Method for spatial mapping of cell targets

Abstract

Provided herein is a method for producing a map of binding events. In some embodiments, this method may comprise: obtaining a sample that comprises uniquely barcoded RCA products that are tethered to the sample, hybridizing a grid oligonucleotide to the uniquely barcoded RCA products while they are tethered to the sample, wherein the 5' and 3' ends of the grid oligonucleotide hybridizes to different RCA products, extending the 5' and 3' ends of the grid oligonucleotide to copy unique molecular identifiers a target identifier sequences from the different RCA products onto the ends of the grid oligonucleotide to make an extended grid oligonucleotide; and sequencing the extended grid oligonucleotide to produce sequence reads. A map of the binding sites of the capture agent and the identities of the target to which the capture agent binds can be generated by analysis of the sequence reads.

Description

[0001] METHOD FOR SPATIAL MAPPING OF CELL TARGETS

[0002] CROSS-REFERENCING

[0003] This application claims the benefit of provisional application serial no. 63 / 625,080, filed on January 25, 2024, which application is incorporated by reference herein.

[0004] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A SEQUENCE LISTING XML FILE

[0005] A Sequence Listing is provided herewith as a Sequence Listing XML, “PIXL-010WO_SEQLIST.xml” created on December 6, 2024 and having a size of 5,398 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

[0006] BACKGROUND

[0007] DNA microscopy methods, which map the relative locations of biomolecules in cells and tissues, rely on the addition of DNA sequences to probes that specifically target proteins or nucleic acids. Many of these methods use capture agents that are coupled to DNA oligonucleotides (e.g., antibody-oligonucleotide conjugates). However, it is difficult to obtain spatial information on the binding of such capture agents by sequencing.

[0008] The present disclosure provides a solution to this problem.

[0009] SUMMARY

[0010] Provided herein is method for producing a map of binding events, comprising: (a) obtaining a sample that comprises uniquely barcoded rolling circle amplification (RCA) products that are tethered to the sample via a capture agent, wherein the RCA products comprise: (i) a unique molecular identifier that distinguishes the RCA products from one another; and (ii) a target identifier sequence that identifies the target to which the capture agent is bound; (b) hybridizing a grid oligonucleotide to the uniquely barcoded RCA products while they are tethered to the sample, wherein the 5’ and 3’ ends of the grid oligonucleotide hybridize to different RCA products; (c) extending the 5’ and 3’ ends of the grid oligonucleotide to copy the unique molecular identifiers and target identifier sequences from the different RCA products onto the ends of the grid oligonucleotide to make an extended grid oligonucleotide; (d) sequencing the extended grid oligonucleotide to produce sequence reads; (e) identifying which unique molecular identifiers and target identifier sequences are paired in the sequence reads; and (f) producing a map of the binding sites of the capture agent and the identities of the target to which the capture agent binds using paired unique molecular identifiers and target identifier sequences.

[0011] Also provided herein is a reagent system comprising: (a) a conjugate comprising a capture agent and an oligonucleotide, wherein the oligonucleotide is tethered to the capture agent by its 5’ end and comprises: a target identifier sequence that identifies the target to which the capture agent binds, and a unique molecular identifier, and (b) a padlock probe, wherein the oligonucleotide of (a) and the padlock probe of (b) hybridize to form a circular complex in which the target identifier sequence and the unique molecular identifier are single-stranded. Methods of using the reagent system to transfer sequence information from an oligonucleotide to a rolling circle amplification product are also provided.

[0012] Also provided is a method comprising hybridizing the padlock probe to the oligonucleotide to form a circular complex in which the target identifier sequence and the unique molecular identifier are single-stranded. These embodiments of the method may further comprise performing a gap- fill ligation reaction to extend the 3’ end of the padlock probe using the oligonucleotide as a template and produce a covalently closed nucleic acid circle comprising a complement of the target identifier sequence and a complement of the unique molecular identifier. This circular product may be amplified by rolling circle amplification using the oligonucleotide as a primer.

[0013] Also provided is a complex made by the method. In these embodiments, the complex may comprise: (a) a conjugate comprising a capture agent and an oligonucleotide, wherein the oligonucleotide is tethered to the capture agent by its 5’ end and comprises: a target identifier sequence that identifies the target to which the capture agent binds, a unique molecular identifier, a 5’ flanking sequence that is 5’ of the target identifier sequence and unique molecular identifier and a 3’ flanking sequence that is 3’ of the target identifier sequence and unique molecular identifier and (b) a covalently closed nucleic acid circle. In this complex, the oligonucleotide and covalently closed nucleic acid circle are hybridized via the target identifier sequence, the unique molecular identifier, a 5’ flanking sequence that is 5’ of the target identifier sequence and unique molecular identifier and a 3’ flanking sequence that is 3’ of the target identifier sequence and unique molecular identifier.

[0014] Also provided is a reagent system comprising the following components: (a) a first conjugate comprising a capture agent and an oligonucleotide, wherein the oligonucleotide is tethered to the capture agent by its 5’ end and comprises a target identifier sequence that identifies the target to which the capture agent binds, a unique molecular identifier, a first 5’ flanking sequence that is 5’ of the target identifier sequence and unique molecular identifier and a first 3’ flanking sequence that is 3’ of the target identifier sequence and unique molecular identifier; (b) a second conjugate comprising a capture agent and an oligonucleotide, wherein the oligonucleotide is tethered to the capture agent by its 5’ end and comprises: a target identifier sequence that identifies the target to which the capture agent binds, a unique molecular identifier, a second 5’ flanking sequence that is 5’ of the target identifier sequence and unique molecular identifier and a second 3’ flanking sequence that is 3’ of the target identifier sequence and unique molecular identifier; wherein at least one of the 5’ and 3’ flanking sequences of the second conjugate is different from the 5’ and 3’ flanking sequences of the first conjugate; (c) a first padlock probe that has: a first 5’ end sequence, wherein the first 5’ end sequence is complementary to the first 5’ flanking sequence of the first conjugate; a first 3’ end sequence, wherein the first 3’ end sequence is complementary to the first 3’ flanking sequence of the first conjugate, and a backbone sequence that connects the 3’ and 5’ end sequences, and (d) a second padlock probe that has: a second 5’ end sequence, wherein the second 5’ end sequence is complementary to the second 5’ flanking sequence of the second conjugate; a second 3’ end sequence, wherein the second 3’ end sequence is complementary to the second 3’ flanking sequence of the second conjugate; and a backbone sequence that connects the 3’ and 5’ end sequences.

[0015] Also provided is a method that comprises hybridizing the first and second padlock probes to the oligonucleotides of the first and second conjugates to form: a first circular complex comprising the first conjugate and the first padlock probe, in which the target identifier sequence and the unique molecular identifier are single-stranded, a second circular complex comprising the second conjugate and the second padlock probe, in which the target identifier sequence and the unique molecular identifier are single-stranded. These complexes may be subjected to a gap-fill ligation reaction and amplified by RCA, as above.

[0016] This method allows one to transfer information (e.g., a unique molecular identifier, or UMI and a target identifier barcode) from one molecule (e.g., a DNA oligonucleotide that is part of an antibody-oligonucleotide conjugate) to another type of molecule (e.g., a rolling circle amplification product or a PCR product) without information loss.

[0017] Finally, a method comprising: (a) creating a complex comprising: a grid oligonucleotide, a gap- fill stop oligonucleotide, a gap-fill primer, a first uniquely barcoded RCA product, and a secondly uniquely barcoded RCA product, wherein: the 3’ end of the grid oligonucleotide is hybridized to a sequence in the first RCA product that is downstream of the unique RCA product identifier sequence of the first RCA product, the 5’ end of the gap-fill stop oligonucleotide is hybridized to a sequence that is upstream of the unique RCA product identifier sequence of the first RCA product, the 5’ end of the grid oligonucleotide is hybridized to a sequence in the second RCA product that is upstream from the unique RCA product identifier sequence of the second RCA product, and the 3’ end of the gap-fill primer oligonucleotide is hybridized to a sequence that is downstream of the unique RCA product identifier sequence of the second RCA product, (b) incubating the product of (a) under gapfill ligation conditions to produce: (i) full length products that comprise the sequences of the grid oligonucleotide, the gap-fill primer, the gap-fill stop oligonucleotide, and the complements of the RCA product identifier sequences from the first and second RCA products, and (ii) incomplete products that do not comprise the sequences of the grid oligonucleotide, the gap- fill primer, the gap-fill stop oligonucleotide, and the complements of the RCA product identifier sequences from the first and second RCA products, and (c) enriching for the full-length products using an exonuclease and / or by affinity is provided. In these embodiments: (i) the 3' end of the gap-fill stop oligonucleotide may be exonuclease resistant, the 5' end of gap-fill primer may be exonuclease resistant, and enrichment of the full-length products is done by treating the product of step (b) with an exonuclease; and / or (ii) the 3' end of the gap-fill stop oligonucleotide or the 5' end of gap-fill primer has a capture moiety, and enrichment of the full-length products is done using a support that has affinity for the capture moiety.

[0018] These and other advantages, aspects and embodiments will be described in greater detail below.

[0019] 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.

[0020] Fig. 1 illustrates a reagent system that can be used in the present method. Fig. 2 schematically illustrates a gap-fill ligation reaction using the components of the reagent system.

[0021] Fig. 3 schematically illustrates how circular products can be amplified in an RCA reaction.

[0022] Fig. 4 shows how RCA may be done using conjugates that are attached to a target. In these embodiments, the RCA product is attached to the target through the capture agent.

[0023] Fig. 5 shows a two-conjugate reagent system that has a first conjugate and a second conjugate, each with an associate padlock probe.

[0024] Fig. 6 schematically illustrates a gap-fill ligation reaction using the components of the present two conjugate reagent system.

[0025] Fig. 7 schematically illustrates how circular products produced by the present two conjugate system can be amplified in an RCA reaction.

[0026] Fig. 8 schematically illustrates a grid oligonucleotide hybridized to two RCA products.

[0027] Fig. 9 schematically illustrates a grid oligonucleotide that has been extended using a dual gap-fill ligation using two RCA products as a template.

[0028] Figs. 10A and 10B illustrate embodiment that add unique molecular identifiers from two RCA products to the ends of a grid oligonucleotide.

[0029] Fig. 11 schematically illustrates a cell covered in RCA products (top) and how they can be linked by grid oligonucleotides (bottom).

[0030] Fig. 12 schematically illustrates how a physical map of binding sites can be generated using sequence information. Fig. 13 schematically illustrates how the present two conjugate reagent system can generate RCA products.

[0031] Fig. 14 shows how information from two RCA products can be copied onto the ends of a grid oligonucleotide.

[0032] Fig. 15 shows a gel of reaction products after hybridization of padlock probe PL1 to COa and gap-fill ligation (top) and after hybridization of padlock probe PL2 to COb and gap-fill ligation (bottom).

[0033] Fig. 16 shows the products of the first gap-fill ligation assay. Circularized products are identified. This data also shows that T4 DNA ligase may perform better than Taq ligase under the conditions tested.

[0034] Fig. 17 shows the results of a PCR assay for detecting RCA products. On the top, the PCR is specific for RCA products generated from the COa - PL1 gap-fill ligation. On the bottom, the PCR is specific for RPC generated from the COb - PL2 gap-fill ligation.

[0035] Fig. 18 shows the results of a reaction in which a grid oligonucleotide is hybridized to both RCA products and extended at both ends in a dual gap-fill ligation reaction that adds the PID and UMI (i.e., PID and UMI) from both RCA products onto the ends of the grid oligonucleotide. A product of the expected size is identified.

