WO2025133768A1

WO2025133768A1 - Transfer of reporter probes from a biological sample to a support by direct contact

Info

Publication number: WO2025133768A1
Application number: PCT/IB2024/061887
Authority: WO
Inventors: Pedro Gonçalo RÉU SIMÕES DE CARVALHO; Liang Zhang; Lars Daniel OLANDER; Nils Gunnar EKSTRAND
Original assignee: Moleculent AB
Current assignee: Moleculent AB
Priority date: 2023-12-21
Filing date: 2024-11-27
Publication date: 2025-06-26
Anticipated expiration: 2026-06-21

Abstract

Provided herein, among other ways, is method for obtaining an imprint of a sample. In these embodiments, the method may comprise directly contacting a planar biological sample comprising reporter probes that comprise a capture tag with a planar support coated in brush molecules that comprise a polymeric linker and a binding agent that recognizes the capture tag, maintaining the sample in contact with the support under conditions by which the capture tag in the sample binds to the binding agent on the support and separating the sample and the support to produce an imprint of the reporter probes on the support, wherein in the imprint the reporter probes are anchored to the support by the capture tag.

Description

TRANSFER OF REPORTER PROBES FROM A BIOLOGICAL SAMPLE TO A SUPPORT BY DIRECT CONTACT

CROSS-REFERENCING

This application claims the benefit of U.S. provisional application serial no. 63/613,701, filed on December 21, 2023, which application is incorporated herein in its entirety for all purposes.

BACKGROUND

Protein expression, RNA expression, and interactions among biomolecules in a tissue can be examined using a variety of methods. However, these conventional methods are often limited for several reasons. For example, the detection of molecules present in a tissue can suffer from optical crowding because the number of molecules that can be resolved in one image is limited. With many molecules crowded in an analyzed area, the detection methods lose resolution thereby making it difficult to produce images with high resolution. Further, amplification-based methods may suffer from spatial crowding, i.e., these methods are limited by the number of molecules that can be placed physically in one area. For example, RCA amplification produces large DNA amplification products that crowd in an area, making it difficult to distinguish them individually.

Moreover, many conventional methods are time consuming and laborious because it takes time for the reactants to diffuse into and out of the tissue section and image the depth of the tissue section using a so-called z-stack. For example, multiplexed assays, e.g., multiplexed assays such as single molecule fluorescence in situ hybridization (smFISH) assays can take several days (see e.g., Shah et al., Neuron 2016 92: 342-357). In addition, because biological specimens often produce a significant amount of background signal, the images obtained from conventional methods are often not very clean which makes the detection of labeled molecules more challenging.

Therefore, new methods for spatial analysis are desirable.

SUMMARY

Provided herein, among other things, is a method for obtaining an imprint of a sample. In these embodiments, the method may comprise directly contacting a planar biological sample comprising reporter probes that comprise a capture tag with a planar support coated in brush molecules that comprise: a polymeric linker and a binding agent that recognizes the capture tag, wherein the linker tethers the binding agent to the support; maintaining the sample in contact with the support under conditions by which the capture tag in the sample binds to the binding agent on the support; and separating the sample and the support to produce an imprint of the reporter probes on the support, wherein in the imprint the reporter probes are anchored to the support by the capture tag. In these embodiments, the method may further comprise detecting the reporter probes on the support, by hybridizing one or more labeled oligonucleotides, directly or indirectly, to reporter probes that are polynucleotides on the support and then analyzing the binding pattern of the labeled oligonucleotides by microscopy.

In some embodiments, a stimulus that releases the reporter probes from the sample may be applied to the sample (an increase in temperature, an enzymatic cleavage, exposure to light, a change in pH or a chemical reaction, etc.) before separating the sample from the support. In these embodiments, at least some of the reporter probes may diffuse to the support. In other embodiments, no stimulus that releases the reporter probes from the sample may be applied to the sample before separating the sample from the support. In these embodiments, the separation of the support from the sample separates that reporter probes from the molecule that they are bound to.

Also provided is an assembly comprising: a planar support coated in brush molecules that comprise: a polymeric linker and a binding agent that recognizes the capture tag, a planar substrate; and a planar biological sample, wherein the planar sample is sandwiched between the substrate and support.

Also provided is a press apparatus comprising: a first holder for a planar substrate, a second holder for planar support, a force generating means that, when activated, pushes the substrate and support together and an alignment element connected to the first and second holders that holds them in alignment when the force generating means is activated.

Also provided is a kit comprising: a planar support coated in brush molecules that comprise: a polymeric linker; and a binding agent that recognizes the capture tag, and reagents for making a planar biological sample comprising reporter probes that comprise a capture tag. This kit may further comprise the press apparatus.

These and other embodiments will be described in greater detail below.

The present method, depending upon how it is implemented, can avoid several problems with the conventional methods. For example, because the nucleic acid reaction products are analyzed after they have been transferred to a support, a major source of background, i.e., the tissue section, can be avoided.

In some cases, a high-resolution image may be obtained by imaging the sample in one plane. Thus, unlike some conventional methods, some of the embodiments disclosed herein avoid taking z-stacks of images during detection since the molecules can be transferred to a planar 2D surface. Making a z-stack is time consuming and, as such, the throughput of methods that use a z-stack is limited. Thus, the method disclosed herein, depending upon how it is implemented, can avoid the need to image z-stacks and potentially save time and cost.

Additionally, in some cases, the present method may involve repeated cycles of label detection. Because, transferred DNA molecules can be attached to a support using very stable chemistries involving covalent attachment or, for example, biotin-avidin interactions which are generally stable even after multiple cycles of labeling and washing, the method may allow molecules to be detected sequentially and combinatorically over a very high number of cycles. Particularly, the DNA molecules attached to the support withstand multiple rounds of labeling and washing. This can be a significant challenge when imaging molecules in tissue materials since the tissue disintegrates slowly over detection and washing cycles. Because only a few barcodes or combinations of barcodes are detected in a cycle, the molecules that are labeled in a particular cycle will be spaced apart more compared to if all analyzed molecules are detected in the same cycle, thereby avoiding optical crowding, i.e., emission of multiple signals from one location. The possibility to use more detection cycles when molecules are firmly immobilized therefore also allows the detection of more (and different) target molecules (higher multiplex detection). The method disclosed herein is more straightforward to multiplex because multiple cycles of labeling and detecting can be directed to different target barcodes or combinations of barcodes that can be included in oligonucleotides conjugated to different binding agent-oligonucleotide conjugates.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings described below are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

Fig. 1 schematically illustrates some principles of the present method. Figs. 2A-2M schematically illustrate several different ways for generating a reporter probe in situ.

Fig. 3 schematically illustrates an assembly of the present disclosure.

Fig. 4 shows two fluorescent microscopy images showing that reporter probes can be transferred to a slide by blotting at 25°C for 75 min or at 65°C for 75min. Note that the 25°C sample is shown twice in different Lookup Tables (LUT). Both temperatures led to singlecell resolution but at 65°C the yield of transfer was higher. These are blots of whole breast tissue.

Fig. 5 three imprints that have single-cell resolution, obtained by incubation of three separate slices of whole breast tissue at 25 °C for Imin.

Fig. 6 is a schematic representation of two possible transfer mechanisms. On the left, the reporter probes are migrating by diffusion and getting captured on the planar support. On the right, the reporter probes are being directly captured on the planar support by direct contact.

Fig. 7 shows single plane DAPI images (tissue section) vs Histone H3 (planar support). A) Top layer of the DAPI staining on the tissue slide. B) Single plane imaging of the histone H3 signal on the planar support. C) Bottom layer of the DAPI staining on the tissue slide.

Fig. 8 is a graph showing a correlation between transfer temperature and transfer signal.

Figs. 9A-9H show an example of a pressing apparatus and its use.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of ordinary skill in the art with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.

A “plurality” contains at least 2 members. In certain cases, a plurality may have at least 2, at least 5, at least 10, at least 100, at least 1000, at least 10,000, at least 100,000, at least 10⁶, at least 10⁷, at least 10⁸ or at least 10⁹ or more members. In certain cases, a plurality may have 2 to 100 or 5 to 100 members.

As used herein, the term “labeling” refers to a step that results in binding of a binding agent to specific sites in a sample (e.g., sites containing an epitope for the binding agent (e.g., an antibody) being used, for example) such that the presence and/or abundance of the sites can be determined by evaluating the presence and/or abundance of the binding agent. The term “labeling” refers to a method for producing a labeled sample in which any necessary steps are performed in any convenient order, as long as the required labeled sample is produced. For example, in some embodiments and as will be exemplified below, a sample can be labeled using labeled probes that can be detected to determine distribution of nucleic acids on a support.

As used herein, the term “planar biological sample” refers to a substantially flat, i.e., two-dimensional, material that comprises cells, including fixed and/or permeabilized cells. A planar biological sample can be made by, e.g., growing cells on a planar support, depositing cells on a planar support, e.g., by centrifugation, or by cutting a three-dimensional object that contains cells into sections and optionally mounting the sections onto a planar support, i.e., producing a tissue section. Cells may be fixed using any number of reagents including formalin, methanol, paraformaldehyde, methanol: acetic acid, glutaraldehyde, bifunctional crosslinkers such as bis(sulfosuccinimidyl)suberate, bis(succinimidyl)polyethyleneglycol, etc. This definition is intended to cover cellular samples (e.g., tissue sections, etc.). Depending on the specific technique used to prepare the section, a planar biological sample can have a thickness of anywhere from 20 to 50 nm and up to 5 to 100 pm.

