Methods and compositions for analyzing molecular components of cells

Abstract

A method for studying molecule constituents of a cell population comprising at least a first and a second cell may include obtaining a labeled cell population wherein the first cell may have a plurality of molecular complexes, each with multiple types of biological molecules. The first plurality of molecular complexes in the first cell may be labeled using a first member of a first group of nucleotide sequences representing different cells. A first type of biological molecule may be labeled using a first member of a second group of nucleotide sequences representing different types of biological molecules. A first molecular complex of the first cell may be labeled using a first member of a third group of nucleotide sequences representing different molecular complexes. The method may determine molecule constituents of the first cell using sequence information of the first, second, and third groups of nucleotide sequences.

Description

Dkt. No.: 10611-P0001 -PCTMETHODS AND COMPOSITIONS FOR ANALYZING MOLECULAR COMPONENTS OF CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application the benefit of U.S. Provisional Patent Application No.63 / 763,778, filed February 26, 2025, entitled “Methods and Compositions for Analyzing Molecular Components of Cells.”BACKGROUND

[0002] Field

[0003] Certain embodiments relate to the field of cellular biology and molecular analysis. Specifically, certain embodiments pertain to methods and compositions for studying biological molecules, such as proteins in or on cells.

[0004] Background

[0005] Understanding the spatial organization and interactions of biological molecules, such as proteins, associated with individual cells, is crucial for deciphering cellular functions and molecular signaling pathways. Various techniques have been developed to investigate protein-protein interactions. However, currently available techniques lack the capability to provide precise information regarding molecular complexes in or on individual cells.Therefore, there is a need for improved methods and compositions that enable accurate proximity analysis of proteins in or on individual cells.SUMMARY

[0006] Embodiments of the present disclosure may include a method for studying molecule constituents of a cell population including at least a first and a second cell, the method including obtaining a labeled cell population including at least a first and a second cell. In some embodiments, the first cell is associated with a plurality of molecular complexes.

[0007] In some embodiments, each molecular complex has a plurality of types of biological molecules. Embodiments may also include determining molecule constituents of the first cell using sequence information of nucleotide sequences associated with molecular complexes.Dkt. No.: 10611-P0001 -PCT

[0008] Certain embodiments described herein disclose methods for analyzing the proximity of surface proteins on cells or intracellular protein inside cells. Methods may include, but are not limited to, one or more steps of antibody binding, rolling circle amplification, and nucleotide-based hybridization, to identify and characterize protein-protein interactions at the cellular level.

[0009] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the embodiments claimed. Thus, it should be understood that, although the present embodiments have been specifically disclosed, modification and variation of the concepts herein disclosed may be implemented by those skilled in the art. Such modifications and variations are considered to be within the scope of embodiments as defined by the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Certain embodiments of present disclosure are described in conjunction with the appended 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.

[0011] FIGS. 1A-1E schematically illustrate a certain implementation of a cell labeling and analysis method according to one embodiment.

[0012] FIGS. 2A-2F schematically illustrate a certain implementation of a cell labeling and analysis method according to one embodiment.

[0013] FIGS. 3A-3F schematically illustrate a certain implementation of a cell labeling and analysis method according to one embodiment.

[0014] FIGS. 4A-4E schematically illustrate a certain implementation of a cell labeling and analysis method according to one embodiment.

[0015] FIGS. 5A-5E schematically illustrate a certain implementation of a cell labeling and analysis method according to one embodiment.

[0016] In the appended figures, similar components and / or features can have the same reference label. Further, various components of the same type can be distinguished by following the reference label by a dash and a second label that distinguishes among theDkt. No.: 10611-P0001 -PCTsimilar components. If only the first reference label is used in the specification, the description is applicable to any one of the similar components having the same first reference label irrespective of the second reference label.DETAILED DESCRIPTIONI. Overview

[0017] Understanding the spatial organization and interactions of biological molecules, such as proteins, associated with individual cells, is important for unraveling cellular functions and molecular signaling pathways. Numerous techniques have been developed to study protein-protein interactions, but existing methods lack the precision required to obtain detailed information about molecular complexes within or on individual cells. Improved methodologies and compositions are provided herein to enable accurate proximity analysis of proteins within or on individual cells.

[0018] Before the various embodiments are described, it is to be understood that the teachings of this disclosure are not limited to the particular embodiments described, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present teachings will be limited only by the appended claims.

[0019] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0020] 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

[0021] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.Dkt. No.: 10611-P0001 -PCT

[0022] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0023] All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.II. Definitions

[0024] Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description.

[0025] 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; and, amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0026] 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 disclosure 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.

[0027] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. For example, the term “a primer” refers to one or more primers, i.e., a single primer and multiple primers. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0028] Additional description of various compositions and methods, such as rolling circle amplification or rolling circle amplification products, can be found at WO 2015 / 047186, the entire contents of which is incorporated by reference herein.Dkt. No.: 10611-P0001 -PCT

[0029] “Rolling circle amplification products (RCPs)” refer to the amplified DNA or RNA molecules generated through the process of rolling circle amplification (RCA). Rolling circle amplification is a molecular biology technique used to amplify specific nucleic acid sequences by exponentially amplifying circular templates. In the RCA process, a circular template, typically a single-stranded DNA or RNA molecule, is hybridized with a complementary primer. This primer is then extended by a DNA or RNA polymerase enzyme, creating a new complementary strand that displaces the original template. The displaced strand then serves as a template for subsequent rounds of primer extension, resulting in the generation of long, concatemeric products composed of repeated copies of the target sequence. These amplified products, known as rolling circle amplification products (RCPs), can be used for various applications, including molecular diagnostics, DNA sequencing, genotyping, and detection of nucleic acid-based biomarkers. RCPs offer advantages such as high amplification efficiency, simplicity, and potential signal amplification, making them valuable tools in molecular biology research and diagnostic assays.

[0030] The RCP described herein comprises multiple copies of a template that can be used to obtain proximity tags and sequences specifically designed to facilitate binding or hybridization with protein tags or cell tags. For example, some sequences present in the RCP are designed to facilitate the interaction between templates for the proximity tags and their corresponding protein tags or cell tags. These sequences possess complementary regions that can hybridize or form specific interactions with the protein tags or cell tags, enabling the precise and specific binding of the protein tags or cell tags to the RCP.