[0036] Figs. 19A-19C show data relating to Example 3. Fig. 19A: Structure of the final amplicon generated using PNA as well as paired-end sequencing requirement (top); 4% agarose gel showing the final product (bottom). Fig. 19B: UMAP of PBMCs in which the major cell types are clustered into separated groups. Fig. 19C: 3D scatter plots of single-cell layouts displaying the abundance of CD 16 on a monocyte cell (top left), CD3e on a T cell (top right) and CD22 on a B cell (bottom left).

[0037] DEFINITIONS

[0038] Unless defined otherwise herein, 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. 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.

[0039] All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0040] 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.

[0041] 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.

[0042] 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 skill 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.

[0043] The term “oligonucleotide” as used herein denotes a single-stranded multimer of nucleotides of from about 2 to 200 nucleotides, up to 500 nucleotides in length. Oligonucleotides may be synthetic or may be made enzymatically, and, in some embodiments, are 30 to 150 nucleotides in length. Oligonucleotides may contain ribonucleotide monomers (i.e., may be oligoribonucleotides) or deoxyribonucleotide monomers. An oligonucleotide may be 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200 nucleotides in length, for example.

[0044] The term “primer” as used herein refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The 3' end of a primer is typically complementary to at least 10 nucleotides (e.g., 10-30 nucleotides) of a template.

[0045] The term “primer extension products” refer to the product of extension of a primer or the product of extension of a molecule that is itself a primer extension product. The term “hybridization” or “hybridizes” refers to a process in which a nucleic acid strand anneals to and forms a stable duplex, either a homoduplex or a heteroduplex, under normal hybridization conditions with a second complementary nucleic acid strand and does not form a stable duplex with unrelated nucleic acid molecules under the same normal hybridization conditions. The formation of a duplex is accomplished by annealing two complementary nucleic acid strands in a hybridization reaction. The hybridization reaction can be made to be highly specific by adjustment of the hybridization conditions (often referred to as hybridization stringency) under which the hybridization reaction takes place, such that hybridization between two nucleic acid strands will not form a stable duplex, e.g., a duplex that retains a region of double-strandedness under normal stringency conditions, unless the two nucleic acid strands contain a certain number of nucleotides in specific sequences which are substantially or completely complementary. "Normal hybridization or normal stringency conditions” are readily determined for any given hybridization reaction. See, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. As used herein, the term "hybridizing” or "hybridization” refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.

[0046] A nucleic acid is considered to be “selectively hybridizable” to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Moderate and high stringency hybridization conditions are known (see, e.g., Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, N.Y.). One example of high stringency conditions includes hybridization at about 42 °C in 50% formamide, 5X SSC, 5X Denhardt’s solution, 0.5% SDS and 100 ug / ml denatured carrier DNA followed by washing two times in 2X SSC and 0.5% SDS at room temperature and two additional times in 0.1 X SSC and 0.5% SDS at 42 °C.

[0047] The term “sequencing”, as used herein, refers to a method by which the identity of at least 10 consecutive nucleotides (e.g., the identity of at least 20, at least 50, at least 100 or at least 200 or more consecutive nucleotides) of a polynucleotide are obtained.

[0048] The term “next-generation sequencing” refers to the so-called parallelized sequencing-by-synthesis or sequencing-by-ligation platforms currently employed by, e.g., Illumina, Life Technologies, BGI Genomics (Complete Genomics technology), Element, and Roche, Pac Bio, Singular, Oxford Nanopore, etc. Next-generation sequencing methods may also include nanopore sequencing methods or electronic-detection based methods such as, e.g., Ion Torrent technology commercialized by Life Technologies.

[0049] The term “duplex,” or “duplexed,” as used herein, describes two complementary polynucleotides that are base-paired, i.e., hybridized together.

[0050] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are used interchangeably herein to refer to forms of measurement, and include determining if an element is present or not. These terms include both quantitative and / or qualitative determinations. Assessing may be relative or absolute.

[0051] The term “ligating”, as used herein, refers to the enzymatically catalyzed joining of the terminal nucleotide at the 5’ end of a first DNA molecule to the terminal nucleotide at the 3’ end of a second DNA molecule.

[0052] The terms “plurality”, “set” and “population” are used interchangeably to refer to something that contains at least 2 members. In certain cases, a plurality may have at least 10, at least 100, at least 1000, at least 10,000, or at least 100,000 members.

[0053] A “primer binding site” refers to a site to which an oligonucleotide hybridizes in a target polynucleotide or fragment. If an oligonucleotide “provides” a binding site for a primer, then the primer may hybridize to that oligonucleotide or its complement.

[0054] The term “strand” as used herein refers to a nucleic acid made up of nucleotides covalently linked together by covalent bonds, e.g., phosphodiester bonds.

[0055] The term “extending”, as used herein, refers to the extension of a nucleic acid by ligation or the addition of nucleotides using a polymerase. If a nucleic acid that is annealed to a polynucleotide is extended, the polynucleotide acts as a template for an extension reaction. In these embodiments, the nucleic acid may be extended by a template-dependent polymerase or by ligation to an oligonucleotide that is complementary to the polynucleotide, where the polynucleotide acts as a splint.

[0056] The term "extending" includes extension at the 3' end or the 5' end. Primer extension, ligation and gap-fill ligation reactions are types of extending.

[0057] The term "extendible 5' or 3' end" refers to a 5' phosphate and 3' hydroxyl, respectively, both of which are extensible by ligation. 3' hydroxyls are also extendible by a polymerase. As used herein, the term “rolling circle amplification” or “RCA” for short refers to an isothermal amplification that generates linear concatemerized copies of a circular nucleic acid template using a strand-displacing polymerase. RCA is well known in the molecular biology arts and is described in a variety of publications including, but not limited to Lizardi et al (Nat. Genet. 1998 19:225-232), Schweitzer et al (Proc. Natl. Acad. Sci. 2000 97:10113- 10119), Wiltshire et al (Clin. Chem. 2000 46:1990-1993) and Schweitzer et al (Curr. Opin. Biotech 2001 12:21-27), which are incorporated by reference herein.

[0058] As used herein, the term “rolling circle amplification products” refers to the concatemerized products of a rolling circle amplification reaction.

[0059] The term "opposite end" refers to the other end of a nucleic acid molecule. The opposite end to a 3' end is the 5' end and the opposite end to a 5' end is the 3' end.

[0060] The term "gap-fill ligation" refers to a reaction in which two oligonucleotide sequences hybridize to nearby sites on a template to define a gap. The gap is filled in by a polymerase and the nick between the primer extension product is sealed by a ligase. See, e.g., Mignardi et al, Nucleic Acids Res. 2015 43: el51. The distance between one end of the first oligonucleotide and the other end of the second oligonucleotide (i.e., the "gap") may be in the range of 4-100 nucleotides (e.g., 5-30 nucleotides) although this distance could be longer or shorter in some cases.

[0061] The term "gap-fill ligation conditions" refers to conditions in which at least one gapfill ligation occurs, which conditions include two oligonucleotide sequences that are hybridized to nearby sites on a template to define a gap, an appropriate polymerase, ligase, dNTPs, buffer, etc. As will be exemplified below, other reactions that do not involve filling in a gap and / or sealing a nick may additionally occur at a different site in a template in a gap-fill ligation reaction (i.e., in addition to a gap-fill ligation reaction itself). Primer extension (without ligation) is an example of such a reaction.

[0062] The terms “antibody” and “immunoglobulin” include antibodies or immunoglobulins of any isotype and fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, minibodies, single-chain antibodies, nanobodies and fusion proteins comprising an antigen-binding portion of an antibody and a non- antibody protein. Also encompassed by the term are Fab’, Fv, F(ab’)2, and / or other antibody fragments that retain specific binding to antigen, and monoclonal antibodies. Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab')2, as well as bi-functional (i.e., bi- specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e. g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988)), which are incorporated herein by reference. (See, generally, Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986)).

[0063] The terms “antibody-oligonucleotide conjugate” and “capture agent that is linked to a oligonucleotide” and the like refers to a capture 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 or the like) linked to a single- stranded oligonucleotide in a way that the capture agent can still bind to its binding site. The oligonucleotide and the capture agent may be linked via a number of different methods, including those that use maleimide or halogen-containing group, which are cysteine-reactive. The capture 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. In some embodiments, the oligonucleotides may be linked to the capture agents by a linker that spaces the oligonucleotide from the capture agents. Oligonucleotides may be linked to capture agents using any convenient method (see, e.g., Gong et al., Bioconjugate Chem. 2016 27: 217-225 and Kazane et al. Proc Natl Acad Sci 2012 109: 3731-3736). In many embodiments, the sequence of an oligonucleotide that is conjugated to a binding agent uniquely identifies the epitope or sequence to which the binding agent binds. For example, if the method is performed using 10 different antibodies, then each antibody is tethered to a different sequence that identifies the epitope to which the antibody binds. This feature allows the method to be multiplexed and, in some embodiments, at least 5, at least 10, at least 20 or at least 50 different antibodies that bind to different markers in or on the surface of a cell can be used in the method. Each antibody is conjugated to a different antibody identifier sequence, and the antibody identifier sequences allow the binding events for a particular antibody to be mapped.

[0064] As used herein, the term “barcoded RCA products” is intended to refer to a population of RCA products that are each separately barcoded with a unique molecular identifier (UMI), i.e., a sequence that is unique to each RCA product that can be distinguished from one another by their unique identifier sequences.

[0065] The term “barcode sequence” or “molecular barcode”, as used herein, refers to a unique sequence of nucleotides used to a) identify and / or track the source of a polynucleotide in a reaction and / or b) count how many times an initial molecule is sequenced (e.g., in cases where substantially every molecule in a sample is tagged with a different sequence, and then the sample is amplified). A barcode sequence may be at the 5'- end, the 3 ’-end or in the middle of an oligonucleotide. Barcode sequences may vary widely in size and composition; the following references provide guidance for selecting sets of barcode sequences appropriate for particular embodiments: Casbon (Nuc. Acids Res. 2011, 22 e81), Brenner, U.S. Pat. No. 5,635,400; Brenner et al, Proc. Natl. Acad. Sci., 97: 1665- 1670 (2000); Shoemaker et al, Nature Genetics, 14: 450-456 (1996); Morris et al, European patent publication 0799897A1; Wallace, U.S. Pat. No. 5,981,179; and the like. In particular embodiments, a barcode sequence may have a length in range of from 4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides.

[0066] The term “capture moiety”, as used herein, refers to moiety that can be used to separate a molecule to which the capture moiety is attached from other molecules that do not contain the affinity tag. A “capture moiety” is a member of a specific binding pair, i.e. two molecules where one of the molecules through chemical or physical means specifically binds to the other molecule. The complementary member of the specific binding pair, referred to herein as a “capture substrate” may be immobilized (e.g., to a chromatography support, a bead or a planar surface) to produce an affinity chromatography support that specifically binds the capture moiety. In other words, an “capture moiety” may bind to a substrate, where the capture moiety specifically binds to the substrate, thereby facilitating the separation of the molecule to which the affinity tag is attached from other molecules that do not contain the affinity tag.

[0067] As used herein, the term “biotin moiety” refers to a capture moiety that includes biotin or a biotin analogue such as desthiobiotin, oxybiotin, 2’ -iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc. Biotin moieties bind to streptavidin with an affinity of at least 10"8M. A biotin affinity agent may also include a linker, e.g., — LC-biotin, — LC-LC-Biotin, — SLC-Biotin or — PEGn-B iotin where n is 3-12.