As used herein, the term “tissue section” refers to a piece of tissue that has been obtained from a subject, optionally fixed, sectioned into slices, and optionally mounted on a planar support, e.g., a microscope slide. A tissue section is a type of planar biological sample. Tissue sections contain multiple cells, e.g., at least 100 or at least 1,000 cells that are connected to one another and in a matrix. While the dimensions of a tissue section may vary, typical tissue sections cover an area of at least 1 mm² to 2 cm² and have a thickness of 3-100 microns, e.g., 3-20 microns.

As used herein, the term “formalin-fixed paraffin embedded (FFPE) tissue section” refers to a piece of tissue, e.g., a biopsy sample that has been obtained from a subject, fixed in formaldehyde (e.g., 3%-5% formaldehyde in phosphate buffered saline) or Bouin solution, embedded in wax, and cut into thin sections.

The phrase “in situ” as used here in refers to a specific position or location in a planar biological sample. For example, “a binding agent that is bound to the sample, in situ,” indicates that the binding agent is bound at a specific location in the planar biological sample.

The term “complementary site” is used to refer to an epitope for an antibody or aptamer, or nucleic acid that has a sequence that is complementary to an oligonucleotide probe. Specifically, if the binding agent is an antibody or aptamer, then the complementary site for the binding agent is the epitope in the sample to which the antibody or aptamer binds. An epitope may be a conformational epitope, or it may be a linear epitope composed of, e.g., a sequence of amino acids. If the binding agent is an oligonucleotide probe, then the complementary site for the binding agent is a complementary nucleic acid (e.g., an RNA or region in a genome).

The term “epitope” as used herein is defined as a structure, e.g., a string of amino acids, on an antigen molecule that is bound by an antibody or aptamer. An antigen can have one or more epitopes. In many cases, an epitope is roughly five amino acids or sugars in size. One skilled in the art understands that generally the overall three-dimensional structure or the specific linear sequence of the molecule can be the main criterion of antigenic specificity.

As used herein, the term “incubating” refers to maintaining a sample and binding agent under conditions (which conditions include a period of time, one or more temperatures, an appropriate binding buffer and a wash) that are suitable for specific binding of the binding agent to molecules (e.g., epitopes or complementary nucleic acids) in the sample.

As used herein, the term “binding agent” refers to an agent that specifically binds to complementary sites in a sample. Exemplary binding agents include oligonucleotide probes, antibodies, and aptamers. If antibodies or aptamers are used, in many cases they may bind to protein epitopes.

As used herein, the term “reading” in the context of reading a fluorescent signal, refers to obtaining an image by scanning or by microscopy, where the image shows the pattern of fluorescence as well as the intensity of fluorescence in a field of view.

As used herein, the term “signal generated by,” in the context of, e.g., reading a fluorescent signal generated by addition of the fluorescent nucleotide, refers to a signal that is emitted directly from the fluorescent nucleotide or a signal that is emitted indirectly via energy transfer to another fluorescent nucleotide (i.e., by fluorescence resonance energy transfer (FRET)).

As used herein, the term “cleavable linker” refers to a linker containing a bond that can be selectively cleaved by a specific stimulus, e.g., a reducing agent such as TCEP or DTT, or UV light.

The phrase “specific binding pair” as used herein comprises “a first binding member” and “a second binding member” that have binding specificity for one another. The binding members of a binding pair may be naturally derived or wholly or partially synthetically produced. A binding member has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organization of the other binding member of a binding pair. Examples of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, nucleic acids that hybridize with each other, and enzyme-substrate.

As used herein, the term “binding agent-oligonucleotide conjugate” or “binding agent conjugate” refers to a binding agent, e.g., an antibody, aptamer or oligonucleotide probe, that is non-covalently (e.g., via a streptavidin/biotin interaction) or covalently (e.g., via a “click” reaction (see, e.g., Evans Aus. J. Chem. 2007 60 : 384-395) or the like) linked to a singlestranded oligonucleotide in a way that the binding agent can still bind to its binding site. The nucleic acid and the binding agent may be linked via a number of different methods, including those that use a cysteine-reactive maleimide or halogen-containing group. The binding agent and the oligonucleotide may be linked proximal to or at the 5’ end of the oligonucleotide, proximal to or at the 3’ end of the oligonucleotide, or anywhere in-between. The linkage between a binding agent and the oligonucleotide in a binding agent- oligonucleotide conjugate can be cleavable so that the nucleic acid reaction product can be released from the corresponding binding agents via cleavage of the cleavable linker. As will be illustrated below, a binding agent-oligonucleotide conjugate can be composed of a single oligonucleotide, where one region of the polynucleotide (the "probe" part of the oligonucleotide which may be in the region of 15-50 bases in length) hybridizes to a target nucleic acid in the sample (e.g., an RNA) and the other region does not hybridize to that target and is free to participate in the other reactions that are described herein.

An oligonucleotide that is linked to a binding agent in a binding agent- oligonucleotide conjugate may be referred to as a "first oligonucleotide" herein.

The phrase “proximity assay” as used herein refers to assays in which a new DNA product (e.g., a ligation product or primer extension product) is produced only if two binding events are proximal. In a proximity assay, oligonucleotides are joined to target specific binding agents, such as antibodies, aptamers or oligonucleotide probes. When the target molecules are DNA or RNAs, oligonucleotides can have sequences complementary to the target nucleic acid. When the binding agents bind to sites in a sample that are proximal, the oligonucleotides that are conjugated to those binding agents (the "first" oligonucleotides) are brought into proximity, which permits the production of a new DNA product. The new DNA product can be produced by a variety of different ways. For example, the new DNA product can be produced by an initial enzymic reaction between one first oligonucleotide and another (by a reaction that, e.g., ligates one end of an oligonucleotide to a nearby oligonucleotide, extends one end of an oligonucleotide using a nearby oligonucleotide as a template, or joins one end of an oligonucleotide to a nearby oligonucleotide via a templated gap-fill/ligation reaction, etc.). Detecting the nucleic acid reaction products indicates that the corresponding binding agent-oligonucleotide conjugates are bound to sites that are proximal. Thus, binding agent-oligonucleotide conjugates are bound to the sample, and then a reaction (e.g., a ligation, gap-fill/ligation and/or primer extension reaction) is performed while the conjugates are bound to a sample. Products are only produced when two binding agent-oligonucleotide conjugates are bound to sites that are proximal. Certain non-limiting examples of proximity assays include a proximity extension assay (PEA) and a proximity ligation assay (PLA). For clarity, a proximity assay may involve an initial enzymatic reaction (e.g., ligations, etc.) that occur between the first oligonucleotides (i.e., the oligonucleotides that are attached to the binding agents) and, optionally, a secondary enzymatic reaction that occurs between other oligonucleotides (e.g., reporter oligonucleotides) that enzymatically react with one another (e.g., ligate with one another) using the products of the initial reactions as a template. Alternatively, a proximity assay may involve an initial enzymatic reaction between other oligonucleotides (e.g., reporter oligonucleotides) that enzymatic react with one another (e.g., ligate with one another) in a reaction that is templated by first oligonucleotides that are proximal to one another, and one or more other oligonucleotides that may act as a splint or provide an overhang.

The phrase “proximity assay reaction products” as used herein refers to the nucleic acids’ products of a proximity assay. As will be explained below, such products contain sequence from two oligonucleotides, or their complements, where the sequences are joined together only in the presence of proximal binding events. The exact nature of a proximity assay reaction product may vary depending on how the assay is performed. In some embodiments, a proximity assay reaction product may be the product of an initial reaction that joins together two first oligonucleotides (by ligation or a gap-fill/ligation reaction). In these embodiments, the proximity assay reaction products contain the same sequences as the two oligonucleotides that have been joined together. In other embodiments, a proximity assay reaction product may be the product of an initial reaction that extends the 3’ end of an oligonucleotide using another oligonucleotide as a template. In these embodiments, the proximity assay reaction products contain the same sequences as one of the oligonucleotides and the complement of the other. In some embodiments, a proximity assay reaction product may be a copy of an initial product. In these embodiments, reporter oligonucleotides may be hybridized to an initial product and then ligated together. In other embodiments, the proximity assay reaction product may contain the sequence of two or three oligonucleotides that are joined to one another in a reaction that is templated by two proximal first oligonucleotides .

The phrase “proximity extension assay” is intended to refer to a proximity assay that relies on primer extension, where one oligonucleotide uses the other as a template. In this assay, the oligonucleotides that are conjugated to two binding agent-oligonucleotide conjugates that are bound to sites that are proximal hybridize with each other via complementary sequences at the 3’ end. The proximity extension assay then involves extending the 3’ ends of the hybridized oligonucleotides, for example, using a polymerase, and using hybridized oligonucleotides as templates, to produce nucleic acid reaction products. The resulting nucleic acid reaction products (or their complements) indicate that the corresponding binding agent-oligonucleotide conjugates are bound to sites that are proximal. Certain details of PEA are described by Di Giusto et al. (2005), Nucleic Acids Research, 33(6, e64):l-7; Lundberg et al. (2011) and Nucleic Acids Research, Vol. 39, No. 15; and Greenwood et al. (2015), Biomolecular Detection and Quantification, Vol. 4:10-16.