[0031] The term “nucleotide” is intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the term “nucleotide” includes those moieties that contain hapten or fluorescent labels and may contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, are functionalized as ethers, amines, or the likes.

[0032] Nucleotide sequences may be incorporated into protein tags, cell tags, proximity tags or complements thereof. For example, protein tags can comprise a nucleotide sequence comprising at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 bp (or any intermediate ranges) designed sequences.Dkt. No.: 10611-P0001 -PCTSimilarly, cell tags can comprise, for example, a nucleotide sequence comprising at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 bp (or any intermediate ranges) designed sequences. Proximity tag or complements thereof can comprise, for example, a nucleotide sequence at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 bp (or any intermediate ranges) degenerate sequences or random sequences.

[0033] “Fluorescent labels” refer to compounds or molecules utilized in various scientific fields and applications, including molecular biology, biochemistry, and imaging techniques. These labels comprise a fluorophore, responsible for fluorescence emission, and a linker molecule connecting the fluorophore to the target molecule of interest, such as a protein or nucleic acid. The fluorophore component of the fluorescent label is capable of absorbing specific wavelengths of light, typically within the ultraviolet (UV) or visible range, and subsequently emitting light at longer wavelengths. This emitted light can be detected and measured using specialized instruments like fluorescence microscopes or plate readers.

[0034] 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 primer may be single-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence or fragment, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The primers herein are selected to be substantially complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5’ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.Dkt. No.: 10611-P0001 -PCT

[0035] 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.

[0036] 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 42C 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.

[0037] 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.

[0038] 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), and RocheDkt. No.: 10611-P0001 -PCTetc. Nextgeneration sequencing methods may also include nanopore sequencing methods or electronic- detection based methods such as, e.g., Ion Torrent technology commercialized by Life Technologies.

[0039] 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.

[0040] The term “extending”, as used herein, refers to the extension of a primer by the addition of nucleotides using a polymerase. If a primer that is annealed to a nucleic acid is extended, the nucleic acid acts as a template for an extension reaction. Extending can also be done by ligation. For clarity: extending can be done by ligation, by a gap-fill ligation reaction, or by a polymerization reaction, as defined above.Ill, Methods

[0041] In certain embodiments, there may be provided a method for studying molecule constituents of a cell population comprising at least a first and a second cell. For example, the cell population can include one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 100, 200, 300, 500, 1000, 10,000, 20,000 or more cells.

[0042] The method may comprise obtaining a labeled cell population comprising at least a first and a second cell. In certain embodiments, the first cell has a plurality of molecular complexes, and each molecular complex has a plurality of types of biological molecules.

[0043] In certain embodiments, the first plurality of molecular complexes in the first cell are labeled using a first member of a first group of nucleotide sequences (for example, cell tags), wherein different members of the first group of nucleotide sequences are designed to represent different cells.

[0044] In certain embodiments, a first type of biological molecule is labeled using a first member of a second group of nucleotide sequences (for example, molecular tags such as protein tags), wherein different members of the second group of nucleotide sequences represents different types of biological molecules.

[0045] In certain embodiments, a first molecular complex of the first cell is labeled using a first member of a third group of nucleotide sequences (for example, proximity tags), wherein different members of the third group of nucleotide sequences represent different molecular complexes.Dkt. No.: 10611-P0001 -PCT

[0046] In further embodiments, the method comprises determining molecule constituents of the first cell using sequence information of the first, second, and third groups of nucleotide sequences in the labeled cell population. The method of determining molecule constituents of the first cell comprises determining that the first type of biological molecule is associated with the first molecular complex if a presence of the first member of the second group of nucleotide sequences (for example, molecular tags) is associated with a presence of the first member of the third group of nucleotide sequences (for example, proximity tags). The method of determining molecule constituents of the first cell comprises determining that the first molecular complex is associated with the first cell if a presence of the first member of the third group of nucleotide sequence (for example, proximity tags) is associated with a presence of the first member of the first group of nucleotide sequences (for example, cell tags).

[0047] In further embodiments, obtaining a labeled cell population comprises labeling a plurality of cells so each cell of the plurality of cells is represented by a different nucleotide sequence or a different combination of nucleotide sequences from the first group of nucleotide sequences to obtain a labelled cell population. In still further embodiments, obtaining a labeled cell population comprises labeling different types of biological molecules, so a different type of biological molecule is represented by a different nucleotide sequence from a second group of nucleotide sequences.

[0048] In further embodiments, obtaining a labeled cell population comprises labeling a subset of biological molecules as belonging to a molecule complex by extending the first member of the second group of nucleotide sequences (for example, molecular tags such as protein tags) along a template in a nucleic acid polymerization action to obtain a first extended product, wherein the first extended product comprises a copy of the first member of the third group of nucleotide sequences; labeling a molecular complex as belonging to a cell by extending the first member of the first group of nucleotide sequences (for example, cell tags) along the same or another copy of the template in a nucleic acid polymerization action to obtain a second extended product, wherein the second extended product comprises a copy of the first member of the third group of nucleotide sequences (for example, proximity tags). In further embodiments, the template is a portion of a rolling circle amplification product.

[0049] In further embodiments, obtaining a labeled cell population comprises: distributing starting cells in a first group of spaces, wherein a particular cell of the starting cells is labeled using a member of the first group of nucleotide sequences; pooling starting cells and re-distributing starting cells in a second group of spaces, wherein the particular cellDkt. No.: 10611-P0001 -PCTof the staring cells is labeled using another member of the first group of nucleotide sequences; and associating molecular complexes of the particular cell with at least two members of a first group of nucleotide sequences that represent a particular cell.

[0050] In other embodiments, obtaining a labeled cell population comprises splitting cells in a group of different spaces, wherein each space has at most one cell and a distinct combination of members of a first group of nucleotide sequences; and associating molecular complexes of a first cell with the distinct combination of members of a first group of nucleotide sequences.

[0051] In certain embodiments, determining molecule constituents of the first cell further comprises sequencing the first, second, third groups of nucleotide sequences in the labeled cell population.

[0052] For example, the first group of nucleotide sequences (for example, cells tags) are attached to particles. In certain embodiments, the first group of nucleotide sequences are attached to particles through a releasable linkage.

