-generation sequencing (NGS), such as sequencing-by-synthesis (SBS). In some embodiments, the capture oligonucleotides can be universal capture oligonucleotides that hybridize to, for example, adapter regions in nucleic acid sequencing libraries or poly-A tails of mRNAs or aptamers. In some embodiments, the capture oligonucleotide can be, for example, a gene-specific capture oligonucleotide that hybridizes to a specifically targeted mRNA or cDNA in a sample, such as a TruSeq™ Custom Amplicon (TSCA) oligonucleotide probe (Illumina, Inc.). In some embodiments, the surface comprises multiple capture oligonucleotides, e.g., multiple identical or different capture oligonucleotides. As described herein, in any of the embodiments of the present disclosure, the methods described herein provide for the simultaneous assay of protein and nucleic acid (e.g., mRNA) from a biological sample. See, e.g., FIG. 2.

[0065] In any of the aspects of the present disclosure, the methods described herein can include extending a surface-attached capture oligonucleotide to which an aptamer, an oligonucleotide, and / or a target nucleic acid is hybridized. In aspects in which the capture oligonucleotide includes a barcode sequence, the resulting extended sequence includes the barcode sequence and a sequence (albeit in a complementary form) from the aptamer, the oligonucleotide, and / or the target nucleic acid. Thus, the extended sequence is a spatially tagged version of the target nucleic acid from a biological sample.

[0066] The sequence of the extended capture oligonucleotide identifies which proteins and / or nucleic acids are present in the biological sample and where the proteins and / or nucleic acids are located in the biological specimen. It will be understood that other sequence elements present in the capture oligonucleotide can also be included in the extended probe. Such elements include, for example, a primer binding site, a cleavage site, other tag sequences (e.g., sample identification tags), capture sequences, or combinations thereof.

[0067] Accordingly, in some aspects, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3' end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; and (b) contacting a plurality of aptamers with the biological sample on the surface, wherein the contacting comprises contacting the plurality of aptamers with the biological sample. (c) contacting the plurality of aptamers with individual proteins in the biological sample, wherein each aptamer in the plurality of aptamers comprises (i) a target nucleotide sequence, (ii) an aptamer barcode nucleotide sequence, and (iii) a cleavage site; (c) removing aptamers in the plurality of aptamers that did not associate with a protein in the biological sample; and (d) cleaving the plurality of aptamers to release (i) the target nucleotide sequence and (ii) the aptamer barcode nucleotide sequence, thereby resulting in association of the target nucleotide sequence with the capture nucleotide sequence, thereby preparing a spatial proteome sequencing library. In some embodiments, the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence. In further embodiments, the blocker nucleic acid is removed from the capture oligonucleotide after step (c). In some embodiments, the plurality of aptamers is cleaved via ultraviolet radiation, enzymes, or chemical cleavage. In further embodiments, the disclosed methods further comprise (e) extending the capture nucleotide sequence to create copies of individual aptamers, thereby creating extended capture oligonucleotides. In further examples, the disclosed methods further comprise (f) adding a template switch oligonucleotide (TSO) to the 3' end of the extended capture oligonucleotide. By way of example, in some embodiments, a reverse transcriptase with terminal transferase activity is used, where the reverse transcriptase adds a non-templated cytosine to the end of the cDNA molecule.In these embodiments, a template switch oligonucleotide (TSO) complementary to the added cytosine is included in the reaction, thereby allowing the TSO to hybridize to the end of the cDNA molecule, which the reverse transcriptase uses as a new template to add a base complementary to the TSO. In some examples, the disclosed methods further include performing single-strand ligation to add a primer landing site for subsequent second-strand synthesis.

[0068] Any suitable enzymatic, chemical, or photochemical cleavage reaction may be used to cleave the cleavable site. The cleavage reaction may result in partial or complete removal of the cleaved strand. Suitable cleavage means include, for example, restriction enzyme digestion, where the cleavable site is a suitable restriction site for the enzyme that induces cleavage of one or both strands of the duplex template; RNase digestion or chemical cleavage of bonds between deoxyribonucleotides and ribonucleotides, where the cleavable site may include one or more ribonucleotides; chemical reduction of disulfide linkages with a reducing agent (e.g., THPP or TCEP), where the cleavable site should include an appropriate disulfide linkage; chemical cleavage of diol linkages with periodate, where the cleavable site should include a diol linkage; generation of an abasic site and subsequent hydrolysis, etc. In some embodiments, the cleavable site is included in the surface-immobilized primer (e.g., within the polynucleotide sequence of the primer). In embodiments, one strand of a double-stranded amplification product (or surface-immobilized primer) can contain a diol linkage that allows cleavage by treatment with periodate (e.g., sodium periodate). It will be understood that more than one diol can be included in the cleavable site. One or more diol units can be incorporated into a polynucleotide using standard methods for automated chemical DNA synthesis. Polynucleotide primers containing one or more diol linkers can be conveniently prepared by chemical synthesis. The diol linker is cleaved by treatment with any substance that promotes cleavage of the diol (e.g., a diol cleaving agent). In embodiments, the diol cleaving agent is a periodate, e.g., aqueous sodium periodate (NaIO). After treatment with a diol cleaving agent (e.g., periodate) to cleave the diol, the cleaved product can be treated with a "capping agent" to neutralize reactive species generated in the cleavage reaction. Suitable capping agents for this purpose include amines, e.g., ethanolamine or propanolamine.