The phrase “proximity ligation assay” or PLA is intended to refer to a proximity assay in which one oligonucleotide is ligated to another oligonucleotide. Such ligation can involve blunt end ligation of single stranded or double stranded oligonucleotides, splint mediated ligation of single stranded oligonucleotides, or ligation of double stranded oligonucleotides having complementary overhangs, for example, overhangs comprising restriction enzyme recognition sites. In certain splint mediated ligations, the oligonucleotides hybridize to a splint in a manner that leaves a gap between the two ends of the oligonucleotides. In such cases, the proximity ligation assay involves sealing the gap using a polymerase in a “gap-fill” reaction and then ligating the 3’ end of the extended oligonucleotide to the 5’ end of the other oligonucleotide. Regardless of the method used to ligate the oligonucleotides, the nucleic acid reaction products resulting from the ligation are analyzed. The resulting nucleic acid reaction products indicate that the corresponding binding agent-oligonucleotide conjugates are bound to sites that are proximal. Certain details of PLA are described by Fredriksson et al. (2002), Nature Biotechnology, 20:473- 477; Gullberg et al. (2004), PNAS, 101(22):8420-8424; Wang et al. (2021), Applied Microbiology and Biotechnology, Vol. 105, pages 923-935; Greenwood et al. (2015), Biomolecular Detection and Quantification, Vol. 4:10-16.

The phrase “preserves the spatial relationship” as used herein characterizes how the nucleic acid reaction products are transferred from a planar biological sample to a support. Particularly, when the nucleic acid reaction products are transferred from a planar biological sample to a support in a manner that preserves the spatial relationship, the relative positions in the x-y plane of different nucleic acid reaction products as present in the planar biological sample do not substantially change when the nucleic acid reaction products are transferred to the support. For example, the relative positions of different nucleic acid reaction products on the support may deviate slightly from the corresponding relative positions in the planar biological sample because of lateral diffusion of the nucleic acid reaction products during the transfer. Accordingly, the positions of the nucleic acid reaction products on the support indicate the positions of the nucleic acid reaction products on the planar biological sample. Molecules (e.g., reaction products or reporter probe) are most commonly transferred from the planar sample to a planar support in a way that preserves the spatial relationship of the molecules in the sample by placing the support on top of the sample (or vice versa) and transferring the molecules directionally onto the support, so that they move in parallel with one another (approximately) out of the sample and onto the support, where they adhere. For clarity, the terms ‘placing the support on top of the sample’, and ‘placing the sample in contact with a planar support” and grammatical equivalents thereof do not imply any directionality. Rather, the term means that the sample is contacted with the support such that the planar faces of the sample and support are in contact with one another. This can be done by, e.g., sandwiching a sample between two slides, placing the sample on a slide or placing a slide on a sample, etc. The products then move from the sample to produce a ‘blot’ or ‘imprint’ of the sample on the support. When imaging the planar support, the transferred molecules will be positioned as a mirror image compared to the original sample. In an exemplary embodiment, this may be done by placing a planar support (e.g., coverslip or other slide) on top of the sample that is mounted on a slide so that the sample is sandwiched between the substrate and slide. The molecules can transfer via diffusion, for example, but the transfer can be aided by electrostatic, electric, magnetic or other forces. In some embodiments, there may be a small gap (e.g., less than 1mm, less than 0.5mm, less than 0.2mm, less than 100 pm, less than 50pm, less than 10 pm less than 5 pm or less than 1 pm) between the sample and the support, which gap may be filled with transfer buffer in some cases. The gap may also be maintained using physical structures, spacers or beads positioned between the surfaces. In other embodiments, the sample and the support may be in direct contact.

The term “proximal” or the phrase “proximally located target sites” as used herein with respect to the location of target sites mean that the target sites are sufficiently close so that the oligonucleotides attached to the binding agent-oligonucleotide conjugates that bind to the target sites interact with each other by for example hybridization or ligation. The target sites can be on the same molecule, for example, two epitopes of one protein. The target sites can also be on different molecules, for example, two epitopes of two different proteins. The target sites can be on different types of molecules, for example, any combination of protein, RNA, DNA, lipid, carbohydrate, etc. The distance between the sites that can be called “proximally located target sites” depends on the length of the oligonucleotides attached to the binding agent-oligonucleotide conjugates and the presence of any linkers between the binding agents and the oligonucleotides. Typically, proximally located target sites are located at a distance that is less 50 nm, for example, less than 30 nm, less than 20 nm, less than 10 nm, or less than 5 nm. The phrase “planar support” as used herein refers to a support to which the nucleic acid reaction products from the analyzed planar biological sample are transferred. A wide variety of different substrates can be used as a planar support. The planar support can be made from any suitable support material, such as glass, modified and/or functionalized glass, hydrogels, films, membranes, plastics (including e.g., acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica- based materials including silicon, silicon wafers, and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene and polycarbonate.

The term “extending” as used herein refers to a ligation reaction (where another oligonucleotide is ligated onto an end of an oligonucleotide), a primer extension reaction (where an oligonucleotide is extended using a polymerase), a gap-fill/ligation reaction, or any combination thereof.

The term “release” as used herein reference refers to an event that places a molecule in solution, not tethered to a support. Release can be done by cleavage of a covalent bond (which may be chemically induced, light induced or enzymatically induced), cleavage of a non-covalent bind, as well as by de-hybridizing the molecule from another molecule, e.g., by heat or using a denaturant.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

Methods

A method for obtaining an imprint of a sample is provided. Some principles of the present method are illustrated in Fig. 1. With reference to Fig. 1 , the initial step of the method may involve obtaining: (i) a planar biological sample comprising nucleic acid reporter probes that comprise a capture tag, and (ii) a planar support coated in brush molecules that comprise: a polymeric linker; and a binding agent that recognizes the capture tag.

In these embodiments, the planar biological sample may be a tissue section, e.g., a section of a human tissue, particularly a soft tissue such as brain, liver, breast, etc. However, as noted above, other types of planar biological sample (e.g., cells that have been spun down onto or filtered through a planar substrate or cells that that have been grown on a planar substrate) may be used. As noted, the planar biological sample comprises nucleic acid reporter probes that comprise a capture tag, where the capture tag specifically binds to the binding agent on the support. In one example, reporter probes may comprise a biotin moiety (e.g., biotin, 2-iminobiotin, desthiobiotin, etc.), however, other capture tags may be used. These molecules are present in the sample in situ (i.e., in and/or on the sample) and, as such, in some embodiments, the method may further comprise an initial step that generates reporter probes in situ. In these embodiments, the method may comprise an initial step that comprises generating reporter probes in situ. This step can be done a variety of different ways.

In some embodiments, the reporter probes may be generated by contacting the sample with one or more exogenous oligonucleotides that comprise the capture tag or a conjugate comprising the same (such as antibody-oligonucleotide conjugate) under conditions by which the exogenous oligonucleotides or conjugates specifically bind to molecules in the sample. This method may be as straightforward as hybridizing a capture tag-linked oligonucleotide (e.g., a biotinylated oligonucleotide) to nucleic acids in the sample (e.g., to endogenous RNA, endogenous DNA, or to a nucleic acid product that was made in situ via a proximity assay, or cDNA) or binding an antibody-oligonucleotide conjugate to the sample, where the oligonucleotide component of the conjugate is doublestranded, where one strand is linked to the antibody and the other strand is linked to the capture tag. In other embodiments, however, the reporter probes may be generated by a two step process that comprises (i) contacting the sample with one or more exogenous oligonucleotides that comprise the capture tag or a conjugate comprising the same (such as antibody-oligonucleotide conjugate) under conditions by which the exogenous oligonucleotides or conjugates specifically bind to molecules in the sample and (ii) performing one or more molecular steps to extend (by primer extension, ligation and/or gapfill ligation) and/or cleave the oligonucleotides. Several examples of such assays are shown in Figs. 2A-2M. In most of these methods, the reporter probes may be generated by contacting the sample with one or more exogenous oligonucleotides (including primers) that comprise the capture tag or a conjugate comprising the same (such as antibody- oligonucleotide conjugate) under conditions by which the exogenous oligonucleotides or conjugates specifically bind to the sample. If oligonucleotides that comprise a capture tag are used, then the oligonucleotides may hybridize to RNA, genomic DNA, or pre-made proximity assay (PEA and or PLA) reaction products, for example. If an antibody- oligonucleotide conjugate is used (where the oligonucleotide comprises the capture tag), then the antibody part of the conjugate may bind to a protein in the sample. In one embodiment, the oligonucleotide component of an antibody-oligonucleotide conjugate may be double-stranded, where one strand is linked to the antibody and the other strand is linked to the capture tag. After binding, the reporter probes may be generated by one or more molecular steps that may include primer extension, gap-fill ligation and/or ligation to produce a ‘new’ product. In other embodiments (e.g., where antibody-oligonucleotide conjugates that have double-stranded oligonucleotides are used), the oligonucleotide does not need to be modified.