[0053] In some embodiments, the method involves a fluorescence detection. For example, the first group of nucleotide sequences are fluorescent labelled, the second group of nucleotide sequences are fluorescent labelled, or the third group of nucleotide sequences are fluorescent labelled.IV. Kits

[0054] A kit comprises a composition for determining constituents of a cell population comprising a first cell, wherein the composition comprises: a plurality of first nucleotide sequences (for example, molecule tags) representing a first type of a biological molecule of the first cell, a plurality of second nucleotide sequences or complements thereof representing the first molecular complex of the first cell (for example, a type of templates for proximity tags), a plurality of third nucleotide sequence or complements thereof representing the second molecular complex of the first cell (for example, another type of templates for proximity tags), a plurality of cell tags, each cell tag comprising a combination of at least two designed nucleotide sequences, wherein the combination of at least two designed nucleotide sequences is designed to represent a first cell origin of the first molecular complex or the second molecular complex as belonging to the first cell.

[0055] In certain embodiments, the first nucleotide sequences are attached to antibodies that selectively bind to the first type of the biological molecule.Dkt. No.: 10611-P0001 -PCT

[0056] In certain embodiments, the second nucleotide sequences have first binding sequences that are allowed to bind (e.g., hybridize selectively) to the first nucleotide sequences, and have second binding sequences that are allowed to bind (e.g., hybridize selectively) to the cell tags. In certain embodiments, the second nucleotide sequences comprise rolling circle amplification products (RCPs).

[0057] In some embodiments, the kit further comprises a plurality of cell tags, each cell tag comprising a combination of at least two designed nucleotide sequences, wherein the combination of at least two designed nucleotide sequences is designed to represent a first cell origin of the first molecular complex, wherein the second nucleotide sequences have second binding sequences that are allowed to bind to the cell tags, e.g., binding sequences that are allowed to ligate or hybridize to the cell tags.

[0058] For example, the kit comprises a container with different spaces, each space has a unique combination of cell tags. In another example, the kit comprises a plurality of particles, and wherein a particular particle comprises a combination of cell tags different from another combination of cell tags on a different particle. In certain embodiments, the kit comprises a plurality of particles, wherein the cell tags are attached to the particles through a releasable linkage. For example, the particles may comprise beads. There may be provided a method for studying molecule constituents of a cell population comprising at least a first and a second cell, the method comprising the use of the kit as described herein.V. Non-limiting Exemplary Embodiments

[0059] As illustrated in FIGS. 1A-1E, certain embodiments are provided for analysis of proteins from cells. FIGS. 2A-2F, FIGS. 3A-3F, FIGS. 4A-4E, and FIGS. 5A-5E are additional embodiments provided for analysis of proteins from cells by using different approaches to process cells.

[0060] As illustrated in FIGS. 1A-1E, cells with surface molecules can be labeled with proximity tags, cell tags, protein tags or any combination thereof. These tags help proteins to be linked to a particular complex and a particular cell. This process may include one or more steps including obtaining cells with surface molecules, using antibody-bound protein tags to label proteins, using rolling circle amplification (RCA) products to identify protein complexes, adding proximity tags, and sequencing and data analysis.

[0061] Step 1 : Obtaining cells with surface molecules

[0062] A plurality of cells are obtained. As illustrated in FIG. 1A, for example, on the surface of a cell (100), there exist various types of surface molecules. Surface molecules canDkt. No.: 10611-P0001 -PCTinclude surface proteins or other biologic molecules. Non-limiting exemplary types of surface proteins are denoted as A, B, C, D, and E. As exemplified and illustrated here, protein A (102) and protein B (104) are found in proximity to each other to form a protein complex, another protein A (106), protein D (108), and protein E (110) are found in proximity to each other to form another protein complex, and protein C (112) exists as an individual molecule.

[0063] Step 2: Using antibody-bound Protein Tags to label proteins

[0064] As illustrated in FIG. IB, three corresponding antibodies labeled with different Protein Tags are allowed to selectively bind to the surface proteins A, protein B, protein C, protein D, and protein E so that different surface proteins A, B, C, D, E are labeled with different Protein Tags. For example, protein tags are a set of designed nucleotide sequences that distinguish different types of proteins by attaching to antibodies that selectively bind to proteins. For example, protein tags can be a nucleotide sequence comprising at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 bps of designed sequences or any intermediate ranges.

[0065] In the first protein complex, protein A and protein B are labeled with Protein Tag A (114) and Protein Tag B (116), respectively. In the second protein complex, another protein A is labeled with Protein Tag A (114), while protein D and protein E are labeled with Protein Tag D (118) and Protein Tag E (120). Protein C is labeled with Protein Tag C (122). This labeling facilitates the subsequent identification and differentiation of the proteins of interest into different types of proteins.

[0066] Step 3 : Using rolling circle amplification (RCA) products to identify protein complexes

[0067] As illustrated in FIG. 1C, three rolling circle amplification products (RCPs) are provided, wherein each RCP carries repeats of a unique Proximity Tag template and one or more universal complementary sequences. The RCPs are allowed to hybridize to the 3’ end of oligonucleotides present on protein tags that are attached to the antibodies using one or more universal complementary sequences. Because of the spatial proximity, proteins in the same protein complex or in close proximity are likely to hybridize to the same RCP and share the same proximity tag sequences.

[0068] For example, to identify the first protein complex, the first RCP (124), carrying repeats of a unique Proximity Tag template (“Proximity Tag 1”, 126), hybridizes to the 3’ end of the oligonucleotides on Protein Tag A and Protein Tag B. To identify the second protein complex, the second RCP (128), carrying repeats of another unique Proximity Tag template (“Proximity Tag 2”, 130), hybridizes to the 3’ end of the oligonucleotides on ProteinDkt. No.: 10611-P0001 -PCTTag A, Protein Tag D, and Protein Tag E. The third RCP (132), carrying repeats of another unique Proximity Tag template (“Proximity Tag 3”, 134), hybridizes to the 3’ end of the oligonucleotides on Protein Tag C.