[0069] "Blocking oligonucleotide," "blocker oligonucleotide," "blocking element," or "blocker nucleic acid" refers to an agent (e.g., polynucleotide, protein, nucleotide) that reduces and / or inhibits hybridization. In embodiments, the blocker nucleic acid is a non-extendable oligomer (e.g., a 3'-blocked oligo). The blocking element on the nucleotide can be reversible, whereby the blocking moiety can be removed or modified to allow the 3' hydroxyl to form a covalent bond with the 5' phosphate of another nucleotide. For example, a reversible terminator may refer to a blocking moiety located, e.g., at the 3' position of a nucleotide, which may be a chemically cleavable moiety such as an allyl group, an azidomethyl group, or a methoxymethyl group. In embodiments, the blocking moiety is not reversible (e.g., a blocking element comprising a blocking moiety irreversibly prevents extension). In embodiments, the blocker nucleic acid comprises an oligo with a 3' dideoxynucleotide or similar modification to prevent extension by a polymerase and is used in conjunction with a non-strand-displacing polymerase. In another exemplary implementation, the blocking element comprises one or more modified nucleotides containing a PEG-containing cleavable linker (e.g., linked to the 5', 3', or nucleobase), thereby blocking extension. In another exemplary implementation, the blocker nucleic acid comprises one or more modified nucleotides linked to biotin to which a protein (e.g., streptavidin) can bind, thereby blocking polymerase extension. In another exemplary implementation, the blocker nucleic acid comprises modified nucleotides, such as isodGTP or isodCTP, that are complementary to each other. In a polymerization reaction lacking the appropriate complementary modified nucleotide, primer extension is halted. In another exemplary implementation, the blocker nucleic acid comprises one or more sequences that are recognized and bound by one or more single-stranded DNA-binding proteins, thereby blocking polymerase extension at the binding site.In another exemplary implementation, the blocker nucleic acid comprises one or more sequences that are recognized and bound by one or more short RNA or PNA oligonucleotides, thereby blocking extension by DNA polymerases that cannot strand-displace the RNA or PNA.

[0070] In a further aspect, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3' end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, the UMI comprising a spatial barcode nucleotide sequence; and (b) contacting a plurality of aptamers with the biological sample on the surface, wherein the contacting comprises contacting a plurality of aptamers with the biological sample on the surface. (c) contacting a plurality of aptamers with individual proteins in the biological sample, each aptamer in the plurality of aptamers comprising (i) a target nucleotide sequence, (ii) an aptamer barcode nucleotide sequence, and (iii) a truncated adaptor nucleotide sequence; (c) removing aptamers in the plurality of aptamers that did not associate with a protein in the biological sample; and (d) eluting the individual aptamers from the individual proteins, thereby resulting in association of the target nucleotide sequence with the capture nucleotide sequence, thereby preparing a spatial proteome sequencing library. In some embodiments, the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence. In further embodiments, the blocker nucleic acid is removed from the capture oligonucleotide after step (c). In some embodiments, the method of the present disclosure further comprises the step of (e) extending the capture nucleotide sequence to create copies of the individual aptamers, thereby creating extended capture oligonucleotides. In a further example, the disclosed method further comprises the step of (f) hybridizing and extending the truncated adapter nucleotide sequence to the full-length adapter nucleotide sequence primer to synthesize the second strand.

[0071] In a further aspect, the disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3′ end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, wherein the UMI comprises a spatial barcode nucleotide sequence; and (b) contacting a plurality of aptamers with the biological sample on the surface, wherein the contacting results in association of each aptamer complex in the plurality of aptamer complexes with each protein in the biological sample, and each aptamer complex in the plurality of aptamer complexes comprises: (1) (i) a capture nucleotide sequence at a 3′ end configured to bind to a target nucleotide sequence; and (ii) an aptamer comprising an aptamer-specific nucleotide sequence; and (2) an oligonucleotide hybridized to the aptamer prior to the contacting, the oligonucleotide comprising (i) a target nucleotide sequence, (ii) a sequence complementary to the aptamer-specific nucleotide sequence, and (iii) an aptamer barcode nucleotide sequence. After association of each aptamer complex in the plurality of aptamer complexes with each protein in the biological sample, the oligonucleotide is released from the aptamer, thereby resulting in association of the target nucleotide sequence of the released oligonucleotide with the capture nucleotide sequence of one capture oligonucleotide of the plurality of capture oligonucleotides, thereby preparing a spatial proteome sequencing library. In some embodiments, the aptamer-specific nucleotide sequence is about 5 to about 20 nucleotides in length. In further embodiments, the aptamer-specific nucleotide sequence is about 10 nucleotides in length. In some embodiments, association of an individual aptamer complex in the plurality of aptamer complexes with an individual protein in the biological sample results in release of the oligonucleotide from the aptamer.In further embodiments, after association of each aptamer complex in the plurality of aptamer complexes with each protein in the biological sample, conditions are changed, thereby resulting in release of the oligonucleotides from the aptamers. In various examples, the conditions are temperature, pH, or salt concentration. In some embodiments, formamide is added, thereby resulting in release of the oligonucleotides from the aptamers.

[0072] In any of the embodiments of the present disclosure, the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence. In some embodiments, the blocker nucleic acid is removed from the capture oligonucleotide after contacting. In some examples, the blocker oligonucleotide is removed from the capture oligonucleotide by exonuclease digestion. In various embodiments, the exonuclease digestion is performed using T7 exonuclease or lambda exonuclease.