Methods for generating the reporter probes in situ are numerous and include: a first method that comprises:

(i) contacting the planar biological sample with exogenous oligonucleotides comprising the capture tag or conjugates comprising the same under conditions by which the exogenous oligonucleotides or conjugates specifically bind to sites in or on the sample; and

(ii) performing one or more steps to extend the exogenous oligonucleotides (by extension, ligation, or gap- fill ligation) to produce the reporter probes; a second method that comprises:

(i) hybridizing oligonucleotides with the sample under conditions by which the oligonucleotides hybridize to endogenous RNA or DNA in the sample, wherein at least some of the oligonucleotides comprise the capture tag; and

(ii) joining together any oligonucleotides that are hybridized to adjacent sites in the RNA or DNA via a ligation or gap-fill/ligation to produce the reporter probes; a third method that comprises:

(i) hybridizing oligonucleotides with the sample under conditions by which the oligonucleotides hybridize to ligation products made by an in situ proximity assay (e.g., a proximity ligation assay), wherein at least some of the oligonucleotides comprise the capture tag; and

(ii) joining together any oligonucleotides that are hybridized to adjacent sites in in the ligation products via a ligation or gap-fill/ligation reaction to produce the reporter probes; a fourth method that comprises contacting the sample with oligonucleotides that comprise a capture tag under conditions by which the oligonucleotides hybridize to an endogenous nucleic acid molecule (e.g., DNA or RNA) in the sample; a fifth method that comprises reverse transcribing RNA (e.g., mRNA) in situ using a primer (e.g., an oligo(dT), random, or gene-specific primer) that comprises the capture tag; or a sixth method that comprises labeling the sample with an antibody that is conjugated to a double-stranded oligonucleotide, where one strand is linked to the antibody and the other strand comprises the capture tag.

In some of these methods, (e.g., the first, second or third method) the oligonucleotide of (i) may exonuclease-sensitive but the product of (ii) may be exonuclease-resistant. In these embodiments, the method comprises treating the sample with an exonuclease before the sample is separated from the support.

Other methods would be readily apparent given these descriptions and Figs. 2A-2M.

As illustrated, the planar support used in the method is coated in brush molecules that comprise a polymeric linker and a binding agent that recognizes the capture tag, e.g., avidin (which term includes streptavidin, neutr avidin and other avidin equivalents). In some embodiments, the polymeric linker may have a length in the range of 3 nm to 3000 nm (e.g., 10 nm to 1000 nm, 1000 nm to 3000 nm, 3 nm to 10 nm, 10 nm to 50 nm, 50 nm to 100 nm, 100 nm to 200 nm, 200 nm to 500 nm, 500 nm to 1000 nm, or 1000 nm to 3000 nm) although longer linkers are envisioned. PEG5000 molecules have a of 33.4nm, PEG20,000 molecules have a length of 126nm, PEG30,000 molecules have a length of 190 nm and 26kPAA has a length of 88.3nm (see, e.g., Chen et al ACS Nano 2021 15: 14022-14048) and, as such, such a linker of such a length should be relatively straightforward so produce. In any embodiment, one end of the polymeric linker may be anchored to the support and the other end may joined to the binding agent. In these embodiments, the polymeric linker is a synthetic polymer (i.e., not a biopolymer) and does not comprise nucleotides or amino acids. In these embodiments, the support may be free of oligonucleotides and does not contain an oligonucleotide array. In any embodiment, the polymeric linker may be a ‘bioinert’ or ‘nonbiofouling’ molecule that does not bind to cellular material, i.e., does not bind cellular protein or nucleic acid, including RNA, cDNA and genomic DNA. Such linkers include, without limitation, poly(ethylene glycol), polyglycerol (PG), polysaccharides, polyoxazoline, poly (propylene sulfoxide), poly (phosphoester), polyvinylpyrrolidone and zwitterionic polymers such as phosphorylcholine and sulfobetaine or carboxybetaine polymers. Poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), carboxybetaine acrylamide and poly(acrylic acid) (PAA) are examples, but there are many others. Methods for making the present support may in some cases be adapted from the biosensor arts, which sometimes use surfaces that are coated in similar brush molecules. See, e.g., US5,242,828, D’Agata (Polymers 2021, 13(12), 1929), Nagasaki et al (Polymer Journal 2011 43: 949-958) and Zhang et al (Ann Biotechnol. 2018; 2: 1006), among many others. In any embodiment, the binding agent may be avidin and the capture tag may be a biotin moiety. A protein (e.g., avidin) can be attached to a polymer on a surface (i.e., polymeric linker) by covalent bonding. In this context, functional groups on the polymer linker such as amino, carboxyl, hydroxyl, aldehyde, carbonyl, epoxy or vinyl groups or others can be used to form covalent bonds with proteins, via reactions with functional groups like amines and carboxylic groups on the protein. A number of coupling agents can be used, including glutaraldehyde, carbodiimides (e.g., EDC), optionally in combination with succinimides (e.g. NHS), hydrazides or hydrazones. Other methods include bioconjugations which involve attaching proteins to polymers using specific chemical reactions. For instance, maleimide-thiol, disulphide-thiol reactions, or various variants of click chemistry (e.g. copper based or copper free) can be employed to create stable covalent bonds between the polymer and protein. To those skilled in the art, a number of additional coupling reactions will be readily obvious. In some embodiments, proteins (e.g. avidin/streptavidin) can be conjugated to the polymeric linker via NHS-ester chemistry, wherein an NHS-ester labeled polymer is conjugated to the protein.

As noted above, the method comprises directly contacting the planar biological sample with the planar support. In this step, the biological sample (e.g., a tissue section) is placed in contact with the support such that the planes are in physical contact with one another, without a gap between the sample and the support. At this point, the reporter probes may be directly or indirectly hybridized to a nucleic acid in the sample and, as such, are not capable of diffusing. In some embodiments, the sample may be placed on the support. However, in some embodiments a force may be applied to facilitate contact between the sample and the support. In some embodiments, the method may comprise mounting the planar biological sample on a planar substrate, generating the reporter probes in situ and contacting the sample with the support while it is mounted on the substrate. In these embodiments, the support or substrate form a sandwich with the sample in between and, depending on whether the support or substrate is on the ‘top’ side, gravity may provide sufficient force to push the sample to the support. In some embodiments, the force may be an external force, applied by an apparatus, as described below. However, if the force is applied, it should be a relatively gentle force that is sufficient to press the sample to the support but not crush the sample. In some embodiments, the force the force applied to the sample can be in the range of 0.01 mN/mm² to 3mN/mm², which approximately corresponds to a weight of in the range of 2g to 600g (e.g., a weight of in the range of 2g to 10g, 10g to 50g, 50g to 200g, or 200g to 600g) evenly applied across the surface of a standard 1 in x 3 in microscope slide or a weight in the range of 0.5g to 150g (e.g., a weight of in the range of 0.5g to 2.5g, 2.5g to 12.5g, 12.5g to 50g, or 50g to 150g) evenly applied across the area of a standard 22mm x 22mm square coverslip, although heavier weights are envisioned. .

Next, the sample and support are maintained in contact with one another under conditions by which the capture tag in the sample binds to the binding agent on the support. This incubation can be for as short as 1 minute. However, in practice, this incubation may be for a period of time in the range of 5 mins to 24 hours, e.g., from 10 mins to 12 hours. The temperature may be any temperature in the range of 20 °C to 90 °C. In any embodiment, the temperature may be in the range of 40-70 °C which, in theory, should facilitate dehybridization of the reporter probes without disrupting the capture tag-binding agent interaction (assuming that the interaction is relatively strong). Indeed, the temperature used could be higher than the Tm of the reporter probe, thereby dehybridizing the reporter probe from the molecule that it is hybridized to. In some embodiments, the sample and support are directly contacted and incubated in the presence of a transfer buffer. In these embodiments, the transfer buffer may be added to the sample (or the support) and the sample and support may be placed in contact. The buffer may facilitate binding between the capture tag and the binding agent. During this incubation, the reporter probes become attached to the binding agent via the capture tag and, in some cases, dehybridize from the molecules that they were hybridized to.

The next step of the method involves separating the sample and the support to produce (i.e., leave behind) an imprint of the reporter probes on the support. In the imprint, the reporter probes are anchored to the support by the capture tag and the relative locations of the reporter probes (in the x-y plane) should be the same as the relative locations in the sample. In some embodiments, the separation (i.e., the physical movement) of the sample and support separates at least some of the reporter probes from the nucleic acids that they are bound to (i.e., it dehybridizes them). However, since the incubation may be heat assisted, the reporter probes may have dehybridized from the targets already. Or the separation could occur at a high temperature to ensure the dehybridization state. Additionally, the molecules that the reporter probes are hybridized to (or the reporter probes themselves) may be cleavable. In these embodiments, the sample may be treated with a cleavage stimulus before the sample and support are separated. This stimulus could be an enzymatic cleavage, exposure to light, a change in pH or a chemical reaction, methods for which are known. For example, if the molecules that the reporter probes are hybridized are RNA, then the RNA can be degraded by an RNAase. If the molecules that the reporter probes are hybridized to are exonuclease sensitive and the reporter probes are not exonuclease sensitive, then the molecules that the reporter probes are hybridized to can be degraded by an exonuclease. If the molecules that the reporter probes are hybridized to are template molecules made in a prior reactions (e.g., a PLA or PEA reaction), then one or more cleavable linkages can be engineered into the template. If the molecules that the reporter probes are hybridized to are attached to an antibody, the molecules may be engineered to contain one or more cleavable linkages can be engineered into the template. Cleavable linkers are known. In these embodiments, the stimulus may release the reporter probes from the sample before the sample is removed.