[0068] The term “terminal nucleotide”, as used herein, refers to the nucleotide at an end a nucleic acid molecule.

[0069] An “oligonucleotide binding site” refers to a site to which an oligonucleotide hybridizes in a target polynucleotide or fragment. If an oligonucleotide “provides” a binding site for a primer, then the primer may hybridize to that oligonucleotide or its complement. The term “covalently linking” refers to the production of a covalent linkage between two separate molecules, e.g., the top and bottom strands of a double stranded nucleic acid. Ligating is a type of covalent linking.

[0070] The term “denaturing,” as used herein, refers to the separation of at least a portion of the base pairs of a nucleic acid duplex by placing the duplex in suitable denaturing conditions. Denaturing conditions are well known in the art. In one embodiment, in order to denature a nucleic acid duplex, the duplex may be exposed to a temperature that is above the melting temperature of the duplex, thereby releasing one strand of the duplex from the other. In certain embodiments, a nucleic acid may be denatured by exposing it to a temperature of at least 90 °C for a suitable amount of time (e.g., at least 30 seconds, up to 30 mins). Nucleic acids may also be denatured chemically (e.g., using urea or NaOH).

[0071] The term "corresponds to" and grammatical equivalents, e.g., “corresponding”, as used herein refers to a specific relationship between the elements to which the term refers. For example, an RCA that corresponds to a sequence in a genome contains the same nucleotide sequence as the sequence in the genome.

[0072] Following convention, the complement of a sequence shown in a formula will be indicated with a prime (') such that the complement of sequence “A” will be “A'”. Moreover, unless otherwise indicated or implicit from the context, a polynucleotide defined by a formula may have an additional sequence, a primer binding site, a molecular barcode, a promoter, or a spacer, etc., at its 3' end, its 5' end or both the 3' and 5' ends. If a polynucleotide defined by a formula is described as being circular then the ends of those molecules are joined together, either directly or indirectly.

[0073] If two nucleic acids (e.g., sequences A and A’) are “complementary”, they hybridize with one another under high stringency conditions. In many cases, two sequences that are complementary have at least 10, e.g., at least 12, at least 15, at least 20 or at least 25 nucleotides of complementarity and in certain cases may have one, two or three non- complementary bases.

[0074] The various component sequences of a polynucleotide may independently have a length in the range of 8-80 nucleotides, e.g., 10-50 nucleotides or 12-30 nucleotides.

[0075] The term “target identifier sequence” (PID) refers to a sequence that identifies the target to which a capture agent binds. The same capture agents (e.g., two aliquots of the same antibody) should be associated with the same PID whereas different capture agents (a first capture agent that recognizes a first target and a second capture agent that recognizes a second target, where the first and second targets are different) should be associate with different PIDs. In some cases, the identity of the target can be identified using a look-up table. In some embodiments, the target may be a protein, e.g., a cell surface protein. In these embodiments, the capture agent may be an antibody or aptamer. In other embodiments, the target may be a sequence of nucleotides in a nucleic acid. In these embodiments, the capture agent may be an oligonucleotide probe.

[0076] The term “unique molecular identifier” or “UMI” refers to sequence that varies in a population of nucleic acid molecules. In embodiments that involve a unique molecular identifier, unless otherwise indicated, it is understood that reference to an oligonucleotide, RCA product, or grid oligonucleotide, etc., refers to a population of oligonucleotide molecules, RCA product molecules, or grid oligonucleotide molecules where the unique molecular identifier in the population contains a sequence that is variable in the population. The term “variable”, in the context of two or more nucleic acid sequences that are variable, refers to two or more nucleic acids that have different sequences of nucleotides relative to one another. In other words, if the polynucleotides of a population have a variable sequence or a particular sequence “varies”, then the nucleotide sequence of the polynucleotide molecules of the population varies from molecule to molecule. The term “variable” is not to be read to require that every molecule in a population has a different sequence to the other molecules in a population. In these embodiments, the unique molecular identifier may have a degenerate sequence and, in some embodiments, a random sequence (e.g., a random sequence of 6-20 nucleotides or more). The complexity of a unique molecular identifier may vary but in many embodiments it will composed of at least 1,000, at least 10,000, at least 100,000 or at least IM different sequences.

[0077] Other definitions of terms may appear throughout the specification.

[0078] DETAILED DESCRIPTION

[0079] As noted above, provided herein is a method for producing a map of binding events, comprising: (a) obtaining a sample that comprises uniquely barcoded RCA products that are tethered to the sample via a capture agent, wherein the RCA products comprise: (i) a unique molecular identifier that distinguishes the RCA products from one another; and (ii) a target identifier sequence that identifies the target to which the capture agent is bound; (b) hybridizing a grid oligonucleotide to the uniquely barcoded RCA products while they are tethered to the sample, wherein the 5’ and 3’ ends of the grid oligonucleotide hybridizes to different RCA products; (c) extending the 5’ and 3’ ends of the grid oligonucleotide to copy the unique molecular identifiers and target identifier sequences from the different RCA products onto the ends of the grid oligonucleotide to make an extended grid oligonucleotide; (d) sequencing the extended grid oligonucleotide to produce sequence reads; (e) identifying which unique molecular identifiers and target identifier sequences are paired in the sequence reads; and (f) producing a map of the binding sites of the capture agent and the identities of the target to which the capture agent binds using paired unique molecular identifiers and target identifier sequences. In any embodiment, the target can be a protein, e.g., a cell surface protein.

[0080] In any embodiment, the extension may be done in a gap-fill ligation reaction that comprises a gap-fill primer that hybridizes to sites in the first and second RCA products that are upstream of the 5’ end of the grid oligonucleotide. In these embodiments and as illustrated in Fig. 10B, the 5’ end of the grid oligonucleotide will be extended by gap-fill ligation and become joined to the gap-fill oligonucleotide, and the 3’ end of the grid oligonucleotide will be extended by primer extension, without ligation. In alternative embodiments (as illustrated in Figs. 8, 9 and 10A), the extension may be done in a gap-fill ligation reaction that comprises a gap-fill primer that hybridizes to sites in the first and second RCA products that are upstream of the 5’ end of the grid oligonucleotide, as well as a gap-fill stop oligonucleotide that hybridize to sites in the first and second RCA products that are downstream of the 3’ end of the grid oligonucleotide. Either way, the unique molecular identifiers and target identifier sequences from the different RCA products onto the ends of the grid oligonucleotide. In view of the above, any embodiment the extension may be done under gap-fill ligation conditions that comprise at least a gap-fill primer that hybridizes to sites in the first and second RCA products that are upstream of the 5’ end of the grid oligonucleotide and, optionally, a gap-fill stop oligonucleotide that hybridize to sites in the first and second RCA products that are downstream of the 3’ end of the grid oligonucleotide. In either of these embodiments, the extension may done under gap-fill ligation conditions that include a gap-fill primer that hybridizes to sites in the first and second RCA products that are upstream of the 5 ’ end of the gap-fill oligonucleotide and, optionally, a gap-fill stop oligonucleotide that hybridize to sites in the first and second RCA products that are downstream of the 3’ end of the grid oligonucleotide. This latter embodiment may be referred to as a “dual gap-fill ligation” and is illustrated in Figs. 8, 9 and 10A. The former embodiment (which involves a single gap-fill ligation) is illustrated on Fig. 10B. In any embodiment, the uniquely barcoded RCA products comprise a first set of RCA products and a second set of RCA products, wherein the first and second sets differ in sequence and the grid oligonucleotide hybridizes to sequences that differ in the first and second sets of RCA products. These embodiments prevent both ends of a single grid oligonucleotide molecule from binding to one RCA product. In these embodiments, the extension copies the unique molecular identifiers and target identifier sequences from the first set of RCA products onto the 3’ end of the grid oligonucleotide and the unique molecular identifiers and target identifier sequences from the second set of RCA products onto the 5’ end of the grid oligonucleotide.

[0081] One implementation of the method is described in greater detail below. The method may make use of the reagent system of Fig. 1 , the principle of which is used in the reagent system of Fig. 6. However, the method may be practiced with other reagents under certain circumstances.

[0082] With reference to Fig. 1 , provided herein is a reagent system 2 comprising the following components: (a) conjugate 4 comprising capture agent 6 and oligonucleotide 8, wherein oligonucleotide 8 is tethered to the capture agent 6 by its 5’ end and comprises: a target identifier sequence (PID) that identifies the target to which the capture agent binds, a unique molecular identifier (UMI), a 5’ flanking sequence (FS1) that is 5’ of the target identifier sequence and unique molecular identifier and a 3’ flanking sequence (FS2) that is 3’ of the target identifier sequence and unique molecular identifier and (b) a padlock probe 10 that has: a 3’ end sequence (FS2’) that hybridizes to the 3’ flanking sequence of oligonucleotide 8, a 5’ end sequence (FST) that hybridizes to the 5’ flanking sequences of oligonucleotide 8, and a backbone sequence that connects the 3’ and 5’ end sequences. As shown, the oligonucleotide 8 and the padlock probe 10 hybridize to form a circular complex 12 in which the target identifier sequence and the unique molecular identifier are singlestranded. In any embodiment, the capture agent may be an antibody, aptamer or oligonucleotide probe. In any embodiment, the UMI of oligonucleotide 8 may be a degenerate (e.g., random) sequence of 6-20 or more nucleotides.

[0083] In any embodiment, the reagent system may comprise reagents for performing a gapfill ligation and a rolling circle amplification, such as a strand-displacing DNA polymerase (optionally strand-displacing DNA polymerase that has a 3’ exonuclease activity), a non- strand-displacing polymerase, dNTPs (including dUTP) and any necessary buffers. With reference to Fig. 2, an embodiment of the present disclosure comprises obtaining the components of the reagent system 2, hybridizing the padlock probe to the oligonucleotide to form a circular complex 12 in which the target identifier sequence and the unique molecular identifier are single-stranded. In some embodiments and as illustrated, this method may comprise performing a gap-fill ligation reaction to extend the 3’ end of the padlock probe using the oligonucleotide as a template to produce a covalently closed nucleic acid circle 20 comprising a complement of the target identifier sequence and a complement of the unique molecular identifier.

[0084] In some embodiments, the method further comprises binding conjugate 4 to a cellular sample (which will be described in greater detail below) such that the conjugate binds to target molecules in the sample via the capture agent 6. In these embodiments, the binding may be done before hybridization of padlock probe 10 to oligonucleotide 8, after hybridization of padlock probe 10 to oligonucleotide 8 but before the gap-fill ligation reaction or after the gap-fill ligation reaction.

[0085] As illustrated in Fig. 3, this method may further comprise: amplifying covalently closed nucleic acid circle 20 by rolling circle amplification (RCA) using oligonucleotide 8 as an initiation primer, thereby producing an RCA product that comprises a concatenate of a sequence that is a copy of the target identifier sequence (PID), a copy of the unique molecular identifier (UMI), and the complements of the backbone sequence, the 3’ end sequence and the 5’ end sequence (not shown). There may be tens, hundreds, or thousands of copies of the sequence in the RCA product.