[0069] As also illustrated in FIG. 1C, the RCPs, carrying repeats of Proximity Tag templates, are also hybridized with oligonucleotides carrying Cell Tags. Each oligonucleotide carries a cell tag. After one or more methods for cell separation into individual spaces, RCPs originating from the same cell will be allowed to hybridize with the same combination of Cell Tags, for example as shown herein, a unique combination of Cell Tag 1 (136), Cell Tag 16 (138), and Cell Tag 52 (140). For example, cell tags can be a nucleotide sequence comprising at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 bps of designed sequences or any intermediate ranges.

[0070] Step 4: Adding Proximity Tags

[0071] Subsequently, the oligonucleotides carrying Cell Tags and Protein Tags hybridized on the RCPs are extended to obtain a corresponding Proximity Tag sequence. This Proximity Tag sequence is a reverse-complement counterpart of or complementary to the corresponding Proximity Tag template. After extension, the Cell Tags and corresponding Proximity Tag sequences are connected on an extended product. Also, the Protein Tags and corresponding Proximity Tag sequences are connected on a different extended product.

[0072] Step 4.1 : Adding Proximity Tags to protein tags.

[0073] As illustrated in FIG. ID, Protein Tags are extended to obtain extension products comprising a corresponding Proximity Tag sequence. For example, in the first complex having Protein A and Protein B, Protein Tag A is extended to obtain an extension product comprising a Proximity Tag 1 sequence and Protein Tag B is also extended to obtain an extension product comprising a Proximity Tag 1 sequence. The Proximity Tag 1 sequence is a reverse-complement counterpart of or complementary to a unique Proximity Tag template (“Proximity Tag 1”, labeled as “126” in FIG. 1C).

[0074] Also as illustrated in FIG. ID, similar to the first complex, in the second complex having Protein A, Protein D, and Protein E, Protein Tag A is extended to obtain a Proximity Tag 2 sequence. Protein Tag D is extended to obtain a Proximity Tag 2 sequence. Protein Tag E is extended to obtain a Proximity Tag 2 sequence. The Proximity Tag 2 sequence is a reverse-complement counterpart of or complementary to a unique Proximity Tag template (“Proximity Tag 2”, labeled as “130” in FIG. 1C).

[0075] Also as illustrated in FIG. ID, similar to the first complex, Protein Tag C in the third complex is extended to obtain a Proximity Tag 3 sequence, which is a reverse-Dkt. No.: 10611-P0001 -PCTcomplement counterpart of or is complementary to a unique Proximity Tag template (“Proximity Tag 3”, labeled as “134” in FIG. 1C).

[0076] Step 4.2: Adding Proximity Tags to Cell Tags.

[0077] As also illustrated in FIG. ID, for example, in the first complex, Cell Tag 1 is extended along the first RCP (124) to obtain an extension product (146) that comprises a Proximity Tag 1 sequence. Cell Tag 16 is extended to obtain an extension product (148) that comprises a Proximity Tag 1 sequence. Cell Tag 52 is extended to obtain an extension product (150) that comprises a Proximity Tag 1 sequence. The Proximity Tag 1 sequence is a reverse-complement counterpart of or complementary to a unique Proximity Tag template (the Proximity Tag template is labeled as “Proximity Tag 1” and 126 in FIG. 1C).

[0078] In the second complex, Cell Tag 1 is extended to obtain a Proximity Tag 2 sequence. Cell Tag 16 is extended to obtain a Proximity Tag 2 sequence. Cell Tag 52 is extended to obtain a Proximity Tag 2 sequence. The Proximity Tag 2 sequence is a reversecomplement counterpart of or complementary to a unique Proximity Tag template (“Proximity Tag 2”, labeled as “130” in FIG. 1C).

[0079] In the third complex that only has protein C, Cell Tag 1 is extended to obtain a Proximity Tag 3 sequence. Cell Tag 16 is extended to obtain a Proximity Tag 3 sequence. Cell Tag 52 is extended to obtain a Proximity Tag 3 sequence. The Proximity Tag 3 sequence is a reverse-complement counterpart of or is complementary to a unique Proximity Tag template (“Proximity Tag 3”, labeled as “134” in FIG. 1C).

[0080] Step 5: Sequencing and Data Analysis

[0081] After sequencing, all the extension products are first sorted based on shared Proximity Tags and then sorted based on shared Cell Tags.

[0082] The extended products sharing the same Proximity Tag sequences are considered to be in proximity, or in the same protein complex.

[0083] And the extended products that have the same combination of Cell Tags are considered to originate from the same cell.

[0084] Brief exemplary explanations for how proteins are associated with a particular complex and a particular cell are provided.

[0085] In the first group that comprises all the extended products that share Proximity Tag 1, there are two subgroups of extension products: the first subgroup includes the first extension product (142) and the second extension product (144), and the second subgroup includes the third (146), fourth (148), and fifth extension product (150).Dkt. No.: 10611-P0001 -PCT

[0086] In the first subgroup, the first extension product (142) contains Protein Tag A and Proximity Tag 1 sequence, and the second extension product (144) contains Protein Tag B and Proximity Tag 1 sequence. So, this grouping indicates that Protein A represented by Protein Tag A and Protein B represented by Protein Tag B belong to the same protein complex represented by Proximity Tag 1 sequence.

[0087] In the second subgroup, the third extension product (146) contains Cell Tag 1 and Proximity Tag 1 sequence, the fourth extension product (148) contains Cell Tag 16 and Proximity Tag 1 sequence, and the fifth extension product (150) contains Cell Tag 52 and Proximity Tag 1 sequence. So, this indicates that the protein complex represented by Proximity Tag 1 sequence is in a cell represented by a combination of Cell Tag 1, Cell Tag 16, and Cell Tag 52. This cell origin information can be combined with the protein complex information above to provide that Protein A and Protein B are in the same protein complex represented by Proximity Tag 1 sequence and is also in a cell represented by a combination of Cell Tag 1, Cell Tag 16, and Cell Tag 52.

[0088] In the second group that comprise the extended products that share Proximity Tag 2, there are also two subgroups of extension products after sorting by proximity tags: the first subgroup includes the first extension product (152), the second extension product (154), and the third extension product (156), and the second group includes the third (158), fourth (160), and fifth extension product (162).