[0073] In a further aspect, the present disclosure provides a method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3′ end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, the UMI comprising a spatial barcode nucleotide sequence; and (b) contacting a plurality of aptamers with the biological sample on the surface. (c) removing aptamers from the plurality of aptamers that did not associate with proteins in the biological sample; and (d) eluting the individual aptamers from the individual proteins, thereby resulting in hybridization of the target nucleotide sequence with the capture nucleotide sequence, thereby preparing a spatial proteome sequencing library. In some embodiments, the plurality of capture oligonucleotides comprise a cleavable site at their 5' ends. In some embodiments, step (d) comprises competitive elution or digestion of proteins in the biological sample with the aptamers. In some embodiments, step (d) further comprises contacting at least one aptamer in the plurality of aptamers with a blocker nucleic acid, thereby forming a blocked aptamer, wherein the blocker nucleic acid is complementary to the target nucleotide sequence, and the blocked aptamer cannot associate with the capture nucleotide sequence. In some embodiments, the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence. In some embodiments, the blocker nucleic acid is removed from the capture oligonucleotide after step (c). In some embodiments, the method further comprises (e) extending the capture nucleotide sequence to create copies of individual aptamers, thereby creating extended capture oligonucleotides.In some embodiments, step (e) further comprises hybridizing a plurality of aptamer-barcoded oligonucleotides to an extended capture oligonucleotide and extending the extended capture oligonucleotide, thereby creating a plurality of barcoded capture oligonucleotides, each of the aptamer-barcoded oligonucleotides comprising at least a portion of an individual aptamer sequence. In some embodiments, the plurality of aptamer-barcoded oligonucleotides comprises a plurality of aptamer blocker nucleic acids, each of the aptamer blocker nucleic acids comprising at least a portion of an individual aptamer sequence.

[0074] In some embodiments, 10% to 90% of the plurality of aptamers specific for a protein (e.g., an abundant protein) are hybridized to an aptamer blocker nucleic acid. In some embodiments, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the plurality of aptamers specific for a protein are hybridized to an aptamer blocker nucleic acid. It will be understood that the amount of aptamer blocker nucleic acid included in the present methods can be adjusted based on the known abundance of a given target protein.

[0075] In some embodiments, the method further comprises contacting a second plurality of aptamers with the biological sample on the surface, wherein the contacting results in association of individual aptamers in the second plurality of aptamers with individual proteins in the biological sample, and each aptamer in the second plurality of aptamers comprises a detectable moiety. In some embodiments, the detectable moiety is a fluorophore. In some embodiments, each aptamer in the second plurality of aptamers comprises a target nucleotide sequence and a truncated adapter nucleotide sequence. In some embodiments, each aptamer in the second plurality of aptamers further comprises an aptamer barcode nucleotide sequence. In some embodiments, after contacting the second plurality of aptamers with the biological sample on the surface, the method further comprises imaging the biological sample, thereby obtaining an image of the biological sample. In some embodiments, the method does not comprise contacting the biological sample with a hematoxylin and eosin (H&E) staining reagent. In some embodiments, at least one aptamer in the second plurality of aptamers is specific to a cell membrane-associated protein. In some embodiments, at least one aptamer in the second plurality of aptamers is specific to a nuclear membrane-associated protein. In some embodiments, at least one aptamer in the second plurality of aptamers is specific to a cell membrane-associated protein, at least one aptamer in the second plurality of aptamers is specific to a nuclear membrane-associated protein, and the at least one aptamer specific to the nuclear membrane-associated protein comprises a different detectable moiety from the at least one aptamer specific to the cell membrane-associated protein. In some embodiments, the at least one aptamer specific to the nuclear membrane-associated protein comprises a different aptamer barcode nucleotide sequence from the at least one aptamer specific to the cell membrane-associated protein.

[0076] In some embodiments, the cell membrane-associated protein is E-cadherin, N-cadherin, or Na + / K +-ATPase. Additional examples of cell membrane-associated proteins that can be targeted by the aptamers of the present disclosure include, but are not limited to, G protein-coupled receptors, epidermal growth factor receptors, P-cadherin, and R-cadherin. In some embodiments, the nuclear membrane-associated protein is a nuclear pore complex protein. Examples of nuclear pore complex proteins and nuclear membrane-associated proteins that can be targeted by the aptamers of the present disclosure include, but are not limited to, Nup62, Nup107-160, Nup155, karyopherin, and Tpr.

[0077] In any of the aspects of the present disclosure, the biological sample is from a mammal. In various examples, the biological sample is from a human. In some aspects, the biological sample is from a mouse.

[0078] In some aspects, the present disclosure also provides methods for identifying a disorder in a subject having or at risk of having the disorder, comprising: i) generating a spatial proteome and / or transcriptome (e.g., mRNA) library from a biological sample from the subject according to the methods of the present disclosure; ii) comparing proteome and / or transcriptome information from the sample proteome and / or transcriptome library with a control proteome and / or transcriptome library; and iii) identifying genetic variations in the sample proteome and / or transcriptome library that are associated with the disease. In some aspects, the disorder is a neurodegenerative disorder, a genetic defect, or cancer. In some aspects, the disorder is Alzheimer's disease.

[0079] In any of the embodiments of the present disclosure, the method for preparing a spatial proteome and / or transcriptome library as described herein further comprises polymerase chain reaction (PCR) amplification of the aptamers or other oligonucleotides captured on the surface. In a further embodiment, the TSO sequence is used as a primer landing site for second strand synthesis. The product is dehybridized from the surface, and PCR is performed on the product. Alternatively, the extended surface oligonucleotides can be cleaved, and PCR can be performed on the product. Sequencing adapters can be added to the product, which is then placed on a second flow cell for standard sequencing (e.g., Illumina sequencing). In any of the embodiments of the present disclosure, following PCR amplification, the amplified product is sequenced to determine the identity and / or location of the protein and / or mRNA in the biological sample.