[0086] In some embodiments, oligonucleotide 8 may be designed such that in the gap-fill ligation product 22 oligonucleotide 8 of the conjugate has a protected 3 ’ end that cannot be extended by a 3 ’ exonuclease-deficient DNA polymerase using the covalently closed nucleic acid circle as a template. In these embodiments, the gap-fill ligation reaction may be done using a 3’ exonuclease-deficient DNA polymerase and the RCA reaction may be done using a strand displacing polymerase that has 3’ exonuclease activity and a polymerase activity, wherein exonuclease activity of the polymerase removes the protected 3 ’ end and, after removal of the protected 3’ end, the polymerase activity of the polymerase extends the oligonucleotide using the covalently closed nucleic acid circle as a template. Klenow fragment of Taq DNA polymerase (which is a 3’ exonuclease-deficient DNA polymerase) could be used for the gap-fill reaction and phi29 DNA polymerase (which has 3’ exonuclease activity) for the RCA reaction, although other choices are available. In these embodiments, the 3’ end of oligonucleotide 8 may have a chemical block or one or more mismatched nucleotides. For example, in some embodiments, the terminal nucleotide at the 3’ end of oligonucleotide 8 may not base pair with the corresponding nucleotide. In some embodiments, the terminal nucleotide at the 3’ end of oligonucleotide 8 and one or two nucleotide next to it may not base pair with the corresponding nucleotide.

[0087] In any embodiment, the RCA may be done in the presence of one or more compaction oligonucleotides or the compaction oligonucleotides may be added during or immediately the grid oligonucleotide hybridization step, which is described below. Strategies for the design of which are described in Clausson et al (Sci Rep 2015 5: 12317). In general, a compaction oligonucleotide comprises a first sequence that hybridizes to a first repeat in the product and a second repeat that hybridizes to a second repeat in the product, wherein the binding site in the repeats are 10-30 nucleotides in length. In some embodiments, the first and second sequences can be the same. Compaction oligonucleotides can be blocked at the 3' end to avoid extension and degradation, e.g., using a 2'0-methyl group. This may reduce the size of the RCA products and increase resolution. The same effect may be achieved using crowding agents such as polyethylene glycol (PEG). In some embodiments, the RCA reaction may be done in the presence of dUTP, thereby creating RCA products that comprise uracil. These RCA products may be degraded at a later time (using USER), if required later in the protocol.

[0088] Also provided is a composition comprising complex 22 (see Fig. 2) that, in some embodiments, comprises: (a) a conjugate 4 comprising capture agent 6 and oligonucleotide 8, wherein the oligonucleotide is tethered to the capture agent by its 5’ end and comprises: (i) a target identifier sequence (PID) that identifies the target to which the capture agent binds, (ii) a unique molecular identifier (UMI), (iii) a 5’ flanking sequence (FS1) that is 5’ of the target identifier sequence and unique molecular identifier, and (iv) a 3’ flanking sequence (FS2) that is 3’ of the target identifier sequence and unique molecular identifier and (b) covalently closed nucleic acid circle 22. As shown, oligonucleotide 22 and covalently closed nucleic acid circle 20 are hybridized via elements (i), (ii), (iii) and (iv), i.e., the PID, UMI, FS1 and FS2, in the complex. In other words, as shown, covalently closed nucleic acid circle 22 has a sequence that contains PID’, UMI’, FS1’ and FS2’, which are complementary to PID, UMI, FS1 and FS2, through which the molecules are hybridized.

[0089] As described above, the capture agent may be an antibody, aptamer or oligonucleotide probe and oligonucleotide 8 may have a protected 3’ end that cannot be extended by a 3 ’ exonuclease-deficient DNA polymerase using the covalently closed nucleic acid circle as a template.

[0090] This complex may be in solution (i.e., dissolved in a solution, where the capture agent is not tethered to a solid). Alternatively, the complex may be bound to an object, e.g., a sample such as a cell or tissue via the capture agent.

[0091] Reagent system 30 is also provided (see Fig. 5). In some embodiments, reagent system 30 may comprise the following components: (a) first conjugate 32 comprising a capture agent 34 and oligonucleotide 36, wherein oligonucleotide 36 is tethered to the capture agent by its 5’ end and comprises: a target identifier sequence (PID1) that identifies the target to which the capture agent binds, a unique molecular identifier (UMI1), a first 5’ flanking sequence (FS1) that is 5’ of the target identifier sequence and unique molecular identifier and a first 3’ flanking sequence (FS2) that is 3’ of the target identifier sequence and unique molecular identifier and (b) second conjugate 38 comprising capture agent 40 and oligonucleotide 42, wherein the oligonucleotide is tethered to the capture agent by its 5’ end and comprises: a target identifier sequence (PID2) that identifies the target to which the capture agent binds (which sequence may be the same as PD1), a unique molecular identifier (UMI2), a second 5’ flanking sequence (FS3) that is 5’ of the target identifier sequence and unique molecular identifier and a second 3’ flanking sequence (FS4) that is 3’ of the target identifier sequence and unique molecular identifier; wherein at least one of the 5’ and 3’ flanking sequences of the second conjugate is different to the 5’ and 3’ flanking sequences of the first conjugate (in any embodiment both the 5’ and 3’ flanking sequences of the second conjugate are different to the 5’ and 3’ flanking sequences of the first conjugate). In any embodiment, the UMI of oligonucleotides 36 and 42 may be a degenerate (e.g., random) sequence of 6-20 or more nucleotides.

[0092] This system may further comprise: (c) first padlock probe 44 that has: a first 5’ end sequence (FST), wherein first 5’ end sequence (FST) is complementary to the first 5’ flanking sequence (FS1) of the first conjugate and a first 3’ end sequence (FS2’), wherein first 3’ end sequence (FS2’) is complementary to the first 3’ flanking sequence (FS2) of the first conjugate, and a backbone sequence that connects the 3’ and 5’ end sequences, and (d) second padlock probe 46 that has: a second 5’ end sequence (FS3’), wherein the second 5’ end sequence (FS3’) is complementary to the second 5’ flanking sequence (FS3) of the second conjugate; a second 3’ end sequence (FS4’), wherein the second 3’ end sequence (FS4’) is complementary to the second 3’ flanking sequence (FS4) of the second conjugate; and a backbone sequence that connects the 3’ and 5’ end sequences. The complex formed by hybridizing the components of the composition together (which may occur en masse) are shown at the bottom of Fig. 5. As for the above, the capture agent of the first conjugate and the capture agent of the second conjugate may be, independently, antibodies, aptamers or oligonucleotide probes.

[0093] In some embodiments, capture agent 34 of the first conjugate and capture agent 40 of the second conjugate may be the same capture agent, linked to different oligonucleotides. In these embodiments, the PID1 and PID2 sequences may be the same in oligonucleotides 36 and 42. In other embodiments, capture agent 34 of the first conjugate and capture agent 40 of the second conjugate may be different. In these embodiments, the capture agents may bind to different proteins or different sites in the same protein. In these embodiments, in these embodiments, the PID1 and PID2 sequences will be different.

[0094] In any embodiment, the system may comprise additional components, a ligase, a strand-displacing DNA polymerase (optionally strand-displacing DNA polymerase that has a 3’ exonuclease activity), and / or a non-strand-displacing polymerase, or other components that may be used in the following method.

[0095] A method that uses the reagent system 30 is also provided. In some embodiments and with reference to Fig. 6, this method may comprise obtaining the components of reagent system 30, hybridizing first and second padlock probes 44 and 46 to the oligonucleotides of the first and second conjugates to form: a first circular complex 50 comprising the first conjugate and the first padlock probe, in which the target identifier sequence (PID1) and the unique molecular identifier (UMI1) are single-stranded, and a second circular complex 52 comprising the second conjugate and the second padlock probe, in which the target identifier sequence (PID2) and the unique molecular identifier (UMI) are single-stranded. Again, this may be an en masse hybridization.

[0096] With reference to Fig. 6, in some embodiments, the method may further comprise performing a gap- fill ligation reaction to extend the 3’ end of the first and second padlock probes using the first and second conjugates as a template to produce: (i) a first covalently closed nucleic acid circle 54 comprising the first padlock probe and complements of the target identifier sequence (PID1’) and unique molecular identifier (UMIF) from the first conjugate and (ii) a second covalently closed nucleic acid circle 56 comprising the second padlock probe and complements of the target identifier sequence (PID2’) and unique molecular identifier (UMI2’) from the second conjugate. Again, this may be an en masse reaction.

[0097] Similar to that described above, the method may further comprise binding the first and second conjugates to a cellular sample such that the conjugates bind to target molecules in the sample via the capture agent and wherein the binding is done before hybridization of the first and second padlock probes to their respective oligonucleotides, after hybridization of the first and second padlock probe to their respective oligonucleotides but before the gapfill ligation reaction or after the gap-fill ligation reaction.

[0098] In any embodiment and as shown in Fig. 7, the method may comprise amplifying the first and second covalently closed nucleic acid circles by rolling circle amplification (RCA) using the oligonucleotides of the first and second conjugates as an initiation primer, respectively, thereby producing: first RCA product 60 that, as shown, comprises: the complements of the backbone sequence, the first 3’ end sequence and the first 5’ end sequence (which complements are the same as the first and second flanking sequences of the first oligonucleotide 36, i.e., FS1 and FS2) of the first padlock probe and copies of the target identifier sequence (PID1) and unique molecular identifier (UMI1) from the first conjugate, and second RCA product 62 that, as shown, comprises: the complements of the backbone sequence, the second 3’ end sequence and the second 5’ end sequence (which complements are the same as the third and fourth flanking sequences of the second oligonucleotide 42, i.e., FS3 and FS4) of the second padlock probe and copies of the target identifier sequence (PID2) and unique molecular identifier (UMI2) from the second conjugate. In any embodiment, the RCA may be done in the presence of one or more compaction oligonucleotides or a crowding agent such as PEG. As described above, in the first and second circular complexes the oligonucleotides of the first and second conjugates have protected 3’ ends that cannot be extended by a 3’ exonuclease-deficient DNA polymerase using the first and second covalently closed nucleic acid circle as a template; the gap-fill ligation reaction is done using a 3’ exonuclease-deficient DNA polymerase; and the RCA reaction is done using a strand displacing DNA polymerase that has 3’ exonuclease activity and a polymerase activity, wherein exonuclease activity of the polymerase removes the protected 3’ ends, and, after removal of the protected 3’ ends, the polymerase activity extends the oligonucleotides using the first and second covalently closed nucleic acid circles as templates. As with the above, the 3’ ends of the first and second oligonucleotides have a chemical block or one or more mismatched nucleotides. Also as with the above, the RCA may be done in the presence of dUTP, thereby creating RCA products that comprise uracil.

[0099] In some embodiments and with reference to Fig. 8, the method may comprise obtaining a grid oligonucleotide 70 that comprises: (i) a 3’ end sequence that hybridizes to the first RCA product but not the second RCA product; and (i) a 5’ end sequence that hybridizes to the second RCA product but not the first RCA product and (b) hybridizing the grid oligonucleotide to the first and second RCA products to produce a complex that links the first and second RCA products together. Fig. 8 shows two examples of how this can be achieved, although there may be other ways to do it. As such, in some embodiments, the method may further comprise: (a) obtaining grid oligonucleotide 70, wherein grid oligonucleotide 70 comprises: a 3’ end sequence (FS2’) that hybridizes to the complement of the first 3’ end sequence (i.e., the complement of FS2’, which is FS2) in the first RCA product; and a 5’ end sequence (FS3’) that hybridizes to the complement of the first 5’ end sequence (i.e., the complement of FS3’, which is FS3) in the second RCA product; or a 5’ end sequence (FST) that hybridizes to the complement of the first 5’ end sequence (i.e., the complement of FS1’, which is FS1) in the first RCA product; and a 3’ end sequence (FS4’) that hybridizes to the complement of the first 5’ end sequence (i.e., the complement of FS4’, which is FS4) in the second RCA product and (b) hybridizing the grid oligonucleotide to the first and second RCA products to produce a complex that links the first and second RCA products together. As shown, the 3’ end sequence of the grid oligonucleotide (FS2’) hybridizes to the complement of the first 3’ end sequence (i.e., the complement of FS2’, which is FS2) in the first RCA product and the 5’ end sequence (FS3’) of the grid oligonucleotide hybridizes to the complement of the first 5’ end sequence (i.e., the complement of FS3’, which is FS3) in the second RCA product. If the other grid oligonucleotide is used, the 5’ end sequence (FST) of the grid oligonucleotide hybridizes to the complement of the first 5’ end sequence (i.e., the complement of FS1’, which is FS1) in the first RCA product; and a 3’ end sequence of the grid oligonucleotide (FS4’) hybridizes to the complement of the first 5’ end sequence (i.e., the complement of FS4’, which is FS4) in the second RCA product. In any embodiment, this hybridization may be done in the presence of one or more compaction oligonucleotides or a crowding agent such as PEG, which should increase resolution.