[0089] In the first subgroup, the first extension product (152) contains Protein Tag A and Proximity Tag 2, the second extension product (154) contains Protein Tag D and Proximity Tag 2 sequence, and the third extension product (156) contains Protein Tag E and Proximity Tag 2 sequence. So, this indicates that Protein A represented by Protein Tag A, Protein D represented by Protein Tag D, Protein E represented by Protein E belong to the same protein complex represented by Proximity Tag 2 sequence.

[0090] In the second subgroup, the third extension product (158) contains Cell Tag 1 and Proximity Tag 2 sequence, the fourth extension product (160) contains Cell Tag 16 and Proximity Tag 2 sequence, and the fifth extension product (162) contains Cell Tag 52 and Proximity Tag 2 sequence. So, this indicates that the protein complex represented by Proximity Tag 2 sequence is in a cell represented by a combination of Cell Tag 1, Cell Tag 16, and Cell Tag 52. This cell origin information can be combined with the protein complex information above to provide that Protein A, Protein D, and Protein E are in a different protein complex represented by Proximity Tag 2 sequence and is also in a cell represented by a combination of Cell Tag 1, Cell Tag 16, and Cell Tag 52.Dkt. No.: 10611-P0001 -PCT

[0091] In the third group that comprises all the extended products that share Proximity Tag 3, there are also two subgroups of extension products: the first subgroup includes the first extension product (164), and the second subgroup includes the second (166), third (168), and fourth extension product (170).

[0092] In the first subgroup, the first extension product (164) contains Protein Tag C and Proximity Tag 3 sequence. So, this indicates that Protein C represented by Protein Tag C belong to a protein complex represented by Proximity Tag 3 sequence.

[0093] In the second subgroup, the second extension product (166) contains Cell Tag 1 and Proximity Tag 3 sequence, the third extension product (169) contains Cell Tag 16 and Proximity Tag 3 sequence, and the fourth extension product (170) contains Cell Tag 52 and Proximity Tag 3 sequence. So, this indicates that the protein complex represented by Proximity Tag 3 sequence is in a cell represented by a combination of Cell Tag 1, Cell Tag 16, and Cell Tag 52. This cell origin information can be combined with the protein complex information above to provide that Protein C is a different protein complex represented by Proximity Tag 3 sequence and is also in a cell represented by a combination of Cell Tag 1, Cell Tag 16, and Cell Tag 52.

[0094] Overall, proteins are identified to be in different protein complexes represented by different proximity tags, and then different protein complexes are also identified to belong to the same cell because different proximity tags are connected to the same combination of cell tags.

[0095] As illustrated in FIGS. 2A-2F, certain embodiments are provided for analysis of proteins from cells using an approach to label individual cells, for example, involving plate splitting.

[0096] Step 1 : Antibody Binding, RCP Hybridization, and First Plate Splitting (FIG. 2A)

[0097] Surface proteins of cells are bound by antibodies, with antibodies labeled with oligonucleotides containing a unique Protein Tag. For example, on the surface of a cell (200), a protein complex (202) comprising a protein A and a protein B. Protein A is bound with an antibody labeled with oligonucleotides containing Protein Tag A (204), and protein B is bound with an antibody labeled with oligonucleotides containing Protein Tag B (206).

[0098] The oligonucleotides labeling antibodies are then hybridized with RCPs, each RCP carrying identical repeats of a unique Proximity Tag template. For example, in an exploded view as illustrated in FIG. 2A, to identify the protein complex having protein A and protein B, a RCP (208) hybridizes to the 3’ end of the oligonucleotides on Protein Tag ADkt. No.: 10611-P0001 -PCT(204) and Protein Tag B (206). The RCP (208) contains repeats of a unique Proximity Tag template.

[0099] The cells are split into a first plate that has a plurality of wells, where each well of the plurality of wells contains different oligonucleotides associated with each well, and each different oligonucleotide carries a particular type of Cell Tags or a particular combination of Cell Tags. For example, all the oligonucleotides in one well contain a type of Cell Tag 1 (210). The oligonucleotides in a well can bind to RCPs from cells within the same well, ensuring that all RCPs from the same well are bound by the same type of Cell Tags (e.g., Cell Tag 1).

[0100] Step 2: Second Plate Splitting (FIG. 2B)

[0101] Cells from all wells are pooled together and split again into another plate carrying a set of Cell Tags different from Cell Tags in the first plate. For example, the cell (200) randomly distributes into a well that contains oligonucleotides carrying another particular type of cell tags Cell Tag 136 (212). Since the pooling and splitting of cells are randomly distributed, this ensures that all RCPs from the same cell 214 are presumably bound by the same set of two Cell Tags (e.g., Cell Tag 1 and Cell Tag 136), allowing for further multiplexed analysis.

[0102] Step 3 : Third Plate Splitting (FIG. 2C)

[0103] Cells are pooled together and are split once again into a third plate, with wells of the third plate carrying a third set of Cell Tags. For example, the cell (200) randomly distributes into a well that contains oligonucleotides carrying another particular type of cell tags Cell Tag 308 (214). Since the pooling and splitting of cells are randomly distributed, this step ensures that each RCP from the same cell is bound by the same set of three different Cell Tags (e.g., Cell Tag 1, Cell Tag 136, and Cell Tag 308), enabling comprehensive multiplexed analysis.

[0104] Step 4: Adding Proximity Tags (FIG. 2D)

[0105] In this step, all the cells are pooled together. All the oligonucleotides (including sequences that have Cell Tags and sequences that have Protein Tags) binding to the RCPs are allowed to extend on the RCPs to obtain a particular type of Proximity Tag sequence corresponding to a particular RCP. Each different RCP has repeats of a different Proximity Tag template sequence.

[0106] For example, on the RCP (208), Protein Tag A, Protein Tag B, Cell Tag 1, Cell Tag 136, and Cell Tag 308 are all extended to obtain a copy of the same Proximity Tag sequence by extending along the RCP (208) to obtain a plurality of extended products.Dkt. No.: 10611-P0001 -PCT

[0107] Step 5 : Extended Products (FIG. 2E)

[0108] The extended products should contain a combination of Protein Tags and Proximity Tags or a combination of Cell Tags and Proximity Tags, allowing for the subsequent analysis and identification of protein complexes in single cells.