[0080] Thus, the methods described herein can be used in conjunction with various nucleic acid sequencing techniques. In embodiments, sequencing includes sequencing by synthesis, sequencing by binding, sequencing by ligation, or pyrosequencing. In any of the embodiments of the present disclosure, as described above, sequencing can occur on a second flow cell rather than the initial surface (e.g., flow cell) on which the biological sample is placed. Particularly applicable techniques are those in which nucleic acids are attached to fixed positions on a surface (e.g., in an array) so that their relative positions do not change, and the array is repeatedly imaged. For example, embodiments in which images are obtained in different color channels corresponding to different labels used to distinguish one nucleotide base type from another are particularly applicable. In some embodiments, the process of determining the nucleotide sequence of a target nucleic acid can be an automated process. Preferred embodiments include sequencing by synthesis ("SBS") techniques. Sequencing by synthesis ("SBS") techniques generally involve enzymatic extension of a nascent nucleic acid strand by the repetitive addition of nucleotides to a template strand. In the conventional method of SBS, a single nucleotide monomer can be provided to the target nucleic acid in the presence of a polymerase in each delivery. However, in the method described herein, two or more types of nucleotide monomers can be provided to the target nucleic acid in the presence of a polymerase during delivery.

[0081] To further illustrate the present disclosure, examples are presented herein. It should be understood that these examples are provided for illustrative purposes and are not to be construed as limiting the scope of the present disclosure. [Example]

[0082] Example 1 Methods for simultaneous spatial protein / mRNA assays Provided herein are methods for assaying the proteome and mRNA content of a biological sample. The methods include methods in which the proteome and mRNA content of a biological sample are assayed simultaneously. See, e.g., Figure 2.

[0083] A schematic workflow of one of the methods is shown in Figure 1. Figure 1 illustrates a spatial proteomics workflow using aptamers tagged with a polyA sequence at the 3' end and a truncated B15 adapter at the 5' end. The truncated adapter can be used to minimize the extra bases added to the aptamer, as these extra bases can affect aptamer-protein binding. The surface capture oligonucleotides possess A14 and ME sequences for downstream library preparation steps, as well as a spatial barcode, UMI, and oligo-dT sequence, enabling capture of polyA-mRNA and the tagged aptamer.

[0084] At the beginning of the protocol, the oligo-dT sequence is optionally blocked using a polyA oligonucleotide. Aptamers are added to the tissue section and allowed to bind to their target proteins. Free aptamers cannot bind to the surface oligonucleotides due to the polyA block. After washing away the free aptamers, the block is removed using a 5' to 3' exonuclease such as T7 or lambda exonuclease. The tagged aptamers are then eluted from their targets by adding excess unlabeled aptamers, which are then diffused onto the surface and captured by the surface-captured oligonucleotides. An enzyme (e.g., reverse transcriptase or polymerase) can then be used to copy the aptamer sequence onto the surface-captured oligonucleotides. In some embodiments, RNA aptamers are used, and the enzyme is reverse transcriptase. In some embodiments, DNA aptamers are used, and the enzyme is polymerase. In further embodiments, a simultaneous DNA aptamer / mRNA assay is performed, and the enzyme is reverse transcriptase. The shortened B15 sequence can then be used as a primer to generate a complementary strand, which can then be dehybridized from the surface. Alternatively, the capture oligonucleotide / aptamer molecule can be cleaved from the surface. PCR is then performed to add the P5 / P7 sequence and sample index to generate the final library. The aptamer sequence (which is unique for each protein target) can then be used to identify the physical location of the target protein using a spatial barcode sequence.

[0085] Another workflow method is shown in Figure 2. Figure 2 illustrates how the disclosed workflow can be modified to accommodate simultaneous protein / mRNA assays. Here, a template-switching reverse transcriptase (RT) is used to add a template-switching oligonucleotide (TSO) sequence to the 3' end of the cDNA or copied aptamer sequence. This TSO sequence then serves as a primer landing site for copying the extended capture oligonucleotide. This second strand is dehybridized from the surface, and the shorter aptamer sequence is separated from the longer cDNA sequence using a solid-phase reversible immobilization (SPRI) step. The short fragment then undergoes PCR to add the remainder of the sequencing adapter, while the long fragment is tagmented and subsequently undergoes PCR to add P5 / P7 and a sample index. In some embodiments, a ligation-based preparation is used.

[0086] A further workflow method is presented in Figure 3. Figure 3 shows a variation of the method provided herein that uses an aptamer containing a cleavable tag. Here, the aptamer contains a cleavage site, followed by a tag sequence unique to the protein target, and a poly-A tail. In this method, after the aptamer binds to the target protein, the tag is cleaved from the aptamer and captured onto a surface oligonucleotide. Cleavage can be achieved through enzymatic cleavage with USER, FPG, or other targeted endonucleases, or through chemical cleavage, for example, using a photocleavable linker in the oligonucleotide activated by UV light. The aptamer is then copied onto the surface-captured oligonucleotide. This method can result in improved elution of the aptamer and avoids aptamer duplication, which can be difficult due to aptamers' stable secondary structure.

[0087] An additional workflow method is shown in Figure 4. Figure 4 shows another variation of the methods provided herein that uses an aptamer bound to single-stranded DNA. Aptamers undergo a conformational change upon binding to their target proteins, and previous studies have shown that a single-stranded DNA reporter bound to an aptamer can be released upon binding of the aptamer to its target protein. In this approach, a single-stranded at least partially complementary DNA (ssDNA) molecule is bound to the aptamer to form an aptamer complex, and the aptamer complex is added to the surface before a biological sample (e.g., a tissue section) is placed on the surface. In some embodiments, a single-stranded at least partially complementary DNA (ssDNA) molecule is bound to the aptamer to form an aptamer complex, and the biological sample (e.g., a tissue section) is added to the surface before the aptamer complex is added to the surface. Binding of aptamers to their target proteins results in the release of complementary ssDNA molecules, which are then captured on the surface by capture oligonucleotides. This approach can be beneficial because it minimizes the length of the sequence that needs to be added to the aptamer, and does not require specific aptamer elution conditions, since binding results in the elution of the ssDNA molecules.