Next, as illustrated, the method may comprise detecting the reporter probes on the support. This step may be done by hybridizing one or more labeled oligonucleotides, directly or indirectly, to the reporter probes on the support and then analyzing the binding pattern of the labeled oligonucleotides by microscopy. The reporter probes themselves could be labeled (e.g., with a fluorophore). In some embodiments, the proximity assay reaction products are detected in or on the support by hybridization to a defined nucleic acid structure composed of a predetermined number of oligonucleotides and a predetermined number of labeled oligonucleotides. In these embodiments, the structure may be nucleated by at least two hybridization events to the proximity assay reaction products. In these embodiments, the at least two hybridization events comprise a first hybridization to a first sequence in a proximity assay reaction product and a second hybridization to a second sequence in the proximity assay reaction product.

In these embodiments, in order to quantify the nucleic acid reaction products as single molecules it may be advantageous to incorporate a defined number of detection labels per nucleic acid reaction product in order to get a reproducible and stable signal from all molecules. Although approaches like RCA or other clonal amplification strategies could be used for detecting the transferred molecules on the planar support, these typically do not incorporate a defined number of labels per molecule and can create uneven signals from different molecules causing crowding if signals are large and undetectable signals if signals are weak. By designing a programmable hybridization, a specific number of hybridization events occur for each detected target resulting in a predetermined and specific number of oligonucleotides and labels to be incorporated into each formed nucleic acid structure. These structures can advantageously be designed so that two or more initial independent hybridization events to the target are required in order to nucleate formation of the nucleic acid structure that is detected. Once the initial hybridization to the nucleic acid reaction product have occurred these will stably attract the hybridizations and formation of the remaining oligonucleotides. The hybridization events forming the nucleic acid structure can advantageously be separated into two or more steps since, in some cases, it might be challenging to design the oligonucleotides so that the entire structure does not spontaneously form if all oligonucleotides are present in the same solution.

The detection reaction is also advantageously designed so that single labels or labelling structures that are present in each step do not generate a detectable signal if the label or labelling structure would adsorb non-specifically to the surface.

In any embodiment, the molecules that are transferred to the support may contain sequences that are complementary to sequences in the probe system being used. These sequences may be in the tails of the reporter oligonucleotides (which become the reporter probes), or they can be built into the oligonucleotides that are conjugated to the binding agents, for example.

Each of these sequences may have multiple binding sites for the probe system, thereby allowing the support to be interrogated by multiple rounds of hybridization, reading, and signal removal. Such sequences may be referred to as “barcode” sequences herein. In some embodiments, the identity of a reporter probe in or on the support may be determined by reading a code that corresponds to whether the product hybridizes or does not hybridize to each probe of a set of probes as described in e.g., Goransson et al (Nucl. Acids Res .2009 37:e7), Moffitt et al (Methods Enzymol. 2016 572: 1-49) and Moffit et al (Proc. Natl. Acad. Sci. 2016 113: 11046-51).

Certain features of the present method e.g., the in situ labeling reactions, fluorescent labeling schema, multiplexing strategies, molecular biology, samples and the production of exonuclease-resistant reporter probes may be described in WO2022269543 and WO2023144684, which are incorporated by reference herein for the disclosure of those features.

Assemblies Also provided is an assembly comprising: (a) a planar support coated in brush molecules that comprise :(i) a polymeric linker and (ii) a binding agent that recognizes the capture tag, as described above, (b) a planar substrate as described above; and (c) a planar biological sample as described above. In these embodiments, the planar sample is sandwiched between the substrate and support. In some embodiments, the planar sample may be fixed to the substrate but not the support so that the sample and support can be separated by pulling the substrate and support away from one another. The sample and support can be microscope slides in some cases.

Press apparatus

Also provided is a press apparatus. An example of such an apparatus is shown in Fig. 9. In these embodiments, the press apparatus may comprise: a first holder for a planar substrate, a second holder for planar support, a force generating means that, when activated, pushes the substrate and support together under a defined force (e.g., a screw, spring or, as illustrated, weight). As noted above, the force can be in the range of 0.01 mN/mm² to 3mN/mm² (e.g., 0.01 mN/mm² to 0.05 mN/mm², 0.05 mN/mm² to 0.2 mN/mm², 0.2 mN/mm² to ImN/mm² or 1 mN/mm² to 3mN/mm²). which approximately corresponds to a weight of in the range of 2g to 600g (e.g., a weight of in the range of 2g to 10g, 10g to 50g, 50g to 200g, or 200g to 600g) evenly applied across the surface of a standard 1 in x 3 in microscope slide or a weight in the range of 0.5g to 150g (e.g., a weight of in the range of 0.5g to 2.5g, 2.5g to 12.5g, 12.5g to 50g, or 50g to 150g) evenly applied across the area of a standard 22mm x 22mm square coverslip, although heavier weights are envisioned. In addition the apparatus contains an alignment element connected to the first and second holders that holds them in alignment when the force generating means is activated (e.g., a pin, guide or, as illustrated, a housing). In some embodiments and as illustrated, the first and second holders may be both dimensioned to fit a standard 1 inch by 3 inch microscope slide.

Kits

Also provided by this disclosure are kits that contain reagents for practicing the subject method, as described above. These various components of a kit may be in separate vessels or mixed in the same vessel. In addition to the above-mentioned components, the subject kit may further include instructions for using the components of the kit to practice the subject method. In some embodiments, the kit may comprise (a) a planar support coated in brush molecules that comprise: (i) a polymeric linker; and (ii) a binding agent that recognizes the capture tag, as described above, and (iii) (b) reagents for making planar biological sample comprising reporter probes that comprise a capture tag (e.g., biotinylated oligonucleotides or antibody conjugates containing the same, etc.). The various components of the kit are described in the methods section above. In these embodiments, the kit may further comprise a press apparatus, as described above.

Utility

The methods, apparatus and kits described herein find general use in a wide variety of applications for analysis of planar biological samples (e.g., in the analysis of tissue sections, sheets of cells, or spun-down cells). The method may be used to analyze any tissue, including tissue that has been clarified, e.g., through lipid elimination, for example. The sample may be prepared using expansion microscopy methods (see, e.g., Chozinski et al. Nature Methods 2016 13: 485-488), which involves creating polymer replicas of a biological system created through selective co-polymerization of organic polymer and cell components. The method can be used to analyze spreads of cells, exosomes, extracellular structures, biomolecules deposited on a solid support or in a gel (Elisa, western blot, dot blot), whole organism, individual organs, tissues, cells, extracellular components, organelles, cellular components, chromatin and epigenetic markers, biomolecules and biomolecular complexes, for example. The binding agents may bind to any type of molecule, including proteins, lipids, polysaccharides, proteoglycans, metabolites, nucleic acid, or artificial small molecules or the like. The method may have many biomedical applications in screening and drug discovery and the like. Further, the method has a variety of clinical applications, including, but not limited to, diagnostics, prognostics, disease stratification, personalized medicine, clinical trials and drug accompanying tests.

The field of spatial analysis technology, the disclosure aims to provide highly multiplex readout of protein-protein interactions and protein modifications in situ. The disclosure also allows single molecule analysis of proteins, protein post-translational modifications, and protein interactions.

The methods disclosed herein could also be used to analyze RNAs or RNA interactions between RNAs and other molecules, such as proteins, in a single assay format.

In some cases, the methods disclosed herein could be used to analyze target RNAs. For example, as discussed above, an RNA target from a planar biological sample can be directly copied into a reporter polynucleotide using reporter probes. Particularly, a proximity assay is not performed to produce a nucleic acid reaction product, but the RNA target is used as a template to produce a reporter polynucleotide. Such step could for example be performed before contacting the sample with binding agents since antigen retrieval steps required for protein analysis may damage RNA but not DNA, or simultaneously with the introduction of detection oligonucleotides ligating to the first products generated by joining the nucleic acids of the binding agents.

Moreover, the methods disclosed herein could be used to analyze interactions of RNA with other biomolecules, such as RNA, protein, DNA, carbohydrates, lipids, etc. In certain such embodiments, a proximity assay can be conducted using one binding agent targeting an RNA and another binding agent targeting a protein, carbohydrate, or lipid. A proximity assay can also be conducted using one binding agent targeting an RNA and another binding agent targeting a different RNA. Such embodiments can be used to analyze interaction of a target RNA to any other biomolecule for which a specific binding agent is available.