[0109] In all the extended products that share the same Proximity Tag, there are two groups of extension products: the first group includes the first extension product (216) and the second extension product (218), and the second group includes the third (220), fourth (222), and fifth extension product (224).

[0110] In the first subgroup, the first extension product (216) contains Protein Tag A and Proximity Tag, and the second extension product (218) contains Protein Tag B and Proximity Tag. So, this indicates that Protein A represented by Protein Tag A and Protein B represented by Protein Tag B belong to the same protein complex represented by the same Proximity Tag.

[0111] In the second subgroup, the third extension product (220) contains Cell Tag 1 and Proximity Tag, the fourth extension product (222) contains Cell Tag 136 and Proximity Tag, and the fifth extension product (224) contains Cell Tag 308 and Proximity Tag. So, this indicates that the protein complex represented by Proximity Tag is in a cell represented by a combination of Cell Tag 1, Cell Tag 136, and Cell Tag 308. This cell origin information can be combined with the protein complex information above to provide that Protein A and Protein B are in the same protein complex represented by Proximity Tag and is also in a cell represented by a combination of Cell Tag 1, Cell Tag 136, and Cell Tag 308.

[0112] Step 6: Sequencing and Data Analysis (FIG. 2F)

[0113] After sequencing, the acquired Proximity Tags are utilized to identify proteins in proximity. Different proteins in proximity can be grouped together based on the shared Proximity Tag sequence.

[0114] Different Proximity Tags belonging to the same cell can be associated by using the same combination of Cell Tags. By comparing the Cell Tags associated with each Proximity Tag, the proteins in proximity within the same cell can be accurately identified, allowing for comprehensive mapping of protein networks within individual cells. For example, Protein Tag B and Protein Tag X are associated with the first Proximity Tag, Protein Tag A and Protein Tag B are associated with the second Proximity Tag, and Protein Tag X is associated with the third Proximity Tag. Also, the three proximity tags are shown to be associated with the same combination of Cell Tags (Cell Tag 1, Cell Tag 136, and Cell Tag 208). This indicates that proteins represented by Protein tags A, Protein Tag B, andDkt. No.: 10611-P0001 -PCTProtein Tag X are in the same cell, and their protein numbers and spatial distribution can be determined based on distribution of Protein Tags.

[0115] As illustrated in FIGS. 3A-3F, certain embodiments are provided for analysis of proteins from cells.

[0116] Step 1 : Distribution of Cells (FIG. 3A)

[0117] Surface proteins of cells are bound by antibodies, each labeled with oligonucleotides containing a unique Protein Tag. The oligonucleotides on the antibodies are then hybridized with RCPs, each carrying a unique Proximity Tag template (for example, one RCP with a unique Proximity Tag template is illustrated). This step ensures specific labeling of protein complexes.

[0118] The cells are distributed into micro wells in a diluted manner, ensuring that each well contains either zero or one cell.

[0119] Step 2: Beads Bound with Cell Tags (FIG. 3B)

[0120] Prior to adding the cells, each well contains a random combination of particles, each associated with oligonucleotides containing a Cell Tag. For example, one well may contain beads associated with Cell Tags 1, 10, and 36, while another well may contain beads associated with Cell Tags 2, 5, and 6. Therefore, each well contains a unique combination of Cell Tags.

[0121] Step 3 : Adding Cell Tags (FIG. 3C)

[0122] After adding the cells to the wells, the oligonucleotides carrying the Cell Tags are released from the beads and bind to the RCPs associated with surface proteins on a single cell. This results in each RCP associated with surface proteins on a single cell being bound by the same combination of Cell Tags in a well. This step enables multiplexed analysis of protein complexes within individual cells.

[0123] Step 4: Adding Proximity Tags (FIG. 3D)

[0124] The oligonucleotides binding to the RCPs are allowed to extend on the RCPs, resulting in the acquisition of the Proximity Tag sequence on an extended product. The extended products should contain a combination of Protein Tags and Proximity Tags or a combination of Cell Tags and Proximity Tags, allowing for the subsequent analysis and identification of protein complexes in single cells.

[0125] Step 5: Proximity Analysis (FIG. 3E)

[0126] After sequencing, the acquired Proximity Tags are utilized to identify proteins in proximity. The Proximity Tags belonging to different proteins in proximity can be grouped together based on the shared Proximity Tag sequence.Dkt. No.: 10611-P0001 -PCT

[0127] Step 6: Cell Tag-Based Cell Identification (FIG. 3F)

[0128] Different Proximity Tags belonging to the same cell can be associated by using the same combination of Cell Tags. By comparing the Cell Tags associated with each Proximity Tag, the proteins in proximity within the same cell can be accurately identified, allowing for comprehensive mapping of protein networks within individual cells.

[0129] As illustrated in FIGS. 4A-4E, certain embodiments are provided for analysis of proteins from cells. Proteins here include intracellular proteins.

[0130] Step 1 : Obtaining Cells (FIG. 4A)

[0131] Cells are contained within a tube.

[0132] Step 2: Cell Lysis (FIG. 4A)

[0133] Cells are distributed into micro wells in a diluted manner to ensure each well contains either zero or one cell. The wells are pre-coated with antibodies that can catch and bind to intracellular proteins. The wells also contain a lysis buffer, which facilitates the release of proteins from the cells and their subsequent capture on the surface of the well by antibodies.

[0134] Step 3: Antibody Binding (FIG. 4A)

[0135] The proteins are captured in cells by binding to antibodies, so that each type of proteins is labeled with oligonucleotides containing a different type of Protein Tags. This step ensures specific labeling of the captured proteins.

[0136] Step 4: RCP Hybridization (FIG. 4A)

[0137] The oligonucleotides on the antibodies are hybridized with RCPs, each carrying repeats of a unique Proximity Tag template. This step allows for the identification of protein complexes by proximity tags.

[0138] Step 5 : Cell Tag Binding (FIG. 4B)

[0139] Each well is randomly distributed with particles, each particle carrying a different type of Cell Tag. Each well contains a random combination of different particles with different Cell Tags. For example, one well may contain particles with Cell Tags 1, 10, and 36. The oligonucleotides on the particles are released and hybridize to the RCPs in the well, ensuring that all RCPs within the well are bound by the same combination of Cell Tags. This enables the association of protein complexes within the same well.