[0088] Another workflow is shown in Figure 5. Figure 5 addresses the issue of large dynamic range and copy number of proteins. Because protein levels are much higher than mRNA levels and span a wider dynamic range, it would be useful to have a mechanism to ensure that mRNA transcripts can be captured along with aptamers. To overcome this issue, the present disclosure contemplates the use of separate capture oligonucleotide sequences for mRNA and aptamer tags (Figure 5, top image). To address the large dynamic range of protein expression, the present disclosure contemplates the use of a mixture of tagged and untagged aptamers in known ratios for highly abundant proteins, so that only a small percentage of these aptamers can be captured on the surface (Figure 5, bottom image). Only tagged aptamers are used for low-abundance proteins.

[0089] Additional workflows are shown in Figures 6A-6F. Figures 6A-6F show an exemplary variation of the disclosed workflow using modified aptamers containing unique barcodes and adapter sequences, with blocker oligonucleotides complementary to the aptamer adapter sequences included to accommodate a large dynamic range of protein abundance (also referred to herein as dynamic range control). Figure 6A is a representative illustration of a cellular environment containing a mixture of proteins (labeled "1," "2," and "X" for mRNA-binding proteins) and mRNA (shown as single-stranded oligonucleotides with poly-A tails). Figure 6B shows the step of permeabilizing cells (e.g., cells in a tissue section) to allow modified aptamers to enter the cells, with the aptamers targeting protein 1 and protein 2. The aptamers targeting each protein may include, from 5' to 3', a protein-specific aptamer sequence, a barcode sequence (e.g., barcode 1 sequence or barcode 2 sequence), and an adapter sequence (e.g., adapter 1 sequence or adapter 2 sequence). A washing step (not shown) removes unbound aptamers, for example, washing with a non-ionic detergent, examples of which include, but are not limited to, Triton™ X-100, Tween® 20, Brij® 35, and Brij® 58.

[0090] Figure 6C shows an immobilized surface primer hybridized to a blocking oligonucleotide. The surface capture oligonucleotide contains A14 and ME sequences for downstream library preparation steps, as well as a spatial barcode, UMI, and oligo-dT sequence to enable capture of polyA-mRNA and tagged aptamers. Figure 6D shows the step of deblocking the surface primer to expose a free 3' end. For example, the block can be removed by dehybridizing the blocker oligo using a 5' to 3' exonuclease, such as T7 or lambda exonuclease, or by exposing the surface of the solid support to denaturing conditions (e.g., heat and / or a chemical denaturant such as formamide).

[0091] Figure 6E illustrates the process of digesting proteins to release mRNA and aptamers, and optionally adding dynamic range compression (DRC) blocker oligonucleotides specific for abundant protein aptamers. Proteins can be digested with a proteinase, such as proteinase K. Additional enzymes, such as lipase, can help facilitate recovery of aptamers from permeabilized tissues / cells. As shown in Figure 6E, the DRC blocker oligonucleotides can be complementary to one or more adapter sequences of the modified aptamer, forming a blocker-aptamer complex that blocks downstream hybridization of the aptamer with immobilized surface primers. In some embodiments, 10% to 90% of the aptamers specific for a protein (e.g., an abundant protein) are hybridized to the blocker oligonucleotides. In some embodiments, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the protein-specific aptamers are hybridized to a blocker oligonucleotide. In some embodiments, the blocker oligonucleotide comprises a locked nucleic acid (LNA), a bis-locked nucleic acid (bisLNAs), a twisted intercalating nucleic acid (TINA), a bridge nucleic acid (BNA), a 2'-O-methyl RNA, a DNA chimeric nucleic acid, a minor groove binder (MGB) nucleic acid, a morpholino nucleic acid, a C5-modified pyrimidine nucleic acid, a peptide nucleic acid (PNA), a phosphorothioate nucleic acid, or a combination thereof.

[0092] In some embodiments, the blocker nucleic acid (i.e., DRC blocker oligo) comprises one or more locked nucleic acids (LNAs), 2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene-functionalized oligonucleotides (TFOs), pyrene-functionalized oligonucleotides (PFOs), peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dC (AP-dC) nucleic acids. In some embodiments, the blocker nucleic acid comprises 10 to 15 locked nucleic acids (LNAs). In some embodiments, the blocker nucleic acid comprises one or more phosphorothioates at the 5'-end. In some embodiments, the blocker nucleic acid comprises one or more LNAs at the 5'-end. In some embodiments, the blocker nucleic acid comprises two or more consecutive LNAs at the 3'-end. In some embodiments, the blocker nucleic acid comprises two or more consecutive LNAs at the 5' end. In some embodiments, the blocker nucleic acid comprises a plurality (e.g., 2-10) of synthetic nucleotides (e.g., LNAs) and a plurality (e.g., 2-10) of canonical or native nucleotides (e.g., dNTPs).

[0093] Figure 6F shows the process of capturing the released mRNA and aptamer barcodes on immobilized surface primers, followed by RT-PCR / PCR to generate covalently attached complements of the mRNA and aptamer barcodes. Aptamers hybridized to DRC blocker oligonucleotides are not captured by the surface primers and can be washed away. PCR is then performed to add P5 / P7 sequences and sample indexes to generate the final library. The aptamer sequences (which are unique for each protein target) can then be used to identify the physical location of the target protein using the spatial barcode sequence. Differences in the barcodes or the sequence itself present in the mRNA capture primers allow the mRNA sequence to be distinguished from the aptamer sequence.