In some cases, the method disclosed herein can be used to identify target sites that are located proximal to each other. For example, a first binding agent-oligonucleotide conjugate binds to a first site and a second binding agent-oligonucleotide conjugate binds to a second site. When the first site and the second site are proximal, the oligonucleotides are brought close to each other. Therefore, the production of a nucleic acid from the oligonucleotides conjugated to the first and the second binding agent-oligonucleotide conjugates indicates that the oligonucleotides are conjugated to binding agents that are bound to sites that are proximal.

Thus, in certain cases, the method disclosed herein can be used to: determine where specific proteins are located in a planar biological sample. In these embodiments, the binding agents bind to different sites on the same protein.

In some cases, the method disclosed herein can also be used to identify where protein-protein interactions occur. In these embodiments, the binding agents bind to different proteins.

Since the relative proximity of targets depends on the absolute concentration and the amount of signal generated from each interaction depends on additional efficiency factors like binding affinity and chemical and enzymatic efficiencies, relating the relative signals within a multiplex experiment to each other will be advantageous. For example, using reference proteins, RNA or DNA targets, or relating the signal from the single individual proteins to the signal from the interaction of the proteins. The signals can for example be analyzed per cell, among a group of cells, for a cell-type determined by the presence of cellular markers or by area.

Also, in some cases, the method disclosed herein can be used to determine posttranslation modification of a biomolecule, such as a protein. In certain such embodiments, one binding agent binds to the post translational modification or to an epitope covering both the post translational modification and the target protein and the other binding agent binds to a different site in the same protein. The production of a nucleic acid from the oligonucleotides conjugated to the first and the second binding agent-oligonucleotide conjugates indicates that the protein has sites that are post-translationally modified. By using binding agents specific for general post translational modifications, the presence of such modifications across a plurality of proteins can be interrogated. The signal can advantageously be analyzed in a relative manner to normalize away the effect of the global presence of modifications, the impact of protein concentrations and assay efficiencies.

In particular embodiments, the sample may be a section of a tissue biopsy obtained from a patient. Biopsies of interest include both tumor and non-neoplastic biopsies of skin (melanomas, carcinomas, etc.), soft tissue, bone, breast, colon, liver, kidney, adrenal, gastrointestinal, pancreatic, gall bladder, salivary gland, cervical, ovary, uterus, testis, prostate, lung, thymus, thyroid, parathyroid, pituitary (adenomas, etc.), brain, spinal cord, ocular, nerve, and skeletal muscle, etc.

In certain embodiments, binding agents specifically bind to biomarkers, including cancer biomarkers, that may be proteinaceous. Exemplary cancer biomarkers, include, but are not limited to carcinoembryonic antigen (for identification of adenocarcinomas), cytokeratins (for identification of carcinomas but may also be expressed in some sarcomas), CD15 and CD30 (for Hodgkin's disease), alpha fetoprotein (for yolk sac tumors and hepatocellular carcinoma), CD117 (for gastrointestinal stromal tumors), CD10 (for renal cell carcinoma and acute lymphoblastic leukemia), prostate specific antigen (for prostate cancer), estrogens and progesterone (for tumor identification), CD20 (for identification of B-cell lymphomas) and CD3 (for identification of T-cell lymphomas).

The above-described method can be used to analyze cells from a subject to determine, for example, whether the cell is normal or not or to determine whether the cells are responding to a treatment. In one embodiment, the method may be employed to determine the degree of dysplasia in cancer cells. In these embodiments, the cells may be a sample from a multicellular organism. A biological sample may be isolated from an individual, e.g., from a soft tissue. In particular cases, the method may be used to distinguish different types of cancer cells in FFPE samples.

The method described above finds particular utility in examining samples using a plurality of antibodies or antibody pairs, each antibody or antibody pair recognizing a different marker. Examples of cancers, and biomarkers that can be used to identify those cancers, are shown below. In these embodiments, one does not need to examine all of the markers listed below to make a diagnosis.

In some embodiments, the method may involve obtaining data (an image) as described above (an electronic form of which may have been forwarded from a remote location), and the image may be analyzed by a doctor or other medical professional to determine whether a patient has abnormal cells (e.g., cancerous cells) or which type of abnormal cells are present. The image may be used as a diagnostic to determine whether the subject has a disease or condition, e.g., a cancer. In certain embodiments, the method may be used to determine the stage of a cancer, to identify metastasized cells, or to monitor a patient’s response to a treatment, for example.

Cell markers, including markers for T-cells, B-cells and neutrophiles (e.g., CD3, CD20, CD15, etc.), can also be investigated. The compositions and methods described herein can be used to diagnose a patient with a disease. In some cases, the presence or absence of a biomarker in the patient’s sample can indicate that the patient has a particular disease (e.g., a cancer). In some cases, a patient can be diagnosed with a disease by comparing a sample from the patient with a sample from a healthy control. In this example, a level of a biomarker, relative to the control, can be measured. A difference in the level of a biomarker in the patient’ s sample relative to the control can be indicative of disease. In some cases, one or more biomarkers are analyzed in order to diagnose a patient with a disease. The compositions and methods of the disclosure are particularly suited for identifying the presence or absence of, or determining expression levels, of a plurality of biomarkers in a sample.

In some cases, the compositions and methods herein can be used to determine a treatment plan for a patient. The presence or absence of a biomarker may indicate that a patient is responsive to or refractory to a particular therapy. For example, a presence or absence of one or more biomarkers may indicate that a disease is refractory to a specific therapy, and an alternative therapy can be administered. In some cases, a patient is currently receiving the therapy and the presence or absence of one or more biomarkers may indicate that the therapy is no longer effective. In some cases, the method may be employed in a variety of diagnostic, drug discovery, and research applications that include, but are not limited to, diagnosis or monitoring of a disease or condition (where the image identifies a marker for the disease or condition), discovery of drug targets (where the a marker in the image may be targeted for drug therapy), drug screening (where the effects of a drug are monitored by a marker shown in the image), determining drug susceptibility (where drug susceptibility is associated with a marker) and basic research (where is it desirable to measure the differences between cells in a sample).

In certain embodiments, two different samples may be compared using the above methods. The different samples may be composed of an “experimental” sample, i.e., a sample of interest, and a “control” sample to which the experimental sample may be compared. In many embodiments, the different samples are pairs of cell types or fractions thereof, one cell type being a cell type of interest, e.g., an abnormal cell, and the other a control, e.g., normal cell. If two fractions of cells are compared, the fractions are usually the same fraction from each of the two cells. In certain embodiments, however, two fractions of the same cell may be compared. Exemplary cell type pairs include, for example, cells isolated from a tissue biopsy (e.g., from a tissue having a disease such as colon, breast, prostate, lung, skin cancer, or infected with a pathogen, etc.) and normal cells from the same tissue, usually from the same patient; cells grown in tissue culture that are immortal (e.g., cells with a proliferative mutation or an immortalizing transgene), infected with a pathogen, or treated (e.g., with environmental or chemical agents such as peptides, hormones, altered temperature, growth condition, physical stress, cellular transformation, etc.), and a normal cell (e.g., a cell that is otherwise identical to the experimental cell except that it is not immortal, infected, or treated, etc.); a cell isolated from a mammal with a cancer, a disease, a geriatric mammal, or a mammal exposed to a condition, and a cell from a mammal of the same species, preferably from the same family, that is healthy or young; and differentiated cells and non-differentiated cells from the same mammal (e.g., one cell being the progenitor of the other in a mammal, for example). In one embodiment, cells of different types, e.g., neuronal and non-neuronal cells, or cells of different status (e.g., before and after a stimulus on the cells) may be employed. In another embodiment of the invention, the experimental material contains cells that are susceptible to infection by a pathogen such as a virus, e.g., human immunodeficiency virus (HIV), etc., and the control material contains cells that are resistant to infection by the pathogen. In another embodiment, the sample pair is represented by undifferentiated cells, e.g., stem cells, and differentiated cells. The images produced by the method may be viewed side-by-side or, in some embodiments, the images may be superimposed or combined. In some cases, the images may be in color, where the colors used in the images may correspond to the labels used.

Cells from any organism, e.g., from bacteria, yeast, plants and animals, such as fish, birds, reptiles, amphibians and mammals may be used in the subject methods. In certain embodiments, mammalian cells, i.e., cells from mice, rabbits, primates, or humans, or cultured derivatives thereof, may be used.

EMBODIMENTS

Embodiment 1. A method for obtaining an imprint of a sample, comprising:

(a) obtaining:

(i) a planar biological sample comprising nucleic acid reporter probes that comprise a capture tag, and

(ii) a planar support coated in brush molecules that comprise: a polymeric linker; and a binding agent that recognizes the capture tag, wherein the linker tethers the binding agent to the support,

(b) directly contacting the planar biological sample with the planar support;

(c) maintaining the sample in contact with the support under conditions by which the capture tag in the sample binds to the binding agent on the support; and

(d) separating the sample and the support to produce an imprint of the reporter probes on the support, wherein in the imprint the reporter probes are anchored to the support by the capture tag.

Embodiment 2. The method of embodiment 1, further comprising:

(e) detecting the reporter probes on the support.