[0140] Step 6: Adding Proximity Tags (FIG. 4C)

[0141] All the oligonucleotides on the RCPs within a well are allowed to extend, resulting in extension products that contain a copy of the Proximity Tag sequence. The extension products from different RCPs within the same well share the same combination ofDkt. No.: 10611-P0001 -PCTCell Tags, facilitating downstream analysis for identification of proteins complexes in the same cells.

[0142] Step 7: Sequencing (FIG. 4D)

[0143] After the extension step, the samples are subjected to sequencing, allowing for the identification and characterization of the Proximity Tags associated with the proteins captured within each micro well, protein tags associated with different types of proteins, and cell tags for cell identification.

[0144] Step 8: Proximity Analysis and Cell Mapping (FIG. 4E)

[0145] After sequencing data is collected, the Proximity Tags on the extended products are utilized to identify proteins in proximity. Different proteins in proximity can be grouped together based on the shared Proximity Tag sequence.

[0146] Different Proximity Tags belonging to the same cell can be associated by using the same combination of Cell Tags. By comparing the Cell Tags associated with each Proximity Tag, the proteins in proximity within the same cell can be accurately identified, allowing for comprehensive mapping of protein complexes within individual cells.

[0147] As illustrated in FIGS. 5A-5E, certain embodiments are provided for analysis of proteins from cells. Proteins here include secreted proteins.

[0148] Step 1 : Obtaining Cells (FIG. 5A)

[0149] Cells are contained within a tube.

[0150] Step 2: Cell Distribution (FIG. 5A)

[0151] Cells are distributed into micro wells in a diluted manner to ensure each well contains either zero or one cell. The micro wells are designed to capture and retain secreted cell materials, such as proteins, on their surface.

[0152] Step 3: Protein Capture (FIG. 5A)

[0153] The proteins secreted by the cells in the well are captured on the surface of the well, which is coated with antibodies specific to the target proteins. The captured proteins in the well are bound by antibodies, each type of protein is labeled with oligonucleotides containing a different type of Protein Tags. This step ensures specific labeling of the captured proteins.

[0154] Step 4: RCP Hybridization (FIG. 5A)

[0155] The oligonucleotides on the antibodies are hybridized with RCPs, each carrying repeats of a unique Proximity Tag template. This step allows for the identification of protein complexes by proximity tags.

[0156] Step 5 : Cell Tag Binding (FIG. 5B)Dkt. No.: 10611-P0001 -PCT

[0157] Each well is randomly distributed with particles, each carrying a different type of Cell Tag. Each well contains a random combination of different particles with different Cell Tags. For example, one well may contain particles with Cell Tags 1, 10, and 36. The oligonucleotides on the particles are released and hybridize to the RCPs in the well, ensuring that all RCPs within the well are bound by the same combination of Cell Tags. This enables the association of protein complexes within the same well.

[0158] Step 6: Adding Proximity Tags (FIG. 5C)

[0159] All the oligonucleotides on the RCPs within a well are allowed to extend, resulting in extension products that contain a copy of the Proximity Tag sequence. The extension products from different RCPs within the same well share the same combination of Cell Tags, facilitating downstream analysis for identification of proteins complexes in the same cells.

[0160] Step 7: Sequencing (FIG. 5D)

[0161] After the extension step, the samples are subjected to sequencing, allowing for the identification and characterization of the Proximity Tags associated with the secreted proteins captured within each micro well, protein tags associated with different types of proteins, and cell tags for cell identification.

[0162] Step 8: Proximity Analysis and Cell Mapping (FIG. 5E)

[0163] After sequencing data is collected, the Proximity Tags on the extended products are utilized to identify proteins in proximity or in the same complexes. Different proteins in proximity can be grouped together based on the shared Proximity Tag sequence.

[0164] Different Proximity Tags belonging to the same cell can be associated by using the same combination of Cell Tags. By comparing the Cell Tags associated with each Proximity Tag, the proteins in proximity within the same cell can be accurately identified, allowing for comprehensive mapping of protein networks within individual cells.

[0165] In non-limiting embodiments, the method disclosed herein offers several advantages. For example, it enables the analysis of surface protein proximity on individual cells, providing insights into cell-specific protein-protein interactions and their spatial distribution. In some embodiments, this approach has broad applications in cellular biology, molecular medicine, drug discovery, and diagnostics, as it allows for the identification of key protein interactions within cells and tissues, facilitating the understanding of disease mechanisms and the development of targeted therapies. In additional embodiments, the disclosed method provides several advantages over existing techniques. For example, it may allow for the comprehensive analysis of secreted cell materials within a micro well-basedDkt. No.: 10611-P0001 -PCTsystem, enabling the identification and characterization of protein-protein interactions specific to individual cells. In some embodiments, the method's simplicity, scalability, and compatibility with high-throughput platforms make it applicable for various research and clinical applications, including cellular biology, drug discovery, diagnostics, and personalized medicine.

[0166] In certain embodiments, the disclosed method allows for proximity analysis of surface proteins on cells and micro well-based proximity analysis of secreted cell materials. In further embodiments, it offers a novel approach to investigate cell-specific protein-protein interactions. In some embodiments, specific antibodies, protein and proximity tags, rolling circle amplification, and a combination of cell distribution, protein capture, antibody labeling, RCP hybridization, Cell Tag binding, sequencing, and proximity analysis can be used or combined to enable the identification, labeling, and mapping of protein networks associated with cells and secreted materials within individual micro wells. These advancements contribute significantly to the fields of cellular biology, molecular analysis, and the understanding of cellular processes and disease mechanisms.IV. Additional Considerations

[0167] The headers and sub-headers between sections and subsections of this document are included solely for the purpose of improving readability and do not imply that features cannot be combined across sections and subsection. Accordingly, sections and subsections do not describe separate embodiments.

[0168] Some embodiments of the present disclosure include a system including one or more data processors. In some embodiments, the system includes a non-transitory computer readable storage medium containing instructions which, when executed on the one or more data processors, cause the one or more data processors to perform part or all of one or more methods and / or part or all of one or more processes disclosed herein. Some embodiments of the present disclosure include a computer-program product tangibly embodied in a non-transitory machine-readable storage medium, including instructions configured to cause one or more data processors to perform part or all of one or more methods and / or part or all of one or more processes disclosed herein.