[0094] Another workflow is shown in Figures 7A-7D. Figures 7A-7D show an exemplary variation of the disclosed workflow, which uses a DRC blocker to maintain abundant aptamers (e.g., SOMAmer®, SomaLogic) in solution rather than being captured on the surface of a solid support. Tissue sections are permeabilized to allow aptamers to enter cells without losing mRNA. After aptamer addition, unbound aptamers are washed away, for example, using a non-ionic detergent. Examples of non-ionic detergents include, but are not limited to, Triton™ X-100, Tween® 20, Brij® 35, and Brij® 58. Cellular proteins are then digested, for example, with proteinase K, to release protein-bound aptamers and any protein-bound mRNA. The immobilized capture oligonucleotides on the solid support are then unblocked, exposing free 3' ends and allowing hybridization to mRNA and aptamer sequences.

[0095] As shown in Figure 7A, after cell permeabilization, aptamer binding, and protein digestion, the released mRNA and SOMAmer are captured and copied onto immobilized surface primers. STUBBY corresponds to the universal adapter sequence at the 3' end of the aptamer (i.e., SOMAmer). This is followed by the addition of a DRC blocker (i.e., a blocker oligo containing a complementary sequence to the STUBBY sequence and a complementary sequence to the SOMAmer sequence) to the SOMAmer. Figure 7B shows the process of washing away unbound mRNA and SOMAmer following replication, leaving behind immobilized extended surface primers containing the cDNA of the mRNA or the complement of the STUBBY and SOMAmer sequences. Figure 7C shows the process of adding a SOMAmer barcode to the extended surface primer containing the complement of the SOMAmer sequence. An oligo containing the SOMAmer-SEQ sequence, barcode sequence, and B-15' adapter sequence is hybridized to the extended surface primer and copied. This oligo may contain, from 5' to 3', a B-15' adapter sequence, a barcode sequence, and a sequence complementary to the SOMAmer sequence (e.g., SOMAMER-SEQ). A "dummy" oligo lacking the barcode and B-15' sequence may also be included, which may act as a DRC blocker to prevent replication and sequencing of highly abundant SOMAmers.

[0096] Figure 7D shows the cleavage step to release the extended surface primer containing the SOMAmer barcode and B-15 adapter sequence, followed by the library preparation step. Cleavage can be achieved through enzymatic cleavage with USER, or FPG, or other targeted endonucleases, or through chemical cleavage, for example, using a photocleavable linker in the oligonucleotide activated by UV light.

[0097] Example 2 Methods for spatial targeting of cell membranes Provided herein are methods for using fluorescently labeled aptamers to assay biological samples, such as cell membranes and nuclear membranes, including methods in which the proteome and mRNA content of a biological sample are simultaneously assayed.

[0098] A schematic workflow of one of the present methods is shown in Figures 8A-8B. Figures 8A-8B show an exemplary variation of the disclosed workflow using a fluorescently labeled aptamer targeted to the cell membrane, e.g., for cell segmentation analysis and image alignment processes. Figure 8A shows the steps of attaching the fluorescently labeled aptamer to a protein, flushing away any unbound aptamer, and then imaging the tissue to visualize the bound aptamer. For example, a fluorescent microscope can be used to image the bound fluorescently labeled aptamer. The labeled aptamer contains a polyA sequence at the 3' end (e.g., next to the fluorophore) and a truncated B15 adaptor at the 5' end. The truncated adaptor can be used to minimize extra bases added to the aptamer, as these extra bases can affect aptamer-protein binding. The surface capture oligonucleotides possess A14 and ME sequences for downstream library preparation steps, as well as spatial barcode, UMI, and oligo-dT sequences to enable capture of polyA-mRNA and tagged aptamers.

[0099] Immobilized surface primers on a solid support are blocked, for example, with complementary blocking oligonucleotides. At the beginning of the protocol, the oligo-dT sequences are optionally blocked using polyA oligonucleotides. Aptamers are added to tissue sections and allowed to bind to their target proteins. Free aptamers cannot bind to the surface oligonucleotides due to the polyA block. Figure 8B shows the process of removing the blocking oligonucleotides from the surface primers, for example, using T7 or lambda exonuclease. The tagged aptamers are then eluted from their targets by adding excess unlabeled aptamers, which are then diffused to the surface and captured by surface-capture oligonucleotides. An enzyme (e.g., reverse transcriptase or polymerase) can then be used to copy the aptamer sequence onto the surface-capture oligonucleotide. In some embodiments, RNA aptamers are used and the enzyme is reverse transcriptase. In some embodiments, DNA aptamers are used and the enzyme is polymerase. In a further embodiment, a DNA aptamer / mRNA simultaneous assay is performed, and the enzyme is reverse transcriptase.Then, a shortened B15 sequence can be used as a primer to generate a complementary strand, and this complementary strand can be dehybridized from the surface.Alternatively, the capture oligonucleotide / aptamer molecule can be cleaved from the surface.Then, PCR is performed to add P5 / P7 sequences and sample indexes to generate the final library.The aptamer sequence (which is unique for each protein target) can then be used to identify the physical location of the target protein using a spatial barcode sequence.

[0100] 9A-9B show various examples of plasma membrane-targeted aptamers and nuclear membrane-targeted aptamers. FIG. 9A shows multiple types of plasma membrane-targeted aptamers containing a common fluorophore and barcode, for example, to enable membrane detection across different tissue types. Each aptamer can contain a plasma membrane barcode and a truncated B15 adapter sequence. FIG. 9B shows exemplary aptamers for co-detection of plasma membrane and nuclear membrane proteins using differentially labeled aptamers (e.g., aptamers with different fluorophore and barcode sequences). In some embodiments, the plasma membrane-associated protein is E-cadherin, N-cadherin, or Na. + / K + In some embodiments, the nuclear membrane protein is a nuclear pore complex protein.