[0100] As illustrated in Fig. 9, both ends of the grid oligonucleotide may be extended in a dual gap-fill ligation reaction to add the complements of the target identifier sequences and unique molecular identifiers to the ends of the grid oligonucleotide. These embodiments may comprise extending both the 3’ end and the 5’ end of the grid oligonucleotide by a gap-fill ligation reaction using the first and second RCA products as a template to add the complements of the target identifier sequence and unique molecular identifier from the first conjugate to one end of the grid oligonucleotide and complements of the target identifier sequence and unique molecular identifier from the second conjugate to the other end of the grid oligonucleotide, to produce an extended grid oligonucleotide 80. Fig. 9 shows two ways in which this can be achieved, although there may be other ways. For example, one could use the single gap-fill ligation method shown in Fig. 10B.

[0101] As illustrated in Fig. 9, the grid oligonucleotide may have another unique molecular identifier (UMI3).

[0102] As illustrated in Fig. 8, the gap-fill ligation may be done in the presence of gap-fill primer 72 or 76 and, optionally, a gap-fill stop oligonucleotide 74 or 78 that hybridize to sites in the first and second RCA products that are on the other side of the identifier sequence and unique molecular identifier relative to the 3’- and 5’ ends of the grid oligonucleotide.

[0103] In any embodiment, the RCA reaction may be terminated by the addition of EDTA. The EDTA may be added immediately before or concurrent with the grid oligonucleotide (and other oligonucleotides that are being used in the method e.g., the gap-fill primer and a gap-fill blocker).

[0104] In some embodiments, the addition of the grid oligonucleotide (and other oligonucleotides that are being used in the method e.g., the gap- fill primer and a gap-fill blocker) may compact the RCA products together. This may decrease the stickiness of the cells which, in turn, may decrease the amount of cell clumping during later centrifugation steps.

[0105] In some embodiments, the method may further comprise amplifying the extended grid oligonucleotide 80. This may be done by polymerase chain reaction, using primers that target the sequences of the gap-fill primer and gap-fill stop oligonucleotide (e.g., using primers that target sequences FS1’ and FS4’, or primers that target FS3’ and FS2’, depending on the orientation of the grid oligonucleotide) or by rolling circle amplification (e.g., by circularizing the extended grid oligonucleotide, hybridizing a primer to the circle and amplifying the circle). Next, the extended grid oligonucleotide or an amplification product thereof may be sequenced. As would be apparent, each sequence read should contain two protein barcodes or their complements (e.g., PID1 and PID2) which provide the identities of the capture agents to which those barcodes were once associated with and three unique molecular barcodes or their complements. Two of these unique molecular barcodes (e.g., UMI1 and UMI2) allow one to differentiate between different molecules of capture agent. The third unique molecular barcode allows one to differentiate between different molecules of grid oligonucleotide.

[0106] In some embodiments, the gap-fill ligation reaction may be done in accordance with the method shown in Fig. 10A. This figure describes a method for enriching for full length products that contain unique molecular identifiers copied from two RCA products. In these embodiments and with reference to Fig 10A, the method may comprise: (a) creating a complex 90 comprising: (i) a grid oligonucleotide 92, (ii) a gap-fill stop oligonucleotide 94, (iii) a gap-fill primer 96, (iv) a first uniquely barcoded RCA product 98, and (v) a secondly uniquely barcoded RCA product 100, wherein, as shown the 3’ end of the grid oligonucleotide is hybridized to a sequence in the first RCA product that is downstream of the unique RCA product identifier sequence of the first RCA product, the 5’ end of the gapfill stop oligonucleotide is hybridized to a sequence that is upstream of the unique RCA product identifier sequence of the first RCA product, the 5’ end of the grid oligonucleotide is hybridized to a sequence in the second RCA product that is upstream from the unique RCA product identifier sequence of the second RCA product, and (b) incubating the product of (a) under gap-fill ligation conditions to produce: (i) full length products 102 that comprise the sequences of grid oligonucleotide 92, the gap-fill primer 96, the gap- fill stop oligonucleotide 94, and the complements of the RCA product identifier sequences from the first and second RCA products (NNNF and NNN2’, respectively) and (ii) incomplete products that do not comprise the full length sequences of grid oligonucleotide, the gap-fill primer, the gap-fill stop oligonucleotide, and the complements of the RCA product identifier sequences from the first and second RCA products (not shown). Next, the method may comprise (c) enriching for the full-length products 102 using an exonuclease and / or by affinity, wherein: (i) the 3' end of the gap-fill stop oligonucleotide is exonuclease resistant, the 5' end of gap-fill primer is exonuclease resistant, and enrichment of the full-length products is done by treating the product of step (b) with an exonuclease; and / or (ii) the 3' end of the gap-fill stop oligonucleotide or the 5' end of gap-fill primer has a capture moiety, and enrichment of the full-length products is done using a support that has affinity for the capture moiety. The enrichment step is not shown in Fig. 10A but should be readily understandable. In some embodiments, the exonuclease resistance can be enabled by the selection of the appropriate exonuclease deficient enzyme and both ends do not need to be chemically protected. As shown, in some embodiments, the grid oligonucleotide may have a unique molecular identifier (UMI). The length of the grid oligonucleotide may vary. In some embodiments, the grid oligonucleotide may be 50-100 nucleotides in length. However, in some embodiments, the grid oligonucleotide may be 100-300 nucleotides in length, depending on the desired resolution.

[0107] For example, an oligonucleotide can be protected from exonuclease activity by using a phosphorothioate linkage or a 2’0Me RNA or 2'-deoxyadenosine-5'-(a-thio) residue, for example. The exonuclease used in the method may be any one or a combination of exonucleases, including, e.g., both exonuclease I and exonuclease III, although one or more other exonucleases, e.g., exonuclease T, exonuclease V, exonuclease VII, T5 exonuclease, T7 exonuclease, RecJ exonuclease, etc., could be used. As would be apparent, the selected exonuclease can be specific either for the 5’ end or the 3’ of DNA, and a mixture could be used. Lambda exonuclease preferentially degrades phosphorylated 5' ends but much less so non-phosphorylated 5' ends. As such, lambda exonuclease can be used in many embodiments, particularly when the 5' end of the full length product has a free 5' end (i.e., not tethered to antibody) that is exonuclease resistant but the partial products do not.

[0108] As would be apparent, unless otherwise stated, an oligonucleotide that is modified at one end (e.g., by an exonuclease resistant linkage or nucleotide) may have hydroxyl or phosphate at the other end (whichever is appropriate).

[0109] Embodiments that employ grid oligonucleotides may be used to spatially map binding events on or in cells.

[0110] In these embodiments, the method may comprise labeling a cell with a first binding agent conjugate and second binding agent conjugate (i.e., two populations of a molecules), as described above, wherein in some embodiments, the capture agent may be a monoclonal antibody or an aptamer, although a polyclonal antibody could be used under certain circumstances. As described above, the padlock probes may be pre-hybridized to the conjugates before they are bound to the sample (in which case the gap-fill ligation reaction may be done in situ) and, in some embodiments, the padlock probes may be pre -hybridized to the conjugates and the initial gap-fill ligation reaction may be performed before the conjugates are bound to the sample. In these embodiments, the cell may be in solution, on a support (e.g., a slide), in a three-dimensional sample of tissue, or in a tissue section. A sample containing cells that are in solution may be a sample of cultured cells that have been grown as a cell suspension, for example. In other embodiments, disassociated cells (which cells may have been produced by disassociating cultured cells or cells that are in a solid tissue, e.g., a soft tissue such as liver of spleen, using trypsin or the like) may be used. In other embodiments, the cells may be found in blood, e.g., cells that are in whole blood or a sub-population of cells thereof. Sub-populations of cells in whole blood include platelets, red blood cells (erythrocytes), platelets and white blood cells (i.e., peripheral blood leukocytes, which are made up of neutrophils, lymphocytes, eosinophils, basophils and monocytes). These five types of white blood cells can be further divided into two groups, granulocytes (which are also known as polymorphonuclear leukocytes and include neutrophils, eosinophils and basophils) and mononuclear leukocytes (which include monocytes and lymphocytes). Lymphocytes can be further divided into T cells, B cells and NK cells. Peripheral blood cells are found in the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver, or bone marrow. If cells that are immobilized on a support are used, then then the sample may be made by, e.g., growing cells on a planar surface, depositing cells on a planar surface, e.g., by centrifugation, cutting a three- dimensional object that contains cells into sections and mounting the sections onto a planar surface, i.e., producing a tissue section. In alternative embodiments, the surface may be made by absorbing cellular components onto a surface. In some embodiments, the cell or sample may be fixed and permeabilized. In other embodiments, the cell may be fixed and not permeabilized.

[0111] After binding, an RCA reaction can be performed which, as described above, will result in two populations of RCA products (i.e., a first population of RCA products derived from the oligonucleotide of the first conjugate and a second population of RCA products derived from the oligonucleotide of the second conjugate, where the RCA products are tethered to sites that are on or in a cell. The individual RCA product molecules become uniquely labeled in this process, because the underlying template has a degenerate (e.g., random) sequence (as described above). A cell that is covered in RCA products is schematically illustrated in Fig. 11. As would be apparent, this figure is a schematic illustration; cells are not perfectly spherical and the RCA products are not perfectly spherical, the same size or evenly distributed in a regular pattern, as shown. After the hybridization of the grid oligonucleotides (which hybridize to adjacent RCA products), extension of the ends of the grid oligonucleotides using the adjacent RCA products as a template (which copies contain the unique identifier sequences from the RCA products and the identities of the capture agent to which they are bound from the RCA products into the grid oligonucleotide) and sequencing the extension products, one can map pairs of binding events (i.e., which capture agents bind close to one another). Because RCA products are a concatemer of sequences, one RCA product can bind to multiple grid oligonucleotides, thereby allowing it to be mapped to multiple neighbors. The grid oligonucleotides join the RCA products together in a matrix or network, as illustrated at the bottom of Fig. 11.

[0112] As noted above, the method may comprise sequencing the extended grid oligonucleotides or an amplification product thereof, to produce sequence reads. As would be apparent, in this embodiment, any of the sequences used in the method may be compatible with use in the sequencing system being used, e.g., Illumina’s reversible terminator method, etc.

[0113] Generally, pairs of RCA products that are proximal can be identified by examining the unique molecular identifiers that have been copied onto the ends of the grid oligonucleotides. This pairwise analysis can be used to map the RCA products relative to one other in two or three dimensions. After the map has been created, the identities of the target molecules to which the RCA products are bound can be added to the map by examining which target identifier sequence is associated with each RCA product.