Embodiment 3. The method of embodiment 2, wherein the detecting is done by hybridizing one or more labeled oligonucleotides, directly or indirectly, to the reporter probes on the support and then analyzing the binding pattern of the labeled oligonucleotides by microscopy.

Embodiment 4. The method of any prior embodiment, wherein one end of the polymeric linker is anchored to the support and the other end is joined to the binding agent.

Embodiment 5. The method of any prior embodiment, wherein the binding agent is avidin and the capture tag is a biotin moiety and the polymeric linker is a synthetic polymer that does not bind cellular material. Embodiment 6. The method of any prior embodiment, wherein step (c) comprises applying an external force that pushes the sample and support together.

Embodiment 6A. The method of embodiment 6, wherein the external force is in the range of 0.01 mN/mm² to 3mN/mm² (e.g., 0.01 mN/mm² to 0.05 mN/mm², 0.05 mN/mm² to 0.2 mN/mm², 0.2 mN/mm² to ImN/mm² or 1 mN/mm² to 3mN/mm²).

Embodiment 7. The method of any prior embodiment, wherein the polymeric linker has a length in the range of 3-3000 nm (e.g., 10 nm to 1000 nm, 1000 nm to 3000 nm, 3 nm to 10 nm, 10 nm to 50 nm, 50 nm to 100 nm, 100 nm to 200 nm, 200 nm to 500 nm, 500 nm to 1000 nm, or 1000 nm to 3000 nm.

Embodiment 8. The method of any prior embodiment, wherein the reporter probes that comprise a capture tag are made by: a first method that comprises:

(ii) performing one or more steps to extend the exogenous oligonucleotides (by extension, ligation, or gap-fill ligation) to produce the reporter probes; a second method that comprises:

(i) hybridizing oligonucleotides with the sample under conditions by which the oligonucleotides hybridize to ligation products made by an in situ proximity assay (e.g., a proximity ligation assay), wherein at least some of the oligonucleotides comprise the capture tag; and (ii) joining together any oligonucleotides that are hybridized to adjacent sites in the ligation products via a ligation or gap-fill/ligation reaction to produce the reporter probes; a fourth method that comprises contacting the sample with oligonucleotides that comprise a capture tag under conditions by which the oligonucleotides hybridize to an endogenous nucleic acid molecule (e.g., DNA or RNA) in the sample; a fifth method that comprises reverse transcribing RNA (e.g., mRNA) in situ using a primer (e.g., an oligo(dT), random, or gene-specific primer) that comprises the capture tag; or a sixth method that comprises labeling the sample with an antibody that is conjugated to a double-stranded oligonucleotide, where one strand is linked to the antibody and the other strand comprises the capture tag.

Embodiment 9. The method of embodiment 8, wherein in the first, second or third method the oligonucleotide of (i) is exonuclease-sensitive but the product of (ii) is exonuclease-resistant, and the method optionally comprises treating the sample with an exonuclease before step (d).

Embodiment 10. The method of any of embodiments 8 or 9, wherein (b) comprises a ligation, gap- fill and/or a primer extension reaction.

Embodiment 11. The method of any prior embodiment wherein the sample and support are directly contacted and maintained in the presence of a transfer buffer.

Embodiment 12. The method of any prior embodiment, wherein the method comprises applying a stimulus that releases the reporter probes from the sample, before step (d).

Embodiment 13. The method of embodiment 12, wherein the stimulus is an increase in temperature, an enzymatic cleavage, exposure to light, a change in pH or a chemical reaction.

Embodiment 14. The method of any prior embodiment, wherein step (a) and step (b) are done by:

(a) mounting the planar biological sample on a planar substrate; and

(b) contacting the sample with the support while it is mounted on the substrate.

Embodiment 15. The method of any prior embodiments, wherein the planar biological sample is a tissue section.

Embodiment 16. An assembly comprising:

(a) a planar support coated in brush molecules that comprise: (i) a polymeric linker; and

(ii) a binding agent that recognizes a capture tag, wherein the linker tethers the binding agent to the support;

(b) a planar substrate; and

(c) a planar biological sample; wherein the planar sample is sandwiched between the substrate and support.

Embodiment 17. The assembly of any prior assembly embodiment, wherein one end of the polymeric linker is anchored to the support and the other end is joined to the binding agent.

Embodiment 19. The assembly of any prior assembly embodiment, wherein step (c) comprises applying an external force that pushes the sample and support together.

Embodiment 20. The assembly of embodiment 19, wherein the force is 0.01 mN/mm² to 3mN/mm² (e.g., 0.01 mN/mm² to 0.05 mN/mm², 0.05 mN/mm² to 0.2 mN/mm², 0.2 mN/mm² to ImN/mm² or 1 mN/mm² to 3mN/mm²)

Embodiment 21. The assembly of any prior assembly embodiment, wherein the polymeric linker has a length in the range of 3-3000 nm (e.g., 10 nm to 1000 nm, 1000 nm to 3000 nm, 3 nm to 10 nm, 10 nm to 50 nm, 50 nm to 100 nm, 100 nm to 200 nm, 200 nm to 500 nm, 500 nm to 1000 nm, or 1000 nm to 3000 nm.

Embodiment 22 The assembly of any prior assembly embodiment, the planar biological sample a comprising a nucleic acid reporter probe that comprise the capture tag.

Embodiment 23. The assembly of any prior assembly embodiment, wherein the binding agent is avidin and the capture tag is a biotin moiety.

Embodiment 24. The assembly of embodiments 22 or 21, wherein the reporter probe is exonuclease-resistant.

Embodiment 25. The assembly of any prior assembly embodiment, wherein the polymeric linker is a synthetic polymer that does not bind cellular material.

Embodiment 26. The assembly of any prior assembly embodiment, wherein the sample and support are in the presence of a transfer buffer.

Embodiment 27. The assembly of any prior assembly embodiment, wherein the planar biological sample is a tissue section.

Embodiment 28. The assembly of any prior assembly embodiment, wherein the support and substrate are microscope slides.

Embodiment 29. A kit comprising:

(ii) a binding agent that recognizes the capture tag, wherein the linker tethers the binding agent to the support; wherein the kit further comprises (b) and/or (c), wherein:

(b) comprises reagents for making planar biological sample comprising reporter probes that comprise a capture tag; and

(c) is a press apparatus comprising: a first holder for the planar substrate; a second holder for a planar support; a force generating means that, when activated, pushes the substrate and support together; and an alignment element connected to the first and second holders that holds them in alignment when the force generating means is activated.

Embodiment 30. The kit of embodiment 29, wherein the kit comprises (a) and (b),

(a) and (c) or (a), (b) and (c).

Embodiment 31. The kit of embodiment 29 or 30, wherein one end of the polymeric linker of (a) is anchored to the support and the other end is joined to the binding agent.

Embodiment 32. The kit of any of embodiments 29-31, wherein in the binding agent of (a) is avidin and the capture tag is a biotin moiety

Embodiment 33. The kit of any of embodiments 29-32, wherein the polymeric linker of (a) is a synthetic polymer that does not bind cellular material.

Embodiment 34. The kit of any of embodiments 29-33, wherein the polymeric linker of (a) has a length in the range of 3-3000 nm (e.g., 10 nm to 1000 nm, 1000 nm to 3000 nm, 3 nm to 10 nm, 10 nm to 50 nm, 50 nm to 100 nm, 100 nm to 200 nm, 200 nm to 500 nm, 500 nm to 1000 nm, or 1000 nm to 3000 nm).

Embodiment 35. The kit of any of embodiments 29-34, wherein the kit further comprises a transfer buffer.

Embodiment 36. The kit of any of embodiments 29-35, wherein the planar sample of

(b) is a tissue section.

Embodiment 37. The kit of any of embodiments 29-36, wherein the substrate and support are microscope slides.

Embodiment 38. The kit of any of embodiments 29-37, wherein the kit components (b) comprise one or more oligonucleotide that comprising the capture tag or a conjugate comprising the same and/or one or more enzymes selected from a DNA polymerase, ligase, and an exonuclease.

Embodiment 39. The kit of any of embodiments 29-38, wherein in (c) the force generating means comprises a screw, spring or weight.

Embodiment 40. The kit of any of embodiments 29-39, wherein in (c) the alignment element comprises a pin, guide or housing.

Embodiment 41. The kit or any of embodiments 29-40, wherein in (c) the first and second holders are both dimensioned to fit a microscope slide and/or the support and substrate are microscope slides.

Embodiment 42. The kit of any of embodiments 29-41, wherein in (c) the force is in the range of 0.01 mN/mm² to 3mN/mm² (e.g., 0.01 mN/mm² to 0.05 mN/mm², 0.05 mN/mm² to 0.2 mN/mm², 0.2 mN/mm² to ImN/mm² or 1 mN/mm² to 3mN/mm²).

In any embodiment, the linker may be biopolymer free. The brush molecules do not comprise oligonucleotides and the support does not have any oligonucleotides tethered to it prior to contact with the sample.

EXAMPLES

To further illustrate some embodiments of the present invention, the following specific examples are given with the understanding that they are being offered to illustrate examples of the present invention and should not be construed in any way as limiting its scope.