[0169] The preceding description provides preferred exemplary embodiments only, and is not intended to limit the scope, applicability or configuration of the disclosure. Rather, the preceding description of the preferred exemplary embodiments will provide those skilled in the art with an enabling description for implementing various embodiments. It is understoodDkt. No.: 10611-P0001 -PCTthat various changes may be made in the function and arrangement of elements without departing from the spirit and scope as set forth in the appended claims.

[0170] Specific details are given in the preceding description to provide a thorough understanding of the embodiments. However, it will be understood that the embodiments may be practiced without these specific details. For example, circuits, systems, networks, processes, and other components may be shown as components in block diagram form in order not to obscure the embodiments in unnecessary detail. In other instances, well-known circuits, processes, algorithms, structures, and techniques may be shown without unnecessary detail in order to avoid obscuring the embodiments.

Claims

Dkt. No.: 10611-P0001 -PCTWhat is claimed is:

1. A method for studying molecule constituents of a cell population comprising at least a first and a second cell, the method comprising:obtaining a labeled cell population comprising at least a first and a second cell, wherein the first cell has a plurality of molecular complexes, and wherein each molecular complex has a plurality of types of biological molecules,wherein the first plurality of molecular complexes in the first cell are labeled using a first member of a first group of nucleotide sequences, wherein different members of the first group of nucleotide sequences represents different cells;wherein a first type of biological molecule is labeled using a first member of a second group of nucleotide sequences, wherein different members of the second group of nucleotide sequences represents different types of biological molecules; andwherein a first molecular complex of the first cell is labeled using a first member of a third group of nucleotide sequences, wherein different members of the third group of nucleotide sequences represents different molecular complexes; anddetermining molecule constituents of the first cell using sequence information of the first, second, and third groups of nucleotide sequences in the labeled cell population, comprising:determining that the first type of biological molecule is associated with the first molecular complex if a presence of the first member of the second group of nucleotide sequences is associated with a presence of the first member of the third group of nucleotide sequences; anddetermining that the first molecular complex is associated with the first cell if a presence of the first member of the third group of nucleotide sequence is associated with a presence of the first member of the first group of nucleotide sequences.Dkt. No.: 10611-P0001 -PCT2. The method of claim 1, wherein obtaining a labeled cell population comprises labeling a plurality of cells so each cell of the plurality of cells is represented by a different nucleotide sequence or a different combination of nucleotide sequences from the first group of nucleotide sequences to obtain a labelled cell population.

3. The method of claim 1, wherein obtaining a labeled cell population comprises labeling different types of biological molecules, so a different type of biological molecule is represented by a different nucleotide sequence from a second group of nucleotide sequences.

4. The method of claim 1, wherein obtaining a labeled cell population comprises:a) labeling a subset of biological molecules as belonging to a molecule complex by extending the first member of the second group of nucleotide sequences along a template in a nucleic acid polymerization action to obtain a first extended product, wherein the first extended product comprises a copy of the first member of the third group of nucleotide sequences; andb) labeling a molecular complex as belonging to a cell by extending the first member of the first group of nucleotide sequences along the same or another copy of the template in a nucleic acid polymerization action to obtain a second extended product, wherein the second extended product comprises a copy of the first member of the third group of nucleotide sequences.

5. The method of claim 4, wherein the template is a portion of a rolling circle amplification product.

6. The method of claim 1, wherein obtaining a labeled cell population comprises:a) distributing starting cells in a first group of spaces, wherein a particular cell of the starting cells is labeled using a member of the first group of nucleotide sequences;b) pooling starting cells and re-distributing starting cells in a second group of spaces, wherein the particular cell of the staring cells is labeled using another member of the first group of nucleotide sequences; andc) associating molecular complexes of the particular cell with at least two members of a first group of nucleotide sequences.

7. The method of claim 1, wherein obtaining a labeled cell population comprises:a) splitting cells in a group of different spaces, wherein each space has at most one cell and a distinct combination of members of a first group of nucleotide sequences; andDkt. No.: 10611-P0001 -PCTb) associating molecular complexes of a first cell with the distinct combination of members of a first group of nucleotide sequences.

8. The method of claim 1, wherein determining molecule constituents of the first cell further comprises sequencing the first, second, third groups of nucleotide sequences in the labeled cell population.

9. The method of claim 1, wherein the first group of nucleotide sequences are attached to particles.

10. The method of claim 9, wherein the first group of nucleotide sequences are attached to particles through a releasable linkage.

11. The method of claim 1, wherein the first group of nucleotide sequences are fluorescent labelled.

12. The method of claim 1, wherein the second group of nucleotide sequences are fluorescent labelled.

13. The method of claim 1, wherein the third group of nucleotide sequences are fluorescent labelled.

14. A kit comprises a composition for determining constituents of a cell population comprising a first cell, wherein the composition comprises:a plurality of first nucleotide sequences representing a first type of a biological molecule of the first cell, wherein the first nucleotide sequences are attached to antibodies that selectively bind to the first type of the biological molecule; a plurality of second nucleotide sequences or complements thereof representing the first molecular complex of the first cell, wherein the second nucleotide sequences have first binding sequences that are allowed to bind to the first nucleotide sequences; anda plurality of cell tags, each cell tag comprising a combination of at least two designed nucleotide sequences, wherein the combination of at least two designed nucleotide sequences is designed to represent a first cell origin of the first molecular complex, wherein the second nucleotide sequences have second binding sequences that are allowed to bind to the cell tags.

15. The kit of claim 14, wherein the kit comprises a container with different spaces, each space has a unique combination of cell tags.

16. The kit of claim 14, wherein the kit comprises a plurality of particles, and wherein a particle comprises a combination of cell tags different from another combination of cell tags on a different particle.Dkt. No.: 10611-P0001 -PCT17. The kit of claim 14, wherein the kit comprises a plurality of particles, wherein the cell tags are attached to the particles through a releasable linkage.

18. The kit of claiml6 or 17, wherein the particles comprise beads.

19. The kit of claim 14, wherein the second nucleotide sequences comprise rolling circle amplification products (RCPs).

20. A method for studying molecule constituents of a cell population comprising at least a first and a second cell, the method comprising the use of the kit in accordance with claim 14.

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