[0114] As illustrated in Fig. 12, after the grid oligonucleotide has been extended to add the UMIs and PIDs from adjacent uniquely barcoded RCA product onto their ends, the extended grid oligonucleotide is sequenced and then analyzed to identify which pairs of unique UMIs have been added onto the grid oligonucleotide. As illustrated in Fig. 12, each extended grid oligonucleotide molecule should have the complement of a first UMI sequence at one end (e.g., sequence UMI1) and the complement of a second UMI sequence at the other (e.g., sequence UMI2). These sequences can be analyzed to compile a list of paired UMIs (e.g., UMI1-UMI3, UMI1-UMI13, etc.) which can be used to make a two- or three-dimensional map of the RCA products. As illustrated in Fig. 12, the method may involve making one or more physical maps (a relational map) of the immobilized particles using the list paired MIDs. As would be apparent, the map may be a map of the surface of one or more cells. In some cases, the physical maps may comprise overlapping and / or non-overlapping maps. Because each of the UMIs is also associated with a target identifier sequence, the targets to which the RCA products bind can also be mapped. As such, the method may comprise producing a map of the binding sites of the capture agent and the identities of the target to which the capture agent binds using paired unique molecular identifiers and target identifier sequences.

[0115] The RCA products contain a UMI and are uniquely barcoded. Barcoded RCA products each contain a unique sequence that is in the repeated sequence. In other words, if there are 1,000 RCA products, each product will have a unique sequence (referred to herein as a unique molecular identifier “UMI”). The UMI for one RCA product is different to the UMIs for other RCA products. In some embodiments, the initial oligonucleotides (the first and second oligonucleotides) may contain a degenerate (e.g., random) sequence of 6-20 nucleotides, or even more random nucleotides dependent on the number of unique RCA products required. Amplification of circularized oligonucleotides that have a degenerate sequence should produce a population of RCA products that each have a unique identifier (i.e., a sequence that is different from the other RCA products in the population).

[0116] The present method provides a way to copy unique identifier sequences and the target identifier sequences from adjacent RCA products, as described above, onto the ends of a grid oligonucleotide. In other words, each extended grid oligonucleotide molecule should have a sequence that identifies a capture agent and a sequence that is unique to a first RCA product at one end and a sequence that identifies a capture agent and a sequence that is unique to a second RCA product at the other. These sequences can be analyzed to compile a list of neighboring RCA products and the capture agent molecules to which they are bound which, in turn, can be used to make a two- or three-dimensional map of the RCA products and the capture agents to which they are bound. In these embodiments, the method may involve making one or more physical maps (a relational map) of the binding events using the paired sequences. As would be apparent, the map may be a map of the surface of one or more cells. In some cases, the physical maps may comprise overlapping and / or nonoverlapping maps. The general principles of how neighboring RCA products can be mapped relative to one another can be found in WO2021084419 if further details are desired.

[0117] In some embodiments, the method may comprise sequencing the extended grid oligonucleotides and analyzing the sequences to identify which pairs of UMI sequences (which uniquely identify the RCA products) have been added onto the grid oligonucleotides. The method may then comprise making a physical map of the RCA products (which are immobilized or in the sample) and / or the identity of the targets which the RCA products are bound to using the pairs of UMI sequences identified by analysis of the sequence reads. Analysis of the unique molecular identifier sequences that are copied onto the ends of the grid oligonucleotide allows the binding sites for each of the capture agents that is bound to the cell to be mapped to a particular RCA product. The identities of the capture agents can then be placed on the map of RCA products described above, thereby providing a two- or three-dimensional map of the binding events, where the map may correspond to the surfaces of one or more cells or within cells.

[0118] As would be apparent, each RCA product contains multiple copies of the same sequence and, as such, multiple binding events can be mapped to a single RCA product. Mapping binding sites to RCA products that, themselves, have been mapped in two or three dimensions provides a way to examine the distribution of binding sites in or on the surface of a cell. This, in turn, provides a way to examine cell polarity without microscopy.

[0119] In any embodiment, the method can be multiplexed in these embodiments, and the method may be performed using at least 10, or at least 20 and up to 50 or 100 or more different antibodies or aptamers, each of which may be conjugated to a first oligonucleotide and a second oligonucleotide, where the target identifier sequence varies with the antibody or aptamer.

[0120] In any embodiment, the method may comprise generating thousands, tens of thousands or hundreds of thousands of uniquely barcoded RCA products (each having a unique molecular identifier and a target identifier sequence) that are tethered to cells (e.g., via an antibody) so that barcoded RCA products coat the cells or are inside the cells. Each cell may be coated in at least 100, at least 1,000 or at least 10,000 RCA products. The barcoded RCA products may be anchored to the cells via an antibody or a nucleic acid probe although other methods are possible. A physical map of the barcoded RCA products can be constructed based on the sequences that have been added to the grid oligonucleotides. The identities of the targets to which the RCA products are bound can be added to the map by examination of the target identifier sequence associated with each unique barcode sequence.

[0121] Kits

[0122] Also provided by this disclosure are kits for practicing the subject methods, as described above. In certain embodiments, the kit may comprise any of the components listed above.

[0123] The kit may additionally contain other agents, including buffers and other components described above. The various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container, as desired. 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.

[0124] EXAMPLES

[0125] The following examples are put forth so as to provide those of ordinary skill in the art with additional disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.

[0126] EXAMPLE 1

[0127] An example of one way that the method can be implemented is illustrated in Figs. 13 and 14. In these figures, relative to the detailed description, COa and COb correspond to the oligonucleotides in the first and second conjugates, PID corresponds to the PID, the UPI corresponds to the UMI, LBS1, LBS1, P5 and P7 correspond to flanking sequences in the oligonucleotides of the first and second conjugate, PL1 is one of the padlock probes and PL2 is the other of the padlock probes.

[0128] With reference to Figs. 13 and 14, the exemplary method is summarized below:

[0129] Two different conjugation oligos (COa and COb) are conjugated to antibodies via their 5’ ends (possibly in random proportions in a mixed conjugation reaction). In this step, COa is composed of a linker binding site 1 (LBS1), a protein barcode (PID), a unique pixel identifier (UPI) and an Illumina region for indexing PCR (P5). COb is composed of an illumina region P7, PID, UPI and linker binding site 2 (LBS2).

[0130] After binding of the conjugates to their targets (which step may be done later, after the padlock probes are hybridized), two different padlock probes (PL1 and PL2) are hybridized to their respective conjugation oligos. PL1 hybridizes to COa and PL2 hybridizes to COb.

[0131] A gap-fill ligation reaction copies the complements of the PID and UPI from the COs into the padlock probes.

[0132] RCA is then performed to produce two types of rolling circle products (RCPs), each anchored to the sample by the COs.

[0133] Next, the different RCPs are connected together using a grid oligo, containing another UMI (referred to as a unique event identifier (UEI)), which binds to the LB SI region in the first RCP (from COa) and the LBS2 region in the second RCP (from COb). At this point, a gap-fill primer (GF primer) and a gap-fill blocker (GF blocker) are also hybridized to the P5 and P7 regions, respectively.

[0134] Next (as illustrated in Fig. 14) a gap-fill ligation reaction is performed to copy the complements of the UPI and PID from both of the RCPs onto the grid oligo.

[0135] The RCPs can be removed using USER enzyme (if desired and if the RCA is done in the presence of dUTP) and the extended grid oligonucleotide can amplified by PCR using standard Illumina primers that target sequences the GFblocker and GFprimer oligonucleotides (P5 and P7).

[0136] Exemplary sequences elements are shown below:

[0137] EXAMPLE 2

[0138] A proof of concept was reduced to practice using the following protocol. PLs hybridization

[0139] A mix containing 0.3 pM of PL1 and PL2 in lx rCUT smart buffer is added to either cells bound by antibody oligonucleotide conjugates or streptavidin beads containing the COs coupled via biotin on their 5’ end instead of an antibody. Streptavidin beads are used as a surrogate for cells. The reaction is carried out for 30 min at 45°C. After incubation, cells / beads are washed twice with 2.0 wash buffer (50 mM NaCL, 1 mM EDTA, 20 mM Tris buffer pH8 and Tween 20 0.05%). Between each wash, cells / beads are centrifuged for 4 min at 700 x g allowing them to pellet at the bottom of the reaction tube. PLs circularization

[0140] A mix containing 1 mM ATP, 0.1 mM dNTPs, 8 U / ul T4 DNA ligase and Hemo Klentaq polymerase in lx rCUTsmart is added to cells / beads and the samples are incubated for 30 min at 37°C. Washes with 2.0 wash buffer are performed as described previously after the incubation. T4 DNA ligase appears to perform better then Taq ligase in this step.

[0141] In some embodiments the base at the 3’ end of the CO may have a mismatch relative to the PL. In these cases, one can perform the gap-fill ligation with a polymerase with no 3’ 5’ exonuclease activity (e.g., Hemo Klentaq).

[0142] RCA

[0143] RCA is performed for 30 min at 30°C by adding a mix containing 0.2 U / pL of Phi29 polymerase, 200 ug / ml BSA, 0.75mM dUACGs in lx Phi29 buffer. Phi29 is then inactivated for 10 min at 65 °C followed by washes with 2.0 wash buffer. dUTPs removal

[0144] Incubation with 1 U of rSAP enzyme for 20 min at 37°C, followed by inactivation of the enzyme for 5 min at 65 °C, allows removal of dUTPs used during RCA. Cells / beads are then washed with 2.0 wash buffer.

[0145] Linker oligo. GFprimer and GFblocker hybridization

[0146] A mix containing 1 pM of linker oligo, 1 pM of GF primer and 1 pM of GF blocker in lx Cutsmart is added to the cells / beads. Hybridization is carried out for 30 min at 45 °C followed by washes with 2.0 wash buffer.

[0147] Gap-fill ligation

[0148] Gap-fill ligation is performed by adding to the cells / beads a mix containing 1 mM ATP, 0.1 mM dNTPs, 8 U / pL T4 DNA ligase and Hemo Klentaq polymerase in lx rCUTsmart. The reaction is carried out for 30 min at 37°C.

[0149] Counting of cells / beads and USER digestion

[0150] Before proceeding to the next steps, an aliquot of the sample is placed on a 96 well plate and the number of cells / beads is quantified. Either 200 or 500 cells / beads are then selected and dispensed in a new reaction tube and 1 U of USER enzyme is added in order to digest the RPCs. After this step, the amplicon formed between RCPs will be floating in the solution; therefore, it is crucial to avoid any washing steps and proceed directly to PCR for amplification and indexing.

[0151] PCR PCR is performed using Illumina forward and reverse UDI indexes that will bind to the P5 and P7 region of the generated amplicon. PCR denaturation step is performed for 60 sec at 98°C, followed by extension for 15 sec at 98°C, 30 sec at 60°C and 40 sec at 72°C repeated for 11 cycles. Final extension is carried out for 5 min at 72°C.

[0152] Results are shown in Figs. 15-17. Fig. 15 shows a gel of reaction products after hybridization of padlock probe PL1 to COa and gap-fill ligation (top) and after hybridization of padlock probe PL2 to COb and gap-fill ligation (bottom). Fig. 16 shows the products of the first gap-fill ligation assay. Circularized products are identified. This data also shows that T4 DNA ligase may perform better than Taq ligase under the conditions tested. Fig. 17 shows the results of a PCR assay for detecting RCA products. On the top, the PCR is specific for RCA products generated from the COa - PL1 gap-fill ligation. On the bottom, the PCR is specific for RPC generated from the COb - PL2 gap-fill ligation. Fig. 18 shows the results of a reaction in which a grid oligonucleotide is hybridized to both RCA products and extended at both ends in a dual gap-fill ligation reaction that adds the PID and UPI (i.e., PID and UMI) from both RCA products onto the ends of the grid oligonucleotide. A product of the expected size is identified.