EXAMPLE 1

Sections of FFPE human breast tissue were submitted to antigen retrieval. Then, they were incubated with a conjugate (antibody conjugated to a DNA oligonucleotide) against Histone H3. Next, oligonucleotides were partially hybridized (as in dsDNA) to the conjugate and a ligation-based assay was used to form a ssDNA reporter probe with a biotin on one end and fluorophore on the other end. The planar substrates with the tissue samples were assembled with their respective planar supports (Fig. 3) and incubated. The temperature and the length of the incubations differed between experiments and are described in the figures and/or in the figure descriptions. The planar supports were then physically separated from their respective planar substrates. Finaly, the blots on the planar supports were imaged with fluorescent microscopy. Transfer imprints with single-cell resolution were obtained at temperatures as low as 25°C (Fig. 4) with an incubation time as short as 1 min (Fig. 5). This data suggests that transfer can occur by direct contact with the planar support (as illustrated in Fig. 6, on the right) as opposed to (or in addition to) diffusion (as illustrated in Fig. 6, on the left). This is also supported by the results indicating that transfer of the reporter probes is mainly occurring from the top layer of the tissue (Fig. 7). Nonetheless, there is a correlation between the quantity of transferred reporter probes (signal in MFI) and the temperature of the reaction (Fig. 8), which can be explained by the fact that even if the reporter probes are bound to the planar support through a streptavidin-biotin bond they are also still hybridized to the conjugated antibodies on the tissue (Fig. 6, on the right). As such, a higher temperature (e.g., 65 °C) should favor dehybridization of the reporter probes without significantly affecting the streptavidin-biotin bond (70°C) (see, e.g., Holmberg et al Electrophoresis. 2005 26: 501-10).

EXAMPLE 2

The following example describes a press apparatus illustrated in Figs. 9A-9H. This apparatus facilitates (1) the alignment of the substrate containing the sample (referred to as the tissue slide) with the planar support (referred to as the transfer chip in this description), (2) the immobilization of the tissue slide, (3) the immobilization of the chip and (4) the controlled lowering of the chip onto the tissue slide.

Fig. 9 A illustrates an example of a transfer apparatus. As illustrated, this apparatus has two cavities that allow the easy placement of the transfer chip, as it will become evident below. It has two other cavities close to the opposite wall where absorbent paper is placed. Said paper can be soaked with 1ml of ultra-pure water (1ml in each position) for humidity production. This device contains a first holder for a microscope slide that contains the sample, a second holder for a planar support that is coated in the brush molecules, a force generating means that, when activated, pushes the substrate and support together (e.g., the weight and/or the latch); and an alignment element connected to the first and second holders that holds them in alignment when the force generating means is activated (the housing).

Fig. 9B shows a tissue slide placed (with the tissue facing up) on the apparatus. The tissue is then aligned with the central square of the apparatus. The central square corresponds to the correct place in the transfer chip.

Fig. 9C shows two magnets that are placed on the tissue slide to immobilize it in the x-y plane. 0.5ml of transfer buffer is added on top of the tissue. Alternatively, the apparatus could be filled with transfer buffer (~50ml) prior to the placement of the tissue slide. In the latter scenario both the tissue slide, and the transfer chip would be fully submerged during the transfer incubation.

Fig. 9D shows the transfer chip placed over the tissue slide without creating air bubbles between the glasses. Fig. 9E shows that the transfer chip does not touch the tissue slide at this stage. There is a distance of 500pm to 1500pm between the slides. If done manually, the assembly may lead to scratches on the chip and the tissue sample caused by inadvertent lateral movements (shaking).

Fig. 9F shows weight (~90g) is placed over the transfer chip to immobilize it in the z- axis, to uniformize the pressure of the sandwich and to facilitate the lowering step.

Fig. 9G shows an apparatus with the lid closed, creating a humidity chamber.

Fig. 9H shows the transfer chip and the weight are lowered by rotating the latch 180°. Now, the transfer chip is in contact with the tissue sample. The transfer incubation can be initiated.

Claims

CLAIMS What is claimed is:

1. A method for obtaining an imprint of a sample, comprising:

(a) obtaining:

(b) directly contacting the planar biological sample with the planar support;

2. The method of claim 1, further comprising:

(e) detecting the reporter probes on the support.

3. The method of claim 2, wherein the detecting is done by hybridizing one or more labeled oligonucleotides, directly or indirectly, to the reporter probes on the support and then analyzing the binding pattern of the labeled oligonucleotides by microscopy.

4. The method of any prior claim, wherein one end of the polymeric linker is anchored to the support and the other end is joined to the binding agent.

5. The method of any prior claim, wherein: the binding agent is avidin, the capture tag is a biotin moiety and the polymeric linker is a synthetic polymer that does not bind cellular material.

6. The method of any prior claim, wherein step (c) comprises applying an external force that pushes the sample and support together.

7. The method of any prior claim, wherein the polymeric linker has a length in the range of 3-3000 nm.

8. The method of any prior claim, wherein the reporter probes that comprise a capture tag are made by: a first method that comprises:

9. The method of claim 8, wherein in the first, second or third method the oligonucleotide of (i) is exonuclease-sensitive but the product of (ii) is exonuclease-resistant, and the method optionally comprises treating the sample with an exonuclease before step (d).

10. The method of any of claims 8 or 9, wherein (b) comprises a ligation, gap-fill and/or a primer extension reaction.

11. The method of any prior claim wherein the sample and support are directly contacted and maintained in the presence of a transfer buffer.

12. The method of any prior claim, wherein the method comprises applying a stimulus that releases the reporter probes from the sample, before step (d).

13. The method of claim 12, wherein the stimulus is an increase in temperature, an enzymatic cleavage, exposure to light, a change in pH or a chemical reaction.

14. The method of any prior claim, wherein step (a) and step (b) are done by:

(a) mounting the planar biological sample on a planar substrate; and

15. The method of any prior claim, wherein the planar biological sample is a tissue section.

16. An assembly comprising:

(a) a planar support coated in brush molecules that comprise:

(i) a polymeric linker; and

(ii) a binding agent that recognizes the capture tag, wherein the linker tethers the binding agent to the support;

(b) a planar substrate; and

17. The assembly of claim 16, wherein an external force pushes the sample and support together.

18. The assembly of claim 17, wherein the force is in the range of 0.01 mN/mm² to 3mN/mm².

19. The assembly of any of claims 16 to 18, wherein: the binding agent is avidin, the capture tag is a biotin moiety and

20. The assembly of any of claims 16-19, wherein the polymeric linker is a synthetic polymer that does not bind cellular material.

21. The assembly of any of claims 16-20, wherein planar tissue sample comprises multiple cells.

22. The assembly of any of claims 16-20, wherein the planar biological sample comprises a nucleic acid reporter probe that comprise the capture tag.

23. The assembly of claim 22, wherein the reporter probe is exonuclease-resistant.

24. The assembly of any of claims 16 to 23, wherein one end of the polymeric linker is anchored to the support and the other end is joined to the binding agent.

25. The assembly of any of claims 16 to 24, wherein the polymeric linker has a length in the range of 3-3000 nm.

26. The assembly of any of claims 16 to 25, wherein the sample is sandwiched between the substrate and support in a transfer buffer.

27. The assembly of any of claims 16 to 25, wherein the sample is a tissue section.

28. The assembly of any of claims 16 to 26, wherein the substrate and support are microscope slides.

29. A kit comprising:

(a) a planar support coated in brush molecules that comprise:

(i) a polymeric linker; and

(ii) a binding agent that recognizes a capture tag, wherein the linker tethers the binding agent to the support; and

(b) and/or (c), wherein:

(b) is reagents for making a planar biological sample comprising reporter probes that comprise the capture tag; and

30. The kit of claim 29, wherein the kit comprises (a) and (b), (a) and (c) or (a), (b) and (c).

31. The kit of claim 29 or 30, wherein one end of the polymeric linker of (a) is anchored to the support and the other end is joined to the binding agent.

32. The kit of any of claims 29-31 , wherein in the binding agent of (a) is avidin and the capture tag is a biotin moiety

33. The kit of any of claims 29-32, wherein the polymeric linker of (a) is a synthetic polymer that does not bind cellular material.

34. The kit of any of claims 29-33, wherein the polymeric linker of (a) has a length in the range of 3-3000 nm.

35. The kit of any of claims 29-34, wherein the kit further comprises a transfer buffer.

36. The kit of any of claims 29-35, wherein the planar sample of (b) is a tissue section.

37. The kit of any of claims 29-36, wherein the substrate and support are microscope slides.

38. The kit of any of claims 29-37, wherein the kit components (b) comprise one or more oligonucleotide that comprising the capture tag or a conjugate comprising the same and/or one or more enzymes selected from a DNA polymerase, ligase, and an exonuclease.

39. The kit of any of claims 29-38, wherein in (c) the force generating means comprises a screw, spring or weight.

40. The kit of any of claims 29-39, wherein in (c) the alignment element comprises a pin, guide or housing.

41. The kit or any of claims 29-40, wherein in (c) the first and second holders are both dimensioned to fit a microscope slide and/or the support and substrate are microscope slides.

42. The kit of any of claims 29-41, wherein in (c) the force is in the range of 0.01 mN/mm² to 3mN/mm².