Claims

1. 1. A method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) a surface, providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3' end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, the UMI comprising a spatial barcode nucleotide sequence and a unique molecular identifier sequence that are common across the oligonucleotides in a spatial feature; (b) contacting a plurality of aptamers with the biological sample on the surface, wherein the contacting results in association of each aptamer in the plurality of aptamers with each protein in the biological sample, each aptamer in the plurality of aptamers comprising (i) the target nucleotide sequence, (ii) an aptamer barcode nucleotide sequence, and (iii) a cleavage site; (c) removing aptamers in the plurality of aptamers that did not associate with proteins in the biological sample; and (d) cleaving the plurality of aptamers to release (i) the target nucleotide sequence and (ii) the aptamer barcode nucleotide sequence, thereby resulting in association of the target nucleotide sequence with the capture nucleotide sequence; thereby preparing said spatial proteome sequencing library.

2. The method of claim 1 , wherein the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence.

3. 3. The method of claim 2, wherein the blocker nucleic acid is removed from the capture oligonucleotide after step (c).

4. The method of any one of claims 1 to 3, wherein the plurality of aptamers are cleaved via ultraviolet radiation, enzyme, or chemical cleavage.

5. 5. The method of any one of claims 1 to 4, further comprising: (e) extending the capture nucleotide sequence to create copies of the individual aptamers, thereby creating extended capture oligonucleotides.

6. 6. The method of claim 5, further comprising: (f) adding a template switch oligonucleotide (TSO) to the 3' end of the extended capture oligonucleotide.

7. 7. The method of claim 6, wherein the TSO is directly ligated to the extended capture oligonucleotide.

8. 1. A method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) a surface, providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3' end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, the UMI comprising a spatial barcode nucleotide sequence and a unique molecular identifier sequence that are common across the oligonucleotides in a spatial feature; (b) contacting a plurality of aptamers with the biological sample on the surface, wherein the contacting results in association of each aptamer in the plurality of aptamers with each protein in the biological sample, each aptamer in the plurality of aptamers comprising (i) the target nucleotide sequence, (ii) an aptamer barcode nucleotide sequence, and (iii) a truncated adaptor nucleotide sequence; (c) removing aptamers in the plurality of aptamers that did not associate with proteins in the biological sample; and (d) eluting the individual aptamers from the individual proteins, thereby resulting in association of the target nucleotide sequence with the capture nucleotide sequence; thereby preparing said spatial proteome sequencing library.

9. The method of claim 8 , wherein the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence.

10. 10. The method of claim 9, wherein the blocker nucleic acid is removed from the capture oligonucleotide after step (c).

11. 11. The method of any one of claims 8 to 10, further comprising (e) extending the capture nucleotide sequence to create copies of the individual aptamers, thereby creating extended capture oligonucleotides.

12. 12. The method of claim 11, further comprising: (f) hybridizing and extending the truncated adapter nucleotide sequence to a full-length adapter nucleotide sequence primer to synthesize a second strand.

13. 1. A method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) a surface, providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3' end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, the UMI comprising a spatial barcode nucleotide sequence and a unique molecular identifier sequence that are common across the oligonucleotides in a spatial feature; (b) contacting a plurality of aptamers with the biological sample on the surface, wherein said contacting results in association of each aptamer complex in the plurality of aptamer complexes with each protein in the biological sample, and each aptamer complex in the plurality of aptamer complexes is (1) an aptamer comprising (i) the capture nucleotide sequence and (ii) an aptamer-specific nucleotide sequence; (2) contacting with an oligonucleotide hybridized to the aptamer prior to the contacting, the oligonucleotide comprising (i) the target nucleotide sequence, (ii) a sequence complementary to the aptamer-specific nucleotide sequence, and (iii) an aptamer barcode nucleotide sequence; after the association of each aptamer complex in the plurality of aptamer complexes with each protein in the biological sample, the oligonucleotide is released from the aptamer, thereby resulting in association of the target nucleotide sequence of the released oligonucleotide with the capture nucleotide sequence of one capture oligonucleotide of the plurality of capture oligonucleotides; Thereby, said spatial proteome sequencing library is prepared.

14. The method of claim 13, wherein the aptamer-specific nucleotide sequence is about 5 to about 20 nucleotides in length.

15. 14. The method of claim 13, wherein the aptamer-specific nucleotide sequence is about 10 nucleotides in length.

16. The method of any one of claims 1 to 15, wherein the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence.

17. 17. The method of claim 16, wherein the blocker nucleic acid is removed from the capture oligonucleotide after the contacting.

18. 18. The method of any one of claims 13 to 17, wherein the association of an individual aptamer complex in the plurality of aptamer complexes with an individual protein in the biological sample results in release of the oligonucleotide from the aptamer.

19. 18. The method of any one of claims 13 to 17, wherein after the association of an individual aptamer complex in the plurality of aptamer complexes with an individual protein in the biological sample, conditions are changed, thereby resulting in release of the oligonucleotide from the aptamer.

20. 20. The method of claim 19, wherein the condition is temperature, pH, or salt concentration.

21. 21. The method of any one of claims 13 to 20, wherein after the association of an individual aptamer complex in the plurality of aptamer complexes with an individual protein in the biological sample, formamide is added, thereby resulting in release of the oligonucleotide from the aptamer.

22. 20. The method of any one of claims 3, 10, or 17, wherein the blocker oligonucleotide is removed from the capture oligonucleotide by exonuclease digestion.

23. 23. The method of claim 22, wherein the exonuclease digestion is carried out using T7 exonuclease or lambda exonuclease.