[0153] EXAMPLE 3

[0154] Materials and methods

[0155] Cell fixation and AOC staining: Cells were resupended in PBS and fixed for 15 min at room temperature in a fixation solution containing 1% v / v PFA in PBS. After fixation, cells were washed once in PBS, followed by addition of a blocking / quenching solution containing 1% FBS, 0.1% BSA, 1 mg / ml of single-stranded DNA, 50 pg / ml of human IgG, 125 mM of glycine, 4 mM of EDTA and 0.04% ProCiin 300 in PBS. Cells were incubated for 15 min at 4 °C, followed by a wash in PBS to remove the blocking / quenching solution.

[0156] Fixed and blocked cells were stained for 16 h at 4°C in a 50 pl reaction containing a cocktail of 164 antibody oligo conjugates (AOCs) each at a concentration of 0.156 ug / ml in a staining buffer comprising 0.2% BSA and 2 mM EDTA in PBS. After four washing steps in wash buffer (50 mM of NaCl, 1 mM of EDTA, 20 mM of Tris-HCl pH8 and 0.05% of Tween- 20) AOCs bound to cells were stabilized using a secondary antibody by incubating the cells for 30 min at 37 °C in a secondary antibody solution consisting of 60 pg / ml of secondary antibody, 0.2% BSA and 2 mM of EDTA in PBS, followed by two washing steps in wash buffer, before proceeding with the protocol. Proximity Network Assay (PNA) protocol: Padlock probes (PL1 and PL2) were hybridized to AOCs on 20,000 stained cells in a 50 pl hybridization reaction containing 0.3 pM of each padlock probe and wash buffer. The reaction was incubated for 30 min at 37°C followed by two washes in wash buffer.

[0157] Padlock probes hybridized on AOCs were circularized via a 50 pl gap-fill ligation reaction consisting of 1 pl Hemo Klentaq (New England Biolabs, cat.no. M0332L), 1 pl (10 U) of E. Coli DNA Ligase (New England Biolabs, cat.no. M0205L), 0.5 pl (0.1 mM) of deoxynucleotide triphosphates (dNTPs) (New England Biolabs, cat.no. N0447L) and 0.5 pl (0.5 mM) of NAD+ (New England Biolabs, cat.no. B9007S) in lx rCutSmart buffer (New England Biolabs, cat.no. B6004S). The reaction was incubated for 30 min at 25°C followed by two washes in wash buffer.

[0158] Rolling circle amplification (RCA) was performed via a 50 pl reaction consisting of 2.5 pl (25U) of Phi29 polymerase (New England Biolabs, cat.no. M0269L), 1.5 pl (0.75 mM) of dUACGs (New England Biolabs, cat.no. N0447L and N0459S), 0.5 pl (200 ug / ml) of recombinant albumin (New England Biolabs, cat.no. B9200S) in lx Phi29 buffer (New England Biolabs, cat.no. M0269L). The reaction was incubated for 10 min at 30°C.

[0159] In order to prevent cell clumping by generating a partial double-strand rolling circle product, a 100 pl solution containing 1.5 pM of proximity oligos (one linker oligo and two gapfill primers) in a low salt - high EDTA buffer (10 mM of NaCl, 45 mM EDTA, 20 mM of Tris-HCl pH8 and 0.05% of Tween-20) was added directly to the 50 pl RCA reaction solution. EDTA at high concentration was used as a chelating agent to deactivate Phi29 polymerase eliminating the need of performing heat inactivation of the enzyme which might damage the cell membrane and affect AOCs stability. The reaction was incubated for 30 min at 45°C followed by three washes to remove excess EDTA which might interfere with downstream reactions.

[0160] UMIs from the rolling circle product were incorporated into a final amplicon by performing a gap- fill ligation reaction in the same conditions as described for the padlock probes circularization step.

[0161] Cells were counted using either a hemocytometer or a Countess automated cell counter (ThermoFisher Scientific) and an aliquot of less than 500 cells was resuspended in a 20 pl volume of Nuclease Free Water.

[0162] PCR and next- generation sequencing: Pre-amplification PCR was performed in a 50 pl reaction containing lx Q5 HotStart HiFi PCR master mix (New England Biolabs, cat.no. M0494L) and 0.25 ,u M (each) of Illumina Truseq and Nextera PCR primers. The denaturation step during the pre- amplification PCR was carried out for 10 min.

[0163] The volume of the pre-amplified product was increased to 100 pl by addition of 50 pl of Nuclease Free Water and a double-sided clean-up (0.6x / lx) was performed using SPRIselect beads (Beckman Coulter, cat.no. B 22318). The purified product was eluted in 50 pl of lOmM Tris, pH 8.0 (ThermoFisher Scientific, cat.no. AM9855G) and could be refrigerated at 4°C for many days or frozen at -20°C for long term storage.

[0164] Optionally, in order to determine the exact number of indexing PCR cycles needed to avoid over-amplification of the samples, qPCR could be performed using 0.5 pM (each) of Illumina Truseq and Nextera PCR primers and lx PowerUp SYBR Green Master Mix (ThermoFisher Scientific, cat.no. A25742).

[0165] Indexing PCR was performed in a 50 pl reaction containing lx Q5 HotStart HiFi PCR master mix (New England Biolabs, cat.no. M0494L), 0.4 pM of Illumina adapter PCR primers (ILM_p5_PCR, ILM_p7_PCR) containing eight nucleotide sample indexes to allow multiplexing and 5 pl of purified pre-amplified sample.

[0166] The PCR products were purified with a lx clean-up step using SPRIselect beads (Beckman Coulter, cat.no. B22318) and eluted in 35 pl of lOmM Tris, pH 8.0 (ThermoFisher Scientific, cat.no. AM9855G); final quantification was performed using Qubit HsDNA assay (Thermo Fisher Scientific, cat.no. Q32854). The purified and quantified products were diluted to either 0.65 nM - for Illumina P2 Xleap kits - or 0.488 nM - for Illumina P3 and P4 Xleap kits - with 15% PhiX (Illumina) spiked in and paired-end sequenced on an Illumina NextSeq2000 sequencing system, using 44 cycles for Read 1 and 78 cycles for Read 2.

[0167] Data was analyzed computationally by connecting the UMI pairs into a 3D spatial network with proteins (AOCs) serving as nodes and the linker oligos as links.

[0168] Results

[0169] The size and composition of the final amplicon generated with PNA is displayed in Fig. 19A and comprises: (1) the Illumina PCR handles (P5 Truseq and P7 Nextera), (2) the unique molecular identifiers (UMIs) incorporated respectively from the rolling circle products of conjugation oligo A (COa) and B (COb), (3) the protein identifiers (PIDs) comprising the information of two proteins in close proximity and (4) the linker region with its unique event identifier (UEI) used as a connection between two AOCs. Paired-end sequencing is performed using 44 cycles for Read 1 and 78 cycles for Read 2 reading the information of the UMIs, PIDs and UEI. The length of the final amplicon is 277 bp as visualized in the 4% agarose gel (Ikb DNA ladder). PNA was performed on fixed PBMCs in suspension using a 164 AOCs plex and Fig. 19B displays cells visualized in a uniform manifold approximation and projection (UMAP), showing separated clusters of the main populations expected at single-cell resolution. It is also possible to generate 3D scatter plots of a single-cell spatial layout where each protein relative position is displayed, showing abundance of specific markers as visible in Fig. 19C. Shows the expression and position of CD 16 on a monocyte cell (top-left), CD3e on a T cell (top-right) and CD22 on a B cell (bottom-left).

Claims

CLAIMSWhat is claimed is:

1. A method for producing a map of binding events, comprising:(a) obtaining a sample that comprises uniquely barcoded rolling circle amplification (RCA) products that are tethered to the sample via a capture agent that is bound to the sample at multiple sites, wherein the RCA products comprise:(i) a unique molecular identifier that distinguishes the RCA products from one another; and(ii) a target identifier sequence that identifies the target to which the capture agent is bound;(b) hybridizing a grid oligonucleotide to the uniquely barcoded RCA products while they are tethered to the sample, wherein the 5’ and 3’ ends of the grid oligonucleotide hybridize to different RCA products;(c) extending the 5’ and 3’ ends of the grid oligonucleotide to copy the unique molecular identifiers and target identifier sequences from the different RCA products onto the ends of the grid oligonucleotide to make an extended grid oligonucleotide;(d) sequencing the extended grid oligonucleotide to produce sequence reads;(e) identifying which unique molecular identifiers and target identifier sequences are paired in the sequence reads; and(f) producing a map of the binding sites of the capture agent and the identities of the target to which the capture agent binds using paired unique molecular identifiers and target identifier sequences.

2. The method of claim 1, wherein the capture agent is an antibody, aptamer or oligonucleotide probe.

3. The method of claim 1 or 2, wherein the target is a protein.

4. The method of any prior claim, wherein the uniquely barcoded RCA products comprise a first set of RCA products and a second set of RCA products, wherein the sets differ in sequence and the grid oligonucleotide hybridizes to sequences that differ in the firstand second sets of RCA products.

5. The method of claim 4, wherein the extension copies the unique molecular identifiers and target identifier sequences from the first set of RCA products onto the 3’ end of the grid oligonucleotide and the unique molecular identifiers and target identifier sequences from the second set of RCA products onto the 5 ’ end of the grid oligonucleotide.

6. The method of any prior claim, wherein the sample comprises cells and the capture agent binds to sites that are in or on the cells.

7. The method of any prior claim, wherein the sample is a sample of tissue or a sample of one or more individual cells.

8. The method of any prior claim, wherein the RCA products are made in situ.

9. A reagent system comprising the following components:(a) an oligonucleotide that is tethered to the capture agent by its 5’ end and comprises: a target identifier sequence that identifies the target to which the capture agent binds, a unique molecular identifier, a 5’ flanking sequence that is 5’ of the target identifier sequence and unique molecular identifier and a 3’ flanking sequence that is 3’ of the target identifier sequence and unique molecular identifier; and(b) a padlock probe that has: a 3’ end sequence that hybridizes to the 3’ flanking sequence, a 5’ end sequence that hybridizes the 3’ flanking sequence, and a backbone sequence that connects the 3’ and 5’ end sequences; wherein the oligonucleotide of (a) and the padlock probe of (b) hybridize to form a circular complex in which the target identifier sequence and the unique molecular identifier are single- stranded.

10. The reagent system of claim 9, wherein the reagent system comprises a conjugate, wherein the conjugate comprises the capture agent and the oligonucleotide,11. The reagent system of claim 10, wherein the capture agent is an antibody, aptamer or oligonucleotide probe.

12. The reagent system of claim 9, 10 or 11, further comprising a ligase, a stranddisplacing DNA polymerase (optionally a strand-displacing DNA polymerase that has a 3’ exonuclease activity), and / or a non-strand-displacing polymerase.

13. A method comprising : obtaining the components of the reagent system of any of claims 9-12; hybridizing the padlock probe to the oligonucleotide to form a circular complex in which the target identifier sequence and the unique molecular identifier are single-stranded.