24. 1. A method for preparing a spatial proteome sequencing library from a biological sample, the method comprising: (a) a surface, providing a surface comprising a plurality of capture oligonucleotides immobilized on the surface, each capture oligonucleotide in the plurality of capture oligonucleotides comprising: (i) a capture nucleotide sequence at a 3' end configured to bind to a target nucleotide sequence; and (ii) a unique molecular identifier (UMI) nucleotide sequence, the UMI comprising a spatial barcode nucleotide sequence and a unique molecular identifier sequence that are common across the oligonucleotides in a spatial feature; (b) contacting a plurality of aptamers with the biological sample on the surface, wherein the contacting results in association of individual aptamers in the plurality of aptamers with individual proteins in the biological sample, each aptamer in the plurality of aptamers comprising (i) the target nucleotide sequence and (ii) an aptamer barcode nucleotide sequence; (c) removing aptamers in the plurality of aptamers that did not associate with proteins in the biological sample; and (d) digesting the proteins in the biological sample, thereby releasing the individual aptamers from the individual proteins, thereby resulting in hybridization of the target nucleotide sequence with the capture nucleotide sequence, thereby preparing the spatial proteome sequencing library.

25. 25. The method of claim 24, wherein the plurality of capture oligonucleotides comprises a cleavable site at the 5' end.

26. The method of claim 24 or 25, wherein step (d) further comprises contacting at least one aptamer in the plurality of aptamers with a blocker nucleic acid, thereby forming a blocked aptamer, wherein the blocker nucleic acid is complementary to the target nucleotide sequence, and the blocked aptamer is unable to associate with the capture nucleotide sequence.

27. 27. The method of any one of claims 24 to 26, wherein the surface further comprises a blocker nucleic acid hybridized to at least a portion of the capture nucleotide sequence.

28. 28. The method of claim 27, wherein the blocker nucleic acid is removed from the capture oligonucleotide after step (c).

29. 29. The method of any one of claims 24-28, further comprising (e) extending the capture nucleotide sequence to create copies of the individual aptamers, thereby creating extended capture oligonucleotides.

30. 30. The method of Claim 29, wherein step (e) further comprises hybridizing a plurality of aptamer-barcoded oligonucleotides to the extended capture oligonucleotide and extending the extended capture oligonucleotide, thereby creating a plurality of barcoded capture oligonucleotides, each of the aptamer-barcoded oligonucleotides comprising at least a portion of an individual aptamer sequence.

31. 31. The method of Claim 30, wherein the plurality of aptamer-barcoded oligonucleotides comprises a plurality of aptamer blocker nucleic acids, each of the aptamer blocker nucleic acids comprising at least a portion of an individual aptamer sequence.

32. 32. The method of any one of claims 24 to 31, further comprising cleaving the cleavable site, thereby releasing the plurality of capture oligonucleotides from the surface.

33. The method of any one of claims 1 to 32, wherein the aptamer comprises a detectable moiety.

34. 34. The method of claim 33, wherein the detectable moiety is a fluorescent dye.

35. 35. The method of any one of claims 1 to 34, wherein the method further comprises contacting a second plurality of aptamers with the biological sample on the surface, wherein the contacting results in association of individual aptamers in the second plurality of aptamers with individual proteins in the biological sample, and wherein each aptamer in the second plurality of aptamers comprises a detectable moiety.

36. 36. The method of claim 35, wherein the detectable moiety is a fluorescent dye.

37. 37. The method of claim 35 or 36, wherein each aptamer in the second plurality of aptamers comprises the target nucleotide sequence and a truncated adaptor nucleotide sequence.

38. 38. The method of claim 37, wherein each aptamer in the second plurality of aptamers further comprises an aptamer barcode nucleotide sequence.

39. 39. The method of any one of claims 35 to 38, wherein after contacting the second plurality of aptamers with the biological sample on the surface, the method further comprises imaging the biological sample, thereby obtaining an image of the biological sample.

40. 40. The method of claim 39, wherein the method does not include contacting the biological sample with a hematoxylin and eosin (H&E) staining reagent.

41. 41. The method of any one of claims 35 to 40, wherein at least one aptamer in the second plurality of aptamers is specific for a cell membrane-associated protein.

42. 41. The method of any one of claims 35 to 40, wherein at least one aptamer in the second plurality of aptamers is specific for a nuclear membrane-associated protein.

43. 41. The method of any one of claims 35 to 40, wherein at least one aptamer in the second plurality of aptamers is specific for a cell membrane-associated protein, at least one aptamer in the second plurality of aptamers is specific for a nuclear membrane-associated protein, and the at least one aptamer specific for the nuclear membrane-associated protein comprises a different detectable moiety than the at least one aptamer specific for the cell membrane-associated protein.

44. 44. The method of any one of claims 38 to 43, wherein the at least one aptamer specific for the nuclear membrane-associated protein comprises an aptamer barcode nucleotide sequence that is different from the at least one aptamer specific for the cell membrane-associated protein.

45. The cell membrane-associated protein is E-cadherin, N-cadherin, or Na ＋ / K ＋ - ATPase.

46. 45. The method of any one of claims 42 to 44, wherein the nuclear membrane-associated protein is a nuclear pore complex protein.

47. The method of any one of claims 1 to 46, wherein the biological sample is from a mammal.

48. The method of any one of claims 1 to 47, wherein the biological sample is from a human.

49. 1. A method for identifying a disorder in a subject having or at risk of having the disorder, comprising: i) generating a spatial proteome and / or transcriptome library from a biological sample from said subject according to a method described herein; ii) comparing the proteomic and / or genetic information from the sample proteomic and / or transcriptomic library with a control proteomic and / or transcriptomic library; and iii) identifying genetic variations in said sample proteome and / or transcriptome library that are associated with disease.

50. 50. The method of claim 49, wherein the disorder is a neurodegenerative disorder.

51. 51. The method of claim 49 or 50, wherein the disorder is Alzheimer's disease.

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