14. The method of claim 13, further comprising: performing a gap-fill ligation reaction to extend the 3’ end of the padlock probe using the oligonucleotide as a template and produce a covalently closed nucleic acid circle comprising a complement of the target identifier sequence and a complement of the unique molecular identifier.

15. The method of claim 13 or 14, wherein the method further comprises: binding the conjugate to a cellular sample such that the conjugate binds to target molecules in the sample via the capture agent and wherein the binding is done: before hybridization of the padlock probe; after hybridization of the padlock probe but before the gap-fill ligation reaction; or after the gap-fill ligation reaction.

16. The method of claim 14 or 15, further comprising: amplifying the covalently closed nucleic acid circle by rolling circle amplification (RCA) using the oligonucleotide of the conjugate as an initiation primer, thereby producing an RCA product that comprises a copy of the target identifier sequence, a copy of the uniquemolecular identifier, the complements of the backbone sequence, the 3’ end sequence and the 5’ end sequence.

17. The method of any of claims 14-16, wherein: in the gap-fill ligation product the oligonucleotide of the conjugate has a protected 3’ end that cannot be extended by a 3’ exonuclease-deficient DNA polymerase using the covalently closed nucleic acid circle as a template; the gap-fill ligation reaction is done using a 3’ exonuclease-deficient DNA polymerase; and the RCA reaction is done using a strand displacing polymerase that has 3’ exonuclease activity and a polymerase activity, wherein exonuclease activity of the polymerase removes the protected 3’ end and, after removal of the protected 3’ end, the polymerase activity of the polymerase extends the oligonucleotide using the covalently closed nucleic acid circle as a template.

18. The method of claim 17, wherein the 3’ end of the oligonucleotide has a chemical block or one or more mismatched nucleotides.

19. The method of any of claims 16-18, wherein the RCA is done in the presence of dUTP, thereby creating RCA products that comprise uracil.

20. A composition comprising a complex comprising:(a) a conjugate comprising a capture agent and an oligonucleotide, wherein the oligonucleotide is tethered to the capture agent by its 5’ end and comprises:(i) a target identifier sequence that identifies the target to which the capture agent binds,(ii) a unique molecular identifier,(iii) a 5’ flanking sequence that is 5’ of the target identifier sequence and unique molecular identifier(iv) 3’ flanking sequence that is 3’ of the target identifier sequence and unique molecular identifier; and(b) a covalently closed nucleic acid circle,wherein the oligonucleotide and covalently closed nucleic acid circle are hybridized via elements (i), (ii), (iii) and (iv) in the complex.

21. The composition of claim 20, wherein the capture agent is an antibody, aptamer or oligonucleotide probe.

22. The composition of claim 20 or 21, wherein the oligonucleotide has a protected 3’ end that cannot be extended by a 3’ exonuclease-deficient DNA polymerase using the covalently closed nucleic acid circle as a template.

23. The composition of any of claims 20-22, wherein the complex is in solution.

24. The composition of any of claims 20-23, wherein the complex is bound to a cell via the capture agent.

25. A reagent system comprising the following components:(a) a first conjugate comprising a capture agent and an oligonucleotide, wherein the oligonucleotide is tethered to the capture agent by its 5’ end and comprises: a target identifier sequence that identifies the target to which the capture agent binds, a unique molecular identifier, a first 5’ flanking sequence that is 5’ of the target identifier sequence and unique molecular identifier and a first 3’ flanking sequence that is 3’ of the target identifier sequence and unique molecular identifier;(b) a second conjugate comprising a capture agent and an oligonucleotide, wherein the oligonucleotide is tethered to the capture agent by its 5’ end and comprises: a target identifier sequence that identifies the target to which the capture agent binds, a unique molecular identifier, a second 5’ flanking sequence that is 5’ of the target identifier sequence and unique molecular identifier anda second 3’ flanking sequence that is 3’ of the target identifier sequence and unique molecular identifier; wherein at least one of the 5’ and 3’ flanking sequences of the second conjugate is different from the 5’ and 3’ flanking sequences of the first conjugate;(c) a first padlock probe that has: a first 5’ end sequence, wherein the first 5’ end sequence is complementary to the first 5’ flanking sequence of the first conjugate; and a first 3’ end sequence, wherein the first 3’ end sequence is complementary to the first 3’ flanking sequence of the first conjugate, and a backbone sequence that connects the 3’ and 5’ end sequences, and(d) a second padlock probe that has: a second 5’ end sequence, wherein the second 5’ end sequence is complementary to the second 5’ flanking sequence of the second conjugate; a second 3’ end sequence, wherein the second 3’ end sequence is complementary to the second 3’ flanking sequence of the second conjugate; and a backbone sequence that connects the 3’ and 5’ end sequences.

26. The reagent system of claim 25, wherein the capture agent of the first conjugate and the capture agent of the second conjugate are, independently, antibodies, aptamers or oligonucleotide probes.

27. The reagent system of claim 25 or 26, wherein the capture agent of the first conjugate and the capture agent of the second conjugate are the same capture agent.

28. The reagent system of claim 25 or 26, wherein the capture agent of the first conjugate and the capture agent of the second conjugate are different, optionally binding to different proteins or different sites in the same protein.

29. The reagent system of any of claims 25-28, further comprising a ligase, a stranddisplacing DNA polymerase (optionally a strand-displacing DNA polymerase that has a 3’ exonuclease activity), and / or a non-strand-displacing polymerase.

30. A method comprising: obtaining the components of the reagent system of any of claims 25-29; hybridizing the first and second padlock probes to the oligonucleotides of the first and second conjugates to form: a first circular complex comprising the first conjugate and the first padlock probe, in which the target identifier sequence and the unique molecular identifier are single-stranded, and a second circular complex comprising the second conjugate and the second padlock probe, in which the target identifier sequence and the unique molecular identifier are single-stranded.

31. The method of claim 30, further comprising performing a gap-fill ligation reaction to extend the 3’ end of the first and second padlock probes using the first and second conjugates as a template to produce: a first covalently closed nucleic acid circle comprising the first padlock probe and complements of the target identifier sequence and unique molecular identifier from the first conjugate; and a second covalently closed nucleic acid circle comprising the second padlock probe and complements of the target identifier sequence and unique molecular identifier from the second conjugate.

32. The method of claim 30 or 31, wherein the method further comprises: binding the first and second conjugates to a cellular sample such that the conjugates bind to target molecules in the sample via the capture agent and wherein the binding is done: before hybridization of the first and second padlock probes; after hybridization of the first and second padlock probes but before the gap-fill ligation reaction; or after the gap-fill ligation reaction.

33. The method of claim 31 or 32, further comprising: amplifying the first and second covalently closed nucleic acid circles by rolling circle amplification (RCA) using the oligonucleotides of the first and second conjugates as an initiation primer, respectively, thereby producing:a first RCA product that comprises: complements of the backbone sequence, the first 3’ end sequence and the first 5’ end sequence of the first padlock probe and copies of the target identifier sequence and unique molecular identifier from the first conjugate, and a second RCA product that comprises: complements of the backbone sequence, the second 3’ end sequence and the second 5’ end sequence of the second padlock probe and copies of the target identifier sequence and unique molecular identifier from the second conjugate.

34. The method of claim 33, wherein: in the first and second circular complexes, the oligonucleotides of the first and second conjugates have protected 3’ ends that cannot be extended by a 3’ exonucleasedeficient DNA polymerase using the first and second covalently closed nucleic acid circle as a template; the gap-fill ligation reaction is done using a 3’ exonuclease-deficient DNA polymerase; and the RCA reaction is done using a strand displacing DNA polymerase that has 3’ exonuclease activity and a polymerase activity, wherein exonuclease activity of the polymerase removes the protected 3’ ends, and, after removal of the protected 3’ ends, the polymerase activity extends the oligonucleotides using the first and second covalently closed nucleic acid circles as templates.

35. The method of claim 34, wherein the 3’ ends of the first and second oligonucleotides have a chemical block or a mismatched nucleotide.

36. The method of any of claims 33-35, wherein the RCA is done in the presence of dUTP, thereby creating RCA products that comprise uracil.

37. The method of any of claims 33-36, further comprising:(a) obtaining a grid oligonucleotide that comprises:(i) a 3’ end sequence that hybridizes to the first RCA product but not the second RCA product; and(i) a 5’ end sequence that hybridizes to the second RCA product but not the first RCA product; and(b) hybridizing the grid oligonucleotide to the first and second RCA products to produce a complex that links the first and second RCA products together.

38. The method of claim 37, further comprising extending both the 3’ end and the 5’ end of the grid oligonucleotide using the first and second RCA products as a template to add the complements of the target identifier sequence and unique molecular identifier from the first conjugate to one end of the grid oligonucleotide and complements of the target identifier sequence and unique molecular identifier from the second conjugate to the other end of the grid oligonucleotide, to produce an extended grid oligonucleotide.

39. The method of claim 37 or 38, wherein the grid oligonucleotide has a unique molecular identifier.

40. The method of claim 38 or 39, wherein the extension is done under gap-fill ligation conditions that comprise at least a gap-fill primer that hybridizes to sites in the first and second RCA products that are upstream of the 5’ end of the grid oligonucleotide and, optionally, a gap-fill stop oligonucleotide that hybridize to sites in the first and second RCA products that are downstream of the 3’ end of the grid oligonucleotide.

41. The method of any of claims 38-40, further comprising amplifying the extended grid oligonucleotide.

42. The method of claim 41, wherein the amplifying is done by polymerase chain reaction, using primers that target the sequences of the gap-fill primer and gap-fill stop oligonucleotide, or by rolling circle amplification.

43. The method of any of claims 38-42, further comprising sequencing the extended grid oligonucleotide or an amplification product thereof.

4. A method comprising:(a) creating a complex comprising:(i) a grid oligonucleotide,(ii) a gap-fill stop oligonucleotide,(iii) a gap-fill primer,(iv) a first uniquely barcoded RCA product, and(v) a secondly uniquely barcoded RCA product, wherein: the 3’ end of the grid oligonucleotide is hybridized to a sequence in the first RCA product that is downstream of the unique RCA product identifier sequence of the first RCA product, the 5’ end of the gap-fill stop oligonucleotide is hybridized to a sequence that is upstream of the unique RCA product identifier sequence of the first RCA product, the 5’ end of the grid oligonucleotide is hybridized to a sequence in the second RCA product that is upstream from the unique RCA product identifier sequence of the second RCA product, and the 3’ end of the gap-fill primer oligonucleotide is hybridized to a sequence that is downstream of the unique RCA product identifier sequence of the second RCA product,(b) incubating the product of (a) under gap-fill ligation conditions to produce:(i) full length products that comprise the sequences of the grid oligonucleotide, the gap-fill primer, the gap-fill stop oligonucleotide, and the complements of the RCA product identifier sequences from the first and second RCA products, and(ii) incomplete products that do not comprise the sequences of the grid oligonucleotide, the gap-fill primer, the gap-fill stop oligonucleotide, and the complements of the RCA product identifier sequences from the first and second RCA products, and(c) enriching for the full-length products using an exonuclease and / or by affinity; wherein:(i) the 3' end of the gap-fill stop oligonucleotide is exonuclease resistant, the 5' end of gap-fill primer is exonuclease resistant, andenrichment of the full-length products is done by treating the product of step (b) with an exonuclease; and / or(ii) the 3' end of the gap-fill stop oligonucleotide or the 5' end of gap-fill primer has a capture moiety, and enrichment of the full-length products is done using a support that has affinity for the capture moiety.

45. The method of claim 44, wherein the grid oligonucleotide has a unique molecular identifier